Permeabilization of cells during animal virus infection

Pharmac. Ther. Vol. 23, pp. 109 to 145, 1983 Printed in Great Britain. All rights reserved

0163-7258/83 $0.00 + 0.50 Copyright © 1983 Pergamon Press Ltd

Specialist Subject Editor: D. SrrOGAR

PERMEABILIZATION OF CELLS DURING VIRUS INFECTION

ANIMAL

L u I s CARRASCO a n d J U A N CARLOS L A C A L

Departamento de Microbiologia, Centro de Biologia Molecular, Universidad Aut6noma and Consejo Superior de Investigaciones Cientificas, Canto Blanco, Madrid-34, Spain

1. INTRODUCTION The plasma membrane of an animal cell has two important functions. It is the physical iLimit of the cell and serves to maintain all the cellular materials in a given space with the appropriate physico-chemical characteristics for their activities and interactions. In the second place, the membrane is used by the cell for communication with the outside medium. It collects signals on its surface that are integrated and transmitted into the cell, giving rise to a metabolic response (Singer, 1974; Rothstein, 1978). With respect to this second function, a number of stimuli and substances interact with the membrane from the outside medium, or even from the cell interior and modulate its function. This is the mechanism for the action of some hormones and growth factors (Dor+e et al., 1978; Aharonov et al., 1978) and the basis for the induction of cell proliferation by a number ofmitogens (Oppenheimer, 1977; Segel and Lichtman, 1978; Kaplan, 1979). The regulative role that ions play in some of these changes is well known (Whittingham and Siracusa, 1978; Hammarstrom and Smith, 1979; Koch and Leffert, 1979; Moolenaar et al., 1981; Hamaguchi and Hiramoto, 1981; Yoshioka and Inoue, 1981). This role is also the basis of the action of some carcinogenic agents that exert a clear effect on the membrane (Moroney et al., 1978). There is now overwhelming evidence showing that cellular differentiation in the immune system is controlled by means of soluble factors (Cantor and Boyse, 1977; Marks and Rifkind, 1978; Harrison, 1982). Although in some of these instances the mechanism of action is not yet fully understood, it seems that the membrane plays an important role. In many cases, the mechanism of cellular response to these agents is a modulation of the transport of amino acids (Foster and Pardee, 1969; Oxender et al., 1977) or other metabolites (Koren and Shohami, 1979; Gazitt, 1979; Nishioka and Magagna, 1981), modifications in plasma membrane potential (Grollman et al., 1977; Kiefer et al., 1980; Beatrice et al., 1980; Tsien et al., 1982), mitochondrial membrane potential (Levenseon et al., 1982), alterations in cellular excitability (Harold, 1977; McGraw et al., 1982) or even modifications in the secretory function (Fallon et al., 1979; Aggeler et al., 1982). Alteration of cellular motility (Cohen, 1980; Potter and Johnson, 1982) or in the ionic gradients needed for the activity of some metabolic processes (Bader et al., 1978; Lazo et al., 1981; Garry et al., 1981) have been observed after treatment with certain compounds that act on the membrane (Lubin, 1967; Orr et al., 1972; Ledbetter and Lubin, 1977; Rubin et al., 1981; Rubin, 1981; Williams, 1982). The importance for the cell of membrane function has been shown in studies where its activity has been modified by means of ionophore antibiotics (Mondovi et al., 1971; Schadt and Haeusler, 1974) and toxins (Duncan and Buckingham, 1977; Brodsky et al., 1979; Barros et al., 1981; De la Pefia et al., 1981). Small local alterations in the lipidic content or the composition of glycoproteins also induce important changes in membrane fluidity and in the activity of some enzymes associated with the membrane (Kimelberg, 1977; Wojtczak and Nalecz, 1979; Nalecz et al., 1980; Hirata and Axelrod, 1978). These changes in 109

110

L. CARRASCOand J. C. LACAL

membrane composition are also important for the relationship between cells (gap junctions) (Smets et al., 1982; Kachar and Reese, 1982) and for cell attachment to surfaces (Juliano and Gagalane, 1979). These studies emphasize the key role of the membrane for the metabolic regulation of the eukaryotic cell. During the process of virus infection, alteration of the membrane takes place. The aim of this review is to point out the importance of some of these modifications if we are to understand several metabolic alterations undergone by cells during virus infection. First, we will discuss the entry of viruses into susceptible cells, since, for this, contact between the viral particle and the cellular membrane is necessary. This is followed by the passage of the viral particle to the cytosol. During early steps of viral infection, the membrane is modified, both structurally and functionally. Late in infection, cellular membranes are changed considerably and finally, for some viruses, the lipidic barrier constituted by the membrane is destroyed and cell lysis ensues. Therefore, cell lysis by viruses should not be regarded as an all or nothing process, but rather as a gradual alteration of membrane permeability, that generally begins before the bulk of viral macromolecular synthesis has taken place. The correlation between these membrane alterations and the cellular metabolic modifications induced by viral infection help us to understand the molecular biology of animal viruses, as well as the mechanisms that regulate the metabolic activity in eukaryotic cells. 2. MODIFICATION OF MEMBRANE PERMEABILITY EARLY DURING VIRUS INFECTION The infection of a cell by a virus commences with the interaction of a viral component and the appropriate receptor located on the cellular membrane. Several articles have thoroughly revised the work done on this subject (Tolmach, 1959; Dales, 1973; Kohn, 1979; Dimmock, 1982). One conclusion reached is the existence, in most of the systems analyzed, of a membrane receptor, specific for each kind of virus, and which is a normal component of cellular membranes. After this first interaction between the virion particle and the cellular membrane, the virus penetrates the host cell. It is important to understand how a virus enters a cell as a step in comprehending how membrane permeability is modified at the early stages of virus infection. Several mechanisms have been put forward to explain how viruses pass through the cytoplasmic membrane. We will comment on some of them briefly. 2.1. MODES OF VIRUS ENTRY INTO CELLS There are at least three different mechanisms by which a virus can pass the membrane of a susceptible animal cell (Fig. 1): (a) direct passage through the membrane, by a mechanism that does not involve a phagocytic vacuole, (b) endocytosis in a vacuole and subsequent release of the viral particle or the nucleocapsid to the cytosol and (c) fusion of the viral envelope with the plasma membrane followed by the delivery of the nucleocapsid to the cytoplasm. 2.1.1. Direct Passage

It is difficult to envisage how a virion particle passes the lipidic bilayer barrier directly. However, for non-enveloped viruses there must be a moment when the viral nucleic acid crosses the membrane and is delivered into the cytoplasm. Direct passage of virion particles through the cytoplasmic membrane has been described for several viruses such as adenovirus (Morgan et al., 1969), poliovirus (Dunnebacke et al., 1969) and for reovirus intermediate subviral particles (Borsa et al., 1979). Increased infectivity of reovirus particles is obtained by proteolytic digestion and convertion into intermediate subviral particles (Spendlove and Schaffer, 1965; Cox and Clinkscales, 1976). Two alternative pathways have been suggested for reovirus entry into L cells. Intermediate subviral particles would pass in mainly by direct penetration, using a mechanism that involves

Permeabilization of cells

111

CELLULAR,~ MEMBRANE ~

@ \\

iN

\\

OUT

IN

B

®(/ OUT

IN

/

IN

® OUT

IN

OUT

\

IN

Fro. 1. Modes of virus entry into animal cells. (A) Translocation or direct entry through the cytoplasmic membrane. (B) Endocytosis through a coated pit mechanism. (C) Fusion of the viral membrane with the cellular membrane.

membrane leakiness, as shown by the release of preloaded 51Cr from cells. Reovirus intact particles would enter mainly by viropexis, with no 5JCr release (Borsa et al., 1979). Whether these intact particles also modify membrane permeability, tested by more sensitive techniques, still remains to be established. The above studies indicate that, at least for reovirus, direct entry is not only possible, but this route of penetration in fact leads to infection as shown by increase in virus titers and reduction of the eclipse phase. Biochemical studies to investigate more deeply into the passage of virions or virion nucleocapsids through biological membranes, particularly for non-enveloped viruses are needed. For instance, we do not yet know if animal viruses use the membrane potential to cross it in the same way as suggested for some bacteriophages (Labedan and Golberg, 1979; Kalasauskaite et al., 1980). 2.1.2. Endocytosis Some macromolecules such as hormones, lectins, etc., enter the cell interior by endocytosis (Allison and Davies, 1974). The precise mechanism of this passage seems to be set in motion by the attachment of the molecule to a specific receptor. This is followed by a clustering of the receptors on the cell surface (coated pit). This clustering is mediated by a protein, clathrin, located at the base of the receptors. This clustering leads to the

112

L. CARgASCOand J. C. LACAL

formation of an endocytotic vacuole (coated vesicle) that migrates to the cytoplasm, where fusion with a primary lysosome forms a secondary lysosome (Goldstein et al., 1979; Pearse, 1980). The low pH in this vacuole induces the passage of the macromolecule to the cytoplasm by a still unknown mechanism. This idea is based on the finding that several amines and compounds which increase the pH in the lysosomes, block the passage of some toxins to the cytosol (Sandvig et al., 1979; Olsnes and Pihl, 1982). There is now reliable evidence that several enveloped animal viruses enter the cell by a mechanism similar to that described above for macromolecules. In the particular case of enveloped viruses, these would bind to receptors, after formation of a coated vesicle and fusion with a lysosome. The low pH in the secondary lysosome would cause the viral envelope to fuse with the lysosomal membrane, releasing the viral nucleocapsid into the cytoplasm. Four different viruses belonging to four different animal virus families have been chosen for these studies: Sendai virus, Semliki Forest virus, vesicular stomatitis virus and influenza virus. It is fairly well established that Sendai virus delivers its nucleocapsid by direct fusion with the plasma membrane, as will be discussed below. Semliki Forest virus, on the other hand, enters the cell by en endocytotic vesicle using a coated pit mechanism. This conclusion has been reached after electron microscopy studies and after experiments in which the lysosomal pH was increased by addition of amines to the medium (Helenius et al., 1980; Lenard and Miller, 1982). The questions that arise after analyzing these experiments are: is this just a viral mechanism to evade digestion by lysosomes? Is this mechanism the only way the virus infects a cell? and does the direct fusion of the virion envelope with the plasma membrane result in productive infection? It could still be argued that some enveloped viruses, such as Semliki Fores virus, do not normally need to fuse with lysosomes but if this occurs, particularly when high multiplicities of infection are used, the virus has a mechanism to avoid lysosome digestion. In fact, low pH leading to membrane fusion could occur under certain conditions in the extracellular medium and in the prelysosomal vacuole, before fusion with the lysosome occurs (Lenard and Miller, 1982). An important finding was that direct fusion with the plasma membrane can occur not only with Sendai virus but also with Semliki Forest virus, influenza virus and vesicular stomatitis virus, provided that a low pH exists in the culture medium (Lenard and Miller, 1982). In the case of Semliki Forest virus this fusion leads to efficient infection (White et al., 1981), whereas for influenza virus and VSV this does not seem to be so. Whether endocytosis is the only route of effective infection by Sindbis virus and vesicular stomatitis virus is still controversial since blockade of this process by cytochalasin B does not inhibit infection by those two viruses (Coombs et al., 1981) or by influenza virus (Patterson et al., 1979). A weak point in most of the studies on this subject is that inhibition of virus infectivity by lysosomotropic amines is taken as evidence that virus entry involves the formation of coated vesicles and subsequent fusion with lysosomes, as discussed above (Helenius et al., 1980; Lenard and Miller, 1982; Schlegel et al., 1982). However, these results must be treated with caution, since amines could also effect direct fusion of the viral membrane, since this is a pH-dependent process. Moreover, amines could affect steps in viral replication other than membrane fusion. This suggestion is reinforced by two places of evidence: first, amantadine blocks virus replication at concentrations below those needed to block virus entry (Schegel et al., 1982) and, second, Sendai virus infection is also blocked by lysosomotropic amines (Neumayer et al., 1965; Skehel et al., 1977; Lenard and Miller, 1982). 2.1.3. Fusion

Virus entry by fusion with the plasma membrane is obviously restricted to enveloped viruses. This is perhaps the easiest way to visualize the delivery of the viral nucleocapsid into the cellular cytoplasm. In addition, this mechanism of virus entry involves virus uncoating taking place in the same step of entry, leaving the free nucleocapsid in the cytoolasm ready to start virus replication.

Permeabilization of cells

113

Sendai virus is the example par excellence of portal entry to the cell by fusion of the viral envelope with the cytoplasmic membrane. The F protein of Sendai virus, a glycoprotein inserted into the viral envelope, is involved in the fusion process (see reviews: Klenk and Rott, 1980; Choppin and Sheid, 1980). The F protein needs activating by proteolytic cleavage in order to bring about virus fusion and infectivity. How the activated l? protein promotes fusion is not yet known, although it is important to point out that the simple contact of the cell membrane or some artificial vesicles is not enough. The active role of some glycoproteins, modulated in a number of instances by the conditions of the medium, are responsible of the fusion process. The increase in intracellular Ca 2+ ions is believed to play an important part in fusion (Kohn, 1979). Once the viral envelope fuses with the cell membrane, the viral glycoproteins that were inbedded in the viral membrane become part of the cell membrane. This has been discovered because these cells are killed by antiviral antibody and complement (Fan and Sefton, 1978). Vesicular stomatitis virus and Sindbis virus, which are thought to enter cells mainly by endocytosis, do not promote cell killing when the infected cells are in the presence of antiviral antibody and complement (Fan and Sefton, 1978). Insertion of these viral components in the cellular membrane early ~n infection would obviously induce modifications in the permeability of these cells, as will be discussed in the next section. 2.2. EARLY MEMBRANE LEAKINESS

Several properties in membrane permeability are modified during virus entry (Fig. 2). The extent and degree of these modifications depends on the virus-cell system under study and the multiplicity of infection used. These alterations are not only physical, but also involve the function of the membrane. The physical changes in the membrane are of two types: (a) appearance of new surface antigens on the plasma membrane, particularly evident for Sendai virus (Fan and Sefton, 1978) and (b) changes in the physical state and composition of the lipidic bilayer, that normally lead to increased membrane fluidity (Kohn, 1979). We will concentrate our discussion on the functional modifications in membrane permeability. 2.2.1. Ionic M o v e m e n t s The cytoplasmic membrane of animal cells maintains a gradient of ions in such a way that the external medium differs widely in its ionic content from the cytosol. Modification of permeability to ions early in infection has been mainly studied with paramyxoviruses. Klemperer in 1960 already observed that Newcastle disease virus induced a leakage of potassium ions from HeLa and ascites cells. This change in potassium permeability occurs within minutes after addition of Sendai virus to cells, and has been confirmed in several laboratories (Fuchs and Giberman, 1973; Pasternack and Micklem, 1974; Fuchs et al., 1980). It seems likely that potassium leakage is accompanied by an increase in sodium permeability, leading to an influx of sodium ions into the cell (Fuchs et al., 1978; Poste and Pasternak, 1978). The increase in sodium concentration in the cytoplasm activates the (Na+/K+)ATPase, so that the level of these two ions is restored a few minutes (20-30 min) after infection. Restoration of ion level is accompanied by the gradual resealing of the membrane. Increase in the concentration of external calcium abolishes membrane leakiness (Imprain et al., 1980), while the presence of 0.5mMATP enhances the leakiness of potassium ions induced by Sendai virus (Fuchs et al., 1980). Increased permeability of divalent cations has also been detected early during animal virus infection (Fuchs et al., 1979). Animal cells maintain a steep gradient of calcium ions whose concentration in the culture medium is around one thousand-fold higher than in the cytoplasm (Carafoli, 1982). Permeability to calcium ions in Sendai virus-infected cells changes at about the same time as the modifications in monovalent cations are observed. This was evidenced by influx and efflux of radioactive 45Ca2+ (Fuchs et al., 1980). The influence that these changes could have for cellular metabolism will be considered later. Viruses other than paramyxoviruses change cellular membranes as can be seen from their IPT 23/I--H

114

L. CARRASCOand J. C. LACAL translation

introeellular ions and metabolites

inhibitor

0 .

" i.(~

"

" .....~ ~

:2; • L:2L •

'

.~...~ ~ , ' - -

r6~. i~

~ ' /

•

fr..~,,

&

. . . .

~

~

~

•

L,t

., •.

p"q

•

..:.~...'t~_"

~

:.,Jl

' "

•

~

. .~

•

.

•

• ~-W

• P~

•

\ ":

. • .

:.' ' v ;-....

•

•.

; .'

." :" : • .., •

•.

•

•

. .' . *

I~1

•

,1~

FIG. 2. Schematic model of the early membrane leakiness to non-permeant inhibitors. Certain translation inhibitors (V) are unable to cross the plasma membrane of animal cells. The presence of viral particles and their attachment to the cell leads to the permeabilization of the membrane to the inhibitor. Subsequently the membrane is resealed.

increase in lipid fluidity (Levanon et al., 1977) and modified permeability to several molecules (Carrasco, 1981) but no early changes in cations have been detected. 2.2.2. Modifications in Membrane Potential Several methods have been used to measure membrane potential in animal cells infected by viruses: microelectrodes inserted in the cell, some dyes, such as 3,3'-dipropyloxadicarbocyanide and the radioactive lipophilic cation tetraphenylphosphonium (TPP ÷) (Heinz, 1981). The microelectrode technique is perhaps more sensitive than that of TPP ÷ and it has the advantage of measuring transient modifications in membrane potential. Since the polarization of cellular membranes depends in part on the monovalent ion gradient that the cytoplasmic membrane maintains (Heinz, 1981), a depolarization of the cellular membrane occurs during the first minutes after infection with Sendai virus (Okada et al., 1975; Fuchs et al., 1978; Imprain et al., 1980). This depolarization is transient and

Permeabilization of cells

v

t

100

115

100 "~

e-~'e/.,~ CONTROL

o

8

en

o,"°',,,!/'\ f',,o'-\ HygromyciBn ~. \

6O E

J

60

X&

E

o

40 ~ -r

20 I.-Z 0

~

~

I

t

I

2

4

6

0

0

--~ o

o,,.._ .y

8 TIME

0

1

I

1

I

2

4

6

8

0

S

o

FZ 0

~

(Hrs.)

FlG. 3. Permeabilization to hygromycin B of HeLa cells infected by poliovirus. Confluent monolayers of HeLa cells were infected with poliovirus type I at a multiplicity of infection of 10PFU/celI. After l hr of adsorption, the cells were pulsed every hour with 0 . 1 # C i / m l [35S]methionine in the presence or absence of 2 mM hygromycin B at the indicated times. The radioactivity incorporated into protein was estimated in a liquid scintillation counter. Note in the right panel that the membrane after the early permeabilization reseals and from the third hour becomes permeabilized again.

after 30min of infection, the cell membrane becomes repolarized. This result is in agreement with the measures of cation influx and etttux during the period of infection. :!.2.3. Entry of Molecules Several molecules which do not normally enter cells have been used to study modifications of membrane permeability. These were non-permeant dyes, fluorescently labeled amino acid derivatives and radioactive compounds such as [~4C]sucrose. However, lhese techniques are not very sensitive, and have led to suggestions that molecules above a certain molecular weight, around 300 (Fuchs et al., 1980) or 1,000 (Wyke et al., 1980) would not pass the membrane of infected cells early in infection. We developed an indirect method to measure permeability changes that is much more sensitive than the above mentioned techniques (Carrasco, 1978; 1979). The method is based on the use of inhibitors of macromolecular synthesis (translation inhibitors have been most widely used) to which cells are impermeable (Fig. 2). An increase in permeability will allow the translation inhibitor to enter the cell and block protein synthesis. The use of this technique showed changes in permeability early in infection with different enveloped (Carrasco and Esteban, 1982) or naked animal viruses (Carrasco, 1981) (Figs 3 and 4). In addition, it was possible to note increased permeability to macromolecules (Yamaizumi et al., 1979; Fern~mdez-Puentes and Carrasco, 1980). The entry of macromolecules was studied by means of protein moieties from certain toxins that effectively block protein synthesis in vitro, but do not pass the membrane by themselves (V~tzquez, 1979). The size limit for molecules that enter infected cells tested by this technique has now increased a lot. Increased permeability is induced by U.V.-inactivated virions, though this does not take place when they are heat-inactivated (Fern~indez-Puentes and Carrasco, 1980; Carrasco and Esteban, 1982). This permeabilization is observed even when most of the virus has entered the cells, though a gradual resealing of the membrane takes place after the virus input has been removed (Fernfindez-Puentes and Carrasco, 1980; Carrasco, 1981). lnhibitors of phagocytosis, such as colchicine or cytocholasin B, do not block the early permeabilization (Carrasco, 1981). At present, the exact molecular mechanism by which this permeabilization occurs is not fully understood, although it is clearly similar to that

116

L. CARRASCOand J. C. LACAL HSV-2

HSV-1

Vaccinio

o K

E g

Adeno-Y

\

10

o

I0 u

\

°~.~o

5

0

5 c e E l

E

0

I

I

0 1

I

I

I I

I

I

I

5

10

0 2

10

20

0

I

I

5 10

I

I

50 I00

i

L

0 0.5

I

I

2.5

5

0

M.0.11. ( P F U / C E L L )

EMC

?

Polio

Mengo

SFV

24

o 24 "

16

16

8

8

o K

"Ira

"o

g

o E i le

E 0

II

I

0210

t

20

I

40

III

04 8

I

40

i

80

M.O.I.

I I

I

I

I

01 3

6

12

II

I

I

0 2 I0 20

I

0

40

(PFU/CELL)

FIG. 4. Early membrane leakiness to hygromycinB induced by several DNA and RNA-containing animal viruses. Cell monolayers were incubated with or without 2mM hygromycin B at the multiplicity indicated in the figure during the adsorption period (1 hr x 37°C). Afterwards the medium was removed and protein synthesis estimated as indicated under Fig. 2. described for some ionophores (see Section 5). However, m a n y more experiments are required in order to advance a possible model. 2.2.4. Exit o f Molecules An increase in permeability as evidenced by entry of some molecules into cells, does not necessarily mean that there is also leakage of molecules from them. For example, the presence of 2 × I0-6M nigericin or 10 P F U E M C virus per cell efficiently permeabilizes cells to the translation inhibitors hygromycin B and alpha-sarcin (Alonso and Carrasco, 1982; Carrasco, 1981) however, under those conditions, no leakage of 86Rb ÷ ions or labeled nucleotides is observed. A higher leakage of molecules from cells is usually seen when the membrane is depolarized as occurs during paramyxovirus infection. A number of labeled metabolites and non-metabolizable compounds are normally used in these studies, generally with similar results for all of them. Exit of labeled choline, amino acids and sugars occurs when cells are infected with Newcastle disease virus or Sendai virus (Poste and Pasternak, 1978; Pasternak and Micklem, 1973, 1981; Imprain et al., 1980; Fuchs et al., 1980). The glycoproteins F and HN, are present in the envelope of paramyxoviruses (Compans and Kemp, 1978). The F glycoprotein is involved in hemolysis (Hsu et al., 1979), virus-cell and cell-cell fusion (Maeda et al., 1977; Scheid and Choppin, 1974; H o s a k a and Shimizu, 1972). For the permeability changes induced by paramyxoviruses, the presence of the intact glycoprotein is required (Wyke et al., 1980). In addition, the environment in which the F glycoproteins are located is important for induction of increased permeability, since Sendai virus, harvested after one day of inoculation of embryonated eggs, although containing

Permeabilization of cells

117 "o

0 ®

I0

A

CONTROL~ CELLS / /"~.....-'=

8

•

B

8

•

///

/

2-

8

I ~

."

I~ 4

oL-

o / /

•

10

/ CELLS

_ ~-

/

../'/n

2~_ -.~ 6

.

6

o.'" X...~,.FEC'rEO CELLS

--:S "1-

./

/

FECTEDcELLS

.

4

~v

2

0

0p

X

E 0~_1

0 05

J

i

K

i

1

2

3

4

0

0 2 4 6 8 lO

15

u

20

TIM~(min) FIG. 5. Inhibition of uridine transport and RNA synthesis in animal cells infected by encephalomyocarditis (EMC) virus. Confluent monolayers of mouse L929 cells were infected with EMC virus at a multiplicity of infection of 20 PFU/cell.

an intact F, still does not induce alterations in permeability. However, it can be rendered active by one of several physical treatments, such as freeze-thawing (Wyke et al., 1980). No experiments have been done with the purified F0 protein or with the two products F1 and F2 that appear after proteolytic treatment (Compans and Kemp, 1978). Exit of molecules early in infection with viruses other than paramyxoviruses has not been found. 2.2.5. Modification of Transport Since cellular membranes are altered during animal virus entry, it is likely that the transport of some molecules would be affected during the first stages of viral infection. There are not yet many studies on the modification of the transport of key metabolites during virus infection. Genty observed that vesicular stomatitis virus reduced the transport of uridine exponentially after infection of primary chick embryo cells. Evidence with VSV mutants suggested that the M protein was involved in this alteration (Genty, 1975). She considered that the inhibition of cellular RNA synthesis after infection is due, at least in part, to this blockade in the transport of the radioactive precursor. Recent studies also indicate that addition of EMC virus to L cells causes inhibition of uridine transport (see Fig. 5) (Castrillo and Carrasco, unpublished observations). The inhibition of the transport of labeled nucleotides during virus infection would obviously contribute to the inhibition of nucleic acid synthesis as measured by incorporation of this radioactive precursor. These studies, however, have not been applied very frequently to other animal viruses. Changes in transport of amino acids and sugars have been analyzed more carefully using Sendai virus-infected Lettr6e cells (Imprain et al., 1980). The uptake of compounds under conditions where their diffusion across the membrane is limiting is enhanced by Sendai virus infection; for instance, choline, 2-deoxyglucose and amino acids at high concentrations. Besides, substances that are accumulated by phosphorylation are not retained because of leakage of the phosphorylated metabolites. Finally, compounds normally accumulated by linkage to a sodium gradient are not taken up, since this gradient is disrupted early in infection (Imprain et al., 1980). 3. MODIFICATION OF MEMBRANE PERMEABILITY LATE IN INFECTION The replication of animal viruses normally leads to the massive synthesis of viral proteins late during infection (Carrasco and Smith, 1980). Many of these late proteins are

HSV-2 VACCINIA RABBIT POX ADENOVIRUS V ADENOVIRUS II SV-40

INFLUENZA A HSV- 1

SFV SINDBIS

VSV

MEASLES

NDV

ECHO-12 SENDAI

MK HeLa, MDBK LI210 cells HeLa, Lettre cells ascites Human peripheral lymphocytes HeLa, L929 CEC (Chick Embryo) HeLa, CEC CEF (Chick Embryo fibroblasts) MDCK, L1210 cells HeLa, Hep 2, Chick embryo Reast cells HeLa HeLa HeLa HeLa Mk CV~

HeLa, L929, Vero BHK-21, Ehrlich HeLa, Vero HeLa, EATcells

EMC

POLIO MENGO

Cells

Virus

Enzymes (48-72 hr) K ÷ (48 hr) Enzymes (48 hr)

Enzymes (8 10 hr)

K ÷ (10 hr)

86Rb+ (7 9 hr)

K + (15 hr) K + (3 hr)

K + (3 hr)

86Rb+, K + (4hr) K + (2.5 hr) Mg 2+ (3.5 hr) Proteins (3 hr) Enzymes (48-72 hr) K ÷/different compounds (5 10min) K+/different compounds (5-10 min) 3-4 h.p.i. 3 h.p.i.

3-4 h.p.i.

Release of ATP

Na ÷ (48 hr)

Na + (12 hr)

Na + (10 br)

Na +, Ca 2+ (15hr) Na + (3 hr)

Na + (3 hr)

Na +

Na + (2.5hr) Na ÷ (3hr)

Na + (3 hr)

Entry of Ions

+(L)

+(E, L)

+(E, L)? +(E, L)

+(E, L)

+(E, L)

+(E, L)

+(L)

+(L)

- +(E,L) +(E,L)

+(Early, Late)

Entry of Inhibitors

TABLE 1. Modification of Membrane Permeability by Viral Infection Release from Cells 86Rb+, K + (1-3 hr)

+ (48 hr)

+(10 hr)

+ (6 hr)

+ (5 hr)

Entry of Dyes

1 (4 hr)

1 (4 hr)

Membrane Potential

Deoxiglucose (1)

sucrose (8-10 hr)

Uptake uridine (1) Uptake a a (1)

Uptake ~A (~)

~. (4~8 hr)

Uptake ~A (1) (immediately) -metil glucosidos (1)

Uptake a~ (1)

Uptake ~ (J.)

Transport

>

©

>

(3o

Permeabilization of cells

119

virion p o l y p e p t i d e s t h a t p a r t i c i p a t e in the a s s e m b l y o f new virus particles. S o m e o f these newly synthesized viral p o l y p e p t i d e s are l o c a t e d on the m e m b r a n e , as occurs with several viral g l y c o p r o t e i n s , whose synthesis is closely a s s o c i a t e d with m e m b r a n e s (Sturgess et al., 1978) (Table 1). A s a consequence, cellular m e m b r a n e s b e c o m e modified, n o t only structurally, b u t in s o m e instances also f u n c t i o n a l l y (Fig. 6). A s will be discussed below, tbr m a n y viruses n o w a n a l y z e d m e m b r a n e p e r m e a b i l i t y is m o d i f i e d at the start o f the late phase, a n d c o n t i n u e s to alter as long as infection progresses. 3.1. RNA-CONTAINING VmUSES M o s t w o r k on m e m b r a n e l e a k a g e late in infection has c o n c e n t r a t e d on viruses c o n t a i n i n g R N A in their genome, p a r t i c u l a r l y picornaviruses. Before starting this discussion, it is w o r t h e m p h a s i z i n g the fact t h a t m a n y o f the changes o b s e r v e d late in infection are n o t the c o n s e q u e n c e o f cell d e a t h , b u t in fact they have been r e g a r d e d r a t h e r as the

•

44,,

'4• ',t-

"4"

-,I-

~-+ •

.,t-

-I.,

4"

4-

• 4o • .. ++ I[°t-~°°'° "~'I)° "--7-" H oII ÷ +

IIo •

oli

+

4,4,• •

÷

,֥

Zt~i~° ,-~,r' '. 0" "~O • : ~ . Vo, ~~, .. 4"

4"

4-

].'o

÷

4"

-

.:ll

.

•

4-

4•

•

4" ,4, •

~"

4" 4"

0 hr$

+

,4-

•~

,÷

p.i.

• ,,..:

a,.

U', ,,-i'3:: Lk,,w:.'~p~'~, , ~ . . . . l

÷

,,JJ

+.":.

-4"

•

4" -t-

4, hr$ p.i.

, , ~'~

+

+ O

+

6 h r s p.i.

,4 - ~

4-

V'~

hr$

4" 4"

p.i.

.,:i~ ;~ .it,: ~ . : ÷ + "..,,..

' "-L__-'____.L__,~.. . . . . . . . : . .

.T'

.

÷

m,,, ,_.._ ÷ ;"H v~_. O ' . ° . . . ~ / - H÷ ~ •.:|L

v.-

+ +!÷

-

.

.•

".

•

,''l"•/I

:rjet-p-1

"..

÷

/i÷~..,,:~;v.+:..+.': , v

5 hrs p.i.

-, 4" + 4 " •.. ,,r~: [ ~ . + ..+,+,,4" oll,

r

•4-• -;t" 4-

÷.

~ la,,, ~ ' * * ~".' , ~ . ; .' . ~ - ~>ATI~ZE] H 4" .. • .

~

4-

2-3

.

........... ~-L..--

+

.t.V

÷

;,~ ~.~+,4- 4-~ ~ + ; ,

O

8 hrs

p.i.

FiG. 6. Schematic model of the late permeabilization of animal cells infected by EMC virus. A number of alterations in the plasma membrane occur during virus infection. One of the first to be detected in the late period is the entry of certain inhibitors (V) unable to pass into uninfected cells. The inhibitors enter the infected cell when viral proteins (O) are being synthesized (black ribosomes) and cellular protein synthesis ((3, white ribosomes) is inhibited. From the fourth hour onwards there is a leakage of cellular metabolites including ATP and ions. From the 6th hour new viral particles are formed and released to the medium. At this time the leakage of the membrane is very acute and the entry of dyes (÷) is also apparent.

120

L. CARRASCO and J. C. LACAL

cause of the cytophatic effect and cell death (Pasternak and Micklem, 1981; Norkin, 1977). Membrane leakiness is detected before cells become stainable with trypan blue, and for many viruses this occurs before the synthesis of the macromolecules that will constitute the virion particles. It must also be remembered that the trypan blue stainability test only measures modifications of membrane permeability. As Norkin (1977) has pointed out "stainability by trypan blue indicates nothing more than a poorly understood change in membrane permeability". 3.1.1. Picornaviruses

The diffusion of monovalent ions was one of the first changes in membrane permeability studied in picornavirus-infected cells. Farnham and Epstein (1963) observed that around the third hour postinfection of L cells by EMC virus, a redistribution of sodium and potassium ions occurred. It was later found that cells loaded with the radioactive potassium analogue, 86Rb+, do not retain this ion from the fourth hour after infection of 3T6 cells with EMC virus (Carrasco and Smith, 1976). This results indicates that the bulk of viral protein synthesis takes place in cells where the ionic content is different from that of a normal cell (Fig. 7). The possibility that cells which lose their membrane integrity are not the ones involved in active macromolecular synthesis was studied as will be explained. Some inhibitors of eukaryotic protein synthesis do not block translation in intact mammalian cells, because the plasma membrane constitutes a barrier for their diffusion (V/tquez, 1979). We reasoned that if the membrane permeability of cells actively engaged in viral protein synthesis is damaged, then the non-permeant inhibitors would pass through the membrane of these cells and block protein synthesis (Carrasco, 1978) (Fig. 2). The use of this approach revealed that cells infected by EMC virus become permeable to the GTP analogue, Gpp CH2p, before the bulk of viral proteins are synthesized. The appearance of late membrane permeability is blocked by compounds that inhibit virus replication, such as interferon (Mufioz and Carrasco, 1981). U.V.-inactivated virus is unable to induce late

TIME POSTINFECTION (Hrs) 1 2 3 4

5

6 78

100 _J 0 n~

80

Z 0 c_)

60 ~x

40 20 0 0

2 Hrs,

4

6

8

10

postinfection

FIG. 7. Protein synthesis and permeability to ions of HeLa cells infected by EMC virus. Cells were infected at a multiplicity of 20 PFU/cell and protein synthesis was measured every hour by estimating the [35S]methionine (0.1 #Ci/ml) incorporated into trichloroacetic acid precipitable material (@). 86Rb uptake was estimated by pulses of 45see (A) and 86Rb content (©) was measured at the indicated times in cells preloaded for 18 hr in the presence of 0.5-1 ~tCi/ml 86Rb. In the right hand panel analysis by polyacrylamide gel electrophoresis of the proteins synthesized at different times postinfection is shown.

Permeabilization of cells

121

membrane leakiness, though it brings about early changes in membrane permeability (Carrasco, 1981). The use of non-permeant inhibitors diminishes the production of infective virus particles at concentrations that do not affect protein synthesis in normal cells (Benedetto et al., 1980; Lacal et al., 1980). A wide screening of translation inhibitors was carried out to select those which inhibit protein synthesis in virus-infected cells selectively (Contreras and Carrasco, 1979). In addition to nucleotide analogues, gougerotin, blasticidin S, edeine, anthelmycin and hygromycin B, specifically inhibit protein synthesis in EMC virus, mengo virus, Semliki Forest virus and SV40-infected cells late in infection (Contreras and Carrasco, 1979). These studies were later extended to other virus-cell .~ystems (Benedetto et al., 1980; Lacal et al., 1980). A correlation was found between increased permeability to these compounds and antiviral action. Curiously enough, zinc ions block membrane leakiness late in infection. Even when the leakiness has developed, addition of zinc ions reverses this membrane ,damage (Carrasco, 1980b). The mechanism by which zinc ions have this effect is probably laot related to the inhibition of cleavage of viral proteins. Whether they are able to reseal :he leaky membrane directly or not still remains to be seen. In a more detailed study, the 86Rb+ content, membrane potential and modification of membrane permeability to hygromycin B in EMC virus and poliovirus infected HeLa cells ~vas carried out (Lacal and Carrasco, 1982). As shown in Fig. 7, permeability to hygromycin B in EMC virus-infected cells increases drastically from the third hour after infection, just before cellular protein synthesis is shut-down. The 86Rb+ content and membrane potential also drop from the fourth hour post-infection, and around the 8-10thhr post-infection, the gradient of monovalent ions that cellular membranes maintain disappears from picornavirus-infected cells. ATP leaks from picornavirusinfected cells also from the third hour (Egberts et al., 1977; Lacal and Carrasco, 1982), and the transport of some amino acids, particularly those dependent on a sodium gradient are altered late in infection (Lacal and Carrasco, 1982). Modifications of divalent cations as measured by depletion of magnesium from cells and diminishing polyamine content is also apparent in mengo virus-infected Ehrlich ascites tumor cells (Egberts et al., 1977). These changes occur after the alterations in monovalent cations, but well before virus particles are formed and released to the medium, and before the cells are stainable by trypan blue (Egberts et al., 1977). Infection of HeLa cells by poliovirus also increase the content of sodium ions late in infection (Nair et al., 1979; Nair, 1981). The increase in this cation occurred from the second hour after infection and was inhibited by the presence of 2 mM guanidine (Nair et al., 1979). This increased membrane permeability to sodium ions is possibly triggered by synthesis of a viral product. In a recent study, protein synthesis, membrane potential, cell volume, Na ÷ and K ÷ levels and (Na ÷, K ÷) ATPase activity were measured in control and poliovirus-infected HeLa cells (Schaefer et al., 1982). The onset of protein synthesis inhibition coincided with a decrease in cell volume. Three hours after infection the sodium and potassium gradients collapsed and membrane potential and amino acid uptake were reduced (Schaefer et al., 1982). Very late in infection, when the progeny virus is released, a number of cellular enzymes can be detected in the culture medium (Gilbert, 1963). Lactic dehydrogenase, for example, is released into the medium after infection of monkey cells with echovirus 12. Curiously enough, the pattern of enzymes released depends on the kind of animal virus studied (Gilbert, 1963). 3.1.2. Togaviruses

Togaviruses are a family of animal viruses, similar to picornaviruses in that they also contain plus-stranded RNA as a genome (Pfefferkorn and Shapiro, 1974). However, the morphology of the virion particle, and the replicative cycle are very different from picornaviruses (Carrasco and Smith, 1980). The first studies on membrane leakiness in togavirus-infected cells were done with

122

L. CARRASCOand J. C. LACAL

01234567%9

TIME

p.i.(h)

w

•

•

•

•

•

r

A

20--" O

v

p62 -

6 Ac:"-

~o-o

4

C-

15 n-

\ o O..o_o o

10 'o

X

E

O.

2I 0o |

u *

*

2

*

*

4

*

*

6

8

0

TI ME (h) FIG. 8. Protein synthesis and 86Rb content in BHK cells infected by Semliki Forest virus (20 PFU/cell). The methods and conditions were as indicated under Fig. 7.

Semliki Forest virus (Carrasco, 1978). They show that the beginning of the late phase (2-3 hr postinfection) is accompanied by a rise in permeability to some translation inhibitors (Contreras and Carrasco, 1979; Lacal et al., 1980). Direct measurement of sodium and potassium ions by flame spectroscopy in Sindbis virus-infected chick cells indicates that there is an increase in the intracellular sodium and a decrease in the intracellular potassium concentration (Garry et al., 1979). These alterations were observed from the second hour after infection at the time when inhibition of cellular protein synthesis commenced. Analysis of protein synthesis and 86Rb+ content in Semliki Forest virus-infected BHK cells is shown in Fig. 8. The shut-off of protein synthesis in this system correlates with the decrease of 86Rb+ content in agreement with the results obtained by Garry et al. (1979) with Sindbis virus. Semliki Forest virus late protein synthesis takes place as from the second hour postinfection and continues beyond the ninth hour in cells with an altered ionic gradient. Analysis of mitochondrial calcium uptake during infection of chicken embryo cells with Semliki Forest virus shows an initial stimulation during the first five hours and a later decline (Peterhans et al., 1979). These results suggest that there is an increase in cytoplasmic calcium early after infection, and a mitochondrial injury late in infection (Peterhans et al., 1979). 3.1.3. Other RNA-Containing Viruses

Late modification of membrane permeability has been studied in more detail in picornaviruses-infected cells. Similar work on other virus species is now emerging, and suggests that some differences exist amongst the various viruses studied. Rhabdoviruses, paramyxoviruses, myxoviruses and reoviruses all modify membrane permeability late in infection as measured by the 'impermeable inhibitor' test (Table 1) (see Figs 9, 10, 1l). Entry of hygromycin B into Sendai virus-infected cells was detected from the eighth hour post-infection, as measured by the inhibition of translation in these cells. This inhibition leads to a decrease in virus yield (Benedetto et al., 1980). Newcastle disease virus also increases membrane permeability to hygromycin B from the eight hour post infection, which is about the time when viral protein synthesis is apparent, as analyzed by polyacrylamide gel electrophoresis (Fig. 9). In the case of NDV-infected cells, it is worth pointing out that no variation in monovalent cation content occurs until 24 hr postinfection, though a general decrease in membrane potential is clear from the beginning of infection. This system exemplifies the situation where great variations in permeability are detected by means of hygromycin B, but with no alterations in 86Rb+ content. This suggests

123

Permeabilization of cells NDV-INFECTED

HeLo

CELLS

t',o

100

100

E "

~ 0

\"o

.-"

x

I 8O

80

='~.= h"..4k, '

60

60

z o

\

?, ~c;

40

40

E i if)

2O

20

~

J o n-

"ok "o I

I

4

8

0

0

I

I

I

I

12 16 20 24

1

I

I

I

0

4

8

12

TIME

4

8

12

I

I

I

16 2 0 24

I

l

0

4

~

I

8

I

I

20 24

(Hrs)

TIME

POSTINFECTION

(Hrs)

16

24

12

20

I

12 16

4

8

16

20

24

HN--

--HN

NP--

--NP

M--

--M

-HB

+2raM HB

FIG. 9. Protein synthesis and alteration of membrane permeability in HeLa cells infected by Newcastle disease virus (NDV) HeLa cells were incubated for 18 hr with 1 # Ci/ml 86Rb+ and then infected with NDV (20 PFU/celI). In parallel cultures, protein synthesis was measured by one hour pulses with 0.1 # Ci/ml pSS]methioninein the absence (0) or the presence (O) of 2 mM hygromycin B (left panel). In the central panel the percentage of inhibition of translation by hygromycin B is represented. The right hand panel shows the membrane potential measured by the [3H]TPP+ technique (11), the intracellular content of 86Rb+ (0) and the activity of the (Na ÷, K ÷) ATPase (O) every hour after infection. The bottom figure shows the proteins synthesized in the presence or absence of hygromycin B.

that the cause o f the change in permeability to h y g r o m y c i n B in N D V - i n f e c t e d cells is n o t a general alteration of the m e m b r a n e , b u t instead is based o n some specific m e c h a n i s m . Finally, m e m b r a n e permeability alterations in influenza virus-infected M D C K cells is s h o w n in Fig. 10. The a p p e a r a n c e o f viral p r o t e i n correlates with the increased permeability to t r a n s l a t i o n inhibitors, with n o v a r i a t i o n in 86Rb+ c o n t e n t or m e m b r a n e potential. The claim that VSV infection does n o t alter the permeability to m o n o v a l e n t cations

124

L. CARRASCOand J. C. LACAL INFLUENZA

VIRUS

A-INFECTED

MDCK

CELLS

i

100 E O. u

80

4

.J o

"0 "o

60

0

z 0 (.,)

0 %

o,,

2

40 I

E

20

1

r~ I

I

I

0

2

4

0

I

I

I

I

i

i

I

I

I

6

8

I0

12

0

2

4

6

8

I

I

I

10 12 0

I

I

I

i

2

4

6

8

I

I

10 12

TIME (Hrs)

TIME

4

8

12

POST INFECTION

]6

20 24

4

(Hrs)

8

12

16

20

24

HA

HA-NP~

NP

M

M

-HB

+2raM

HB

FIG. 10. Protein synthesis and alteration of membrane permeability in MDCK cells infected by influenza virus. Conditions and symbols are as indicated under Fig. 9.

(Francoeur and Stanners, 1978) was disproved by more careful studies in which both sodium and potassium concentrations were measured after vesicular stomatitis virus infection. Garry and Waite (1979) found an increase in the permeability of infected cells to both cations from the second hour after infection. Increased permeabilization to hygromycin B also occurs from the third hour postinfection (Benedetto et al., 1980). 3.2. DNA-CONTAINING VIRUSES The few studies n o w available o n m e m b r a n e leakiness in D N A virus-infected cells indicate that some q u a n t i t a t i v e a n d qualitative differences exist a m o n g s t the viruses studied.

Permeabilization of cells

125

Papovaviruses

3.2.1.

Infection of monkey cells with SV40 is found to impair membrane formation and function (Norkin, 1977). Incorporation of choline into phosphatidylcholine decreases 40 hr after infection, at about the same time as cells become stainable by trypan blue and release enzymes into the medium. Though the viability of SV40-infected CV 1 cells decreases from the 48th hr postinfection, no inhibition of A T P content in the cells is detected (Norkin, 1977). The SV40 replication cycle lasts for several days and 48 hr after infection, the cells ~ynthesize massive amounts of the VP1 protein. At this time, a very acute permeability change to translation inhibitors takes place (Contreras and Carrasco, 1979). More recently, the ionic content of these cells has been measured (Eggleton and Norkin, 1981). By 48 hr post infection, an increase in sodium ions and a decrease in potassium ions was seen. The

HSV-1-

m

INFECTED

HeLo

CELLS

,o-s 7\

50

b E

100

~o o-~.~.,, 8O

4O

._1

o 60

30

o ~0,..0 •

\

40

hI'Z

0 ¢_)

20

E

'%.

~

i

i

i

i

0

4

8

12

0

i

~o %-w~,

16 2 0 2 4

0

4

8

TIME

TIME 4

8

12

16

20

12

16 2 0 2 4

0

8

12

16 2 0

24

(Hrs)

POSTINFECTION 24

4

4

8

(Hrs) 12

16

20

24

m

1-2 '3

4-5-7 --

4-5 7

]-2-3

Ac~

Ac

21--

21

23 --

__ 23

-HB

+2raM

HB

FIG. 11. Protein synthesis and alteration of membrane permeability in HeLa cells infected by HSV-1. Conditions and symbols were as indicated under Fig. 9.

126

L. CARRASCO and J. C. LACAL

authors indicate that alterations in membrane permeability can be involved in cell killing by SV40. 3.2.2. Herpesviruses Because herpesviruses are clinically important viruses, it was of interest to test whether herpes simplex virus (HSV)-infected cells also became more permeable to inhibitors, as compared to the uninfected control cells. Benedetto et al. (1980) showed that the HSV-1 infected cells are permeable to hygromycin B when late herpesvirus proteins are synthesized, and hence this compound reduced the production of new infective viruses. By contrast, a lower permeability in HSV-I infected cells was reported as estimated by the 51Cr-release technique (Schlehofer et al., 1979). However, this technique is not very sensitive when measuring permeability changes, and only when the membrane is extensively modified is a release of 51Cr detected. Measurement of monovalent ions at different times after HSV-1 infection indicates a decrease in 86Rb+ uptake from the fourth hour post-infection, but the total sodium and potassium content seven hours after infection is only slightly altered (Hackstadt and Mallavic, 1982). The results shown in Fig. 11 correlate viral protein synthesis, permeability alterations to hygromycin B, 86Rb+ transport, 86Rb+ content and membrane potential throughout infection of HeLa cells by HSV-1. A parallelism is observed between the inhibition of protein synthesis and the alteration of membrane permeability to hygromycin B. However, in agreement with Hackstadt and Mallavic's results (1982) no serious alteration in 86Rb+ content is detected, though its transport is reduced and membrane potential also drops from the beginning of infection. 3.2.3. Poxviruses Infection of cells by poxviruses such as vaccinia virus induces a drastic inhibition of protein synthesis and severe cytophatic changes (Bablanian, 1975). A leakage of proteins from rabbit poxvirus-infected cells was detected from the 10th hour postinfection (Schiiperli et al., 1978). This release was not connected with the exit of virus to the medium, since incubation at low temperature inhibits virus release, but not protein leakage. Analysis of monovalent cation content by flame photometry, in vaccinia virus-infected HeLa cells, showed a drop in the intracellular potassium from around 100 mM to 80 mM between the second and fifth hour post-infection. An increase in sodium ions occurred at around 5-6 hr postinfection and a drastic change in sodium and potassium concentration from the 10th hour postinfection (Norrie et al., 1982). No correlation between the early inhibition of host protein synthesis and variations in monovalent ions was apparent. Our unpublished studies on 86Rb+ leakage from vaccinia virus-infected L cells indicate a slight decrease in this cation from the beginning until the 6th hr and a drastic leakage from the 6th hr onwards. 4. CONSEQUENCES OF MEMBRANE MODIFICATION AFTER VIRAL INFECTION The membrane is a key structure for the maintenance and regulation of cellular processes. It seems evident that a change in its function would have repercussions on cellular metabolism. The extent and duration of these modifications would have a more or less profound effect on a particular cellular function. We described in Section 2 and 3 how viruses modify the cell membrane profoundly, though we are only starting to understand the different membrane functions that change during viral infection. The little we do know, however, is helping us to get closer to the causes of the modification of metabolic processes, the changes that take place in cell morphology and even the mechanisms by which cells are killed by viral infection. 4.1. EFFECT ON CELLULAR METABOLISM

The alterations in membrane permeability have consequences not only for cellular functions but also for several viral processes that depend on, or are regulated by, the extent

Permeabilization of cells

127

to which the membrane is altered. The following sections describe some viral and cellular functions that are influenced by membrane modification. 4.1.2. Inhibition o f Cellular Protein Synthes& It is well known that viral infection interferes with several cellular functions. A number of recent reviews have covered this topic in detail (Bablaniam, 1975; Carrasco and Smith, L980; Agol, 1980). This section will concentrate on the blockade of translational that many cytopathic viruses induce after infection, emphasizing that viral protein synthesis is resistant to inhibition by high salt concentrations and to membrane-active compounds lSaborio et al., 1974; Nuss and Koch, 1976; Carrasco, 1979; Alonso and Carrasco, L981c, d). The general conclusions that we can draw from all the available evidence on modifications of the ionic gradients after viral infection are that a number of viruses that cause shut-off of cellular protein synthesis, modify membrane permeability to ions in such a way that the gradient of sodium and potassium concentration maintained by the membrane is broken, and the concentrations of these ions gradually reach an equilibrium between the outside medium and the cytosol (see Section 3). Two different questions should be kept in mind when considering the implications of these changes in protein synthesis: lhe first is whether these changes in ionic concentrations occur while viral protein synthesis is taking place; and the second whether these alterations are involved in the inhibition of protein synthesis in the infected cell. Let us consider this second possibility. In 1977, we put forward the hypothesis that the inhibition of cellular functions after infection could be a consequence of the modification of membrane permeability to ions (Carrasco, 1977). Since then, the work of several laboratories has concentrated on measuring monovalent ions at different times postinfection. There are, however, other ions, such as calcium, which are important for cellular metabolism, and have not yet been analyzed in detail in the infected cells. In addition, a number of key metabolites that might act as regulatory signals can vary when membrane permeability changes. This is still an unexplored field. With regard to monovalent ion alterations, viral systems exists in which a very striking parallelism is found between the inhibition of host protein synthesis and the alteration of monovalent ion content in the infected cell. This occurs in cells infected with EMC virus or togaviruses (SFV or Sindbis virus) (Garry et al., 1979; Lacal and Carrasco, 1982). In the case of EMC virus, the inhibition of cellular translation depends on the concentration of sodium ions in the outside medium (Carrasco, 1979; Alonso and Carrasco, 1981d). It can be reversed if NaC1 is removed from the medium (Alonso and Carrasco, 1981d) and is favored by the ionophore amphotericin B (Alonso and Carrasco, 1981c). In addition, the leakiness of the infected cells to 86Rb+ ions and the blockade of protein synthesis are parallel processes. A striking similarity exists between the inhibition of protein synthesis by EMC virus and by nigericin, an ionophore that acts specifically on the membrane iPressman, 1976; Ovchinnikov, 1979) and block protein synthesis by altering the monovalent ion concentration in the treated cell (Alonso and Carrasco, 1981). It seems likely lhat, at least in the case of EMC virus, SFV and Sindbis virus, a viral compound exists that modifies the membrane and inhibits protein synthesis in this indirect way. For other viruses such as poliovirus (Nair, 1981; Lacal and Carrasco, 1982; Schaefer et al., 1982), herpesvirus (Hackstadt and Mallavic, 1982) and vaccinia (Norrie et al., 1982), the inhibition of cellular translation precedes the modification of membrane permeability to sodium and potassium. However, in these systems, alterations in membrane permeability to other compounds have been detected prior to the blockade of translation lCarrasco, 1981; Carrasco and Esteban, 1982). Whether shut-off in other systems is produced by some modifications, as yet overlooked, of the membrane, or leads to the generation of a translation inhibitor that either increase the concentration of other ions, still remains to be tested. It is worth mentioning, however, that in these systems the leakiness to monovalent ions occurs soon after inhibition of protein synthesis. For poliovirus, a drop in 86Rb+ ions takes place one hour after the beginning of shut-off of translation (Nair, 1981; Lacal and Carrasco, 1982). For herpesvirus-infected cells, a 30~o

128

L. CARRASCOand J. C. LACAL

reduction in potassium concentration is observed from the second to the fifth hour post infection, and a total redistribution of monovalent ions starts 10 hr postinfection (Hackstadt and Mallavic, 1982). In addition to permeability changes as the cause of inhibition of protein synthesis in virus-infected cells, other possibilities exist for different virus-cell systems. For instance, some in vitro evidence suggests that poliovirus infection inactivates an initiation factor activity necessary to translate mRNAs with a Y-terminal cap structure (Rose et al., 1978; Trachsel et al., 1980; Hansen and Ehrenfeld, 1981). However, this seems unlikely, since in vivo evidence indicates that capped mRNAs from viruses are translated in cells double-infected with poliovirus and another virus such as Semliki Forest virus (Alonso and Carrasco, 1982b). Competition between viral mRNAs and cellular mRNAs for translation components such as ribosomes or initiation factors, has also been suggested as the cause of inhibition of cellular protein synthesis (Lawrence and Thach, 1974; Golini et al., 1976; Brendler et al., 1981; Walder et al., 1981). It seems unlikely to be the cause of the shut-off by vesicular stomatitis virus, for two main reasons: (a) total synthesis of proteins decreases throughout infection. This would not be expected if a cellular mRNA were simply replaced by a more efficient viral mRNA, and (b) the shut-off occurs even under conditions in which viral transcription and translation are very much inhibited, i.e. by pretreatment of cells with interferon (Simili et al., 1980). 4.1.3. Inhibition o f R N A Synthesis Rapid inhibition of cellular RNA synthesis occurs in several virus-cell systems studied. Perhaps the most representative systems are the infection of mouse cells by VSV or EMC virus. Infection of Krebs-2 cells by VSV leads to a rapid shut-off of cellular RNA synthesis, depending on the method of investigation used (Huang and Wagner, 1965). This inhibition is caused by the infective virus input, since it occurs even when viral gene expression is blocked by U.V.-irradiation, or puromycin, or when DI particles are used to infect cells (Huang and Wagner, 1965; Yamazaki and Wagner, 1970; Yaoi et al., 1970; Wertz and Youngner, 1972; Baxt and Bablanian, 1976). Even some purified virion components, such as the virion glycoprotein G, block RNA synthesis in BHK21 cells, at least partially (McSharry and Choppin, 1978). The cause of this inhibition is not yet known. Genty (1975) found that VSV virus induces an inhibition in the transport of uridine, and suggested that the reduced transport of this nucleoside influences inhibition of RNA synthesis. Evidence with ts mutants pointed to the M protein as being responsible for this inhibition (Genty, 1975). The results of McSharry and Choppin (1978) with the isolated G glycoprotein indicated that inhibition of RNA synthesis was higher than the blockade of uridine transport two hours after addition of the G protein. Six hours after addition of the G protein to BHK21 cells, inhibition of RNA synthesis was around 25~, whereas transport of the radioactive nucleoside precursor was reduced by 36~o. The authors, however, concluded that the inhibition of RNA synthesis was due to direct action of the G glycoprotein on macromolecular synthesis rather than to an indirect effect on the membrane. Picornavirus infection also blocks cellular RNA synthesis, but in this instance, cardioviruses (EMC virus, mengovirus) are much more active in inhibition than enteroviruses (poliovirus) which is contrary to the results obtained on protein synthesis (Bienz et al., 1978). As an example, Fig. 4 shows the rapid and drastic inhibition of RNA synthesis in L cells infected by EMC virus, compared to the inhibition induced by poliovirus in HeLa cells (Castrillo and Carrasco, unpublished results). For some time no agreement was reached as to whether the input virus was sufficient to cause this shut-off of RNA synthesis, or whether partial viral replication was needed (Franklin and Baltimore, 1962; Collins and Roberts, 1972). Our recent, unpublished experiments with U.V.-inactivated virus and several inhibitors of virus replication, favour the results of Collins and Roberts (1972). We

Permeabilization of cells

129

found that the input virions per se were responsible for the inhibition. No experiments have been done with picornavirus to isolate the viral component involved in this inhibition so its mechanism is still controversial. Miller and Penhoet (1972) found a decrease in the activity of RNA polymerase II. However, it was later seen that the functioning of this enzyme was unchanged after injection (Apriletti and Penhoet, 1974; Schwartz et al., 1974). Fig. 4 shows uridine transport in EMC virus-infected L cells. An inhibitory effect is observed early after infection. It is not yet certain that this is the only mechanism involved in the inhibition of cellular RNA synthesis in this system, but it provides another example in which macromolecular synthesis could be indirectly affected by an effect on cellular membranes. 4.1.4. Membrane Proliferation in Virus-Infected Cells Not all cellular functions are blocked after virus infection: some are stimulated. For instance, poliovirus infection of HeLa cells leads to an increased incorporation of choline into phosphatidylcholine (Penman, 1965). This stimulation is accompanied by a proliferation of smooth endoplasmic membranes (Mosser et al., 1972). Recent experiments indicate that the cytidylyl transferase reaction is the rate-limiting step in the synthesis of phosphatidylcholine in HeLa cells. This reaction is regulated by the concentration of CTP. Poliovirus infection stimulates this reaction by raising the amount of free CTP in the cell several-fold (Vance et al., 1980; Choy et al., 1980). The increase of CTP is observed early after infection, but it is not yet clear if the proliferation of membranes is connected with the changes in membrane permeability produced by virus infection. 4.1.5. Cell Killing and Cytopathic Effect It is surprising that the exact mechanisms by which cells are killed during virus infection are very poorly known. One of the reasons could simply be that the term 'killing' is rather vague in molecular terms. For instance, cells could stop synthesis of macromolecules but still remain 'alive' as regards to cell respiration, ATP synthesis and so on. Generally a cell is considered to be alive, if it is still capable of excluding certain dyes, such as trypan blue. however, as was already pointed out by Norkin (1977), this test does not given an accurate idea of the extent of a change in membrane permeability. This change can occur in cells actively synthesizing ATP and even synthesizing considerable amounts of viral macromolecules. In a recent review, Pasternak and Micklem (1981) stressed the point that the cytopathic effects induced by viral infection have, as a common cause, the alteration of cell plasma membranes. Another view is that cell killing, by viruses such as SV40 is similar to that by different agents such as ionophores (Eggleton and Norkin, 1981). In this latter case, killing is ~Lhought to be triggered by a common mechanism that involves calcium ions (Trump et al., 1971; Schanne et al., 1979). Merely to conclude that cell death is the result of virus multiplication evades the main question, because it is well known that virus replication occurs in some instances with no cellular degeneration, such as in persistent infection systems. To review the morphological lesions that viruses cause in their host cells is beyond the ~cope of this chapter. For this information the reader is referred to the review by Bablanian (1975). Here we would simply stress the fact that cellular membranes are profoundly modified during the course of virus infection, and those modifications resemble those caused by other toxic proteins and compounds that act on cellular membranes and also lead to cell death. 4.1.6. Cell Transformation The infection of mammalian cells by certain DNA and RNA-containing viruses results, under some conditions, in cell transformation (Tooze, 1981; Weiss et al., 1981). RetroJPT 23/1--1

130

L. CARRASCO and J. C. LACAL

viruses induce this modification particularly efficiently. In most cases studied the expression of a single gene is responsible for such a profound change in cell behavior. For retroviruses, expression of the src gene in sarcoma viruses leads to cellular transformation. This gene codes for a protein kinase. The role of this protein remains a mystery in spite of all the efforts concentrated on its study (Bishop and Varmus, 1982). Immunofluorescence experiments indicate that it is located on the membrane and the modification of a membrane function could account for the alterations in cell metabolism (Willingham et al., 1979, 1980; Krueger et al., 1980; Levinson et al., 1981). Cells transformed by animal viruses show an increase in the transport of several metabolites (Hatanaka and Hanafusa, 1970; Lage-Davila et al., 1979; Lang and Weber, 1978). With regard to ions, conflicting reports exist as to whether transformed cells contain a similar or different sodium and potassium concentration as compared to normal cells (Banerjee et al., 1977; Smith et al., 1978; Johnson and Weber, 1979). Cone (1971) advanced the theory that a sustained depolarization of the membrane would lead to continuous cell proliferation. This suggestion was reinforced by the finding that central nervous system cells in culture are committed to divide if the membrane is depolarized by certain ionophores (Cone and Cone, 1976). Recent theories on cell proliferation suggest that the first step triggered by several mitotic agents is the opening of a specific sodium channel in the membrane (Rozengurt et al., 1979; Mendoza et al., 1980). This results in increased influx of sodium ions in the cytoplasm, followed by a rise in the (Na*/K ÷) ATPase activity that stimulates the transport of some metabolites (Fagan and Racker, 1978), with the consequent increase of cellular macromolecular synthesis. If the expression of the sarc protein were able to maintain a higher permeability to sodium ions, this would stimulate the increase in (Na÷/K ÷) ATPase activity and cellular metabolism. However, this does not seem to be the case since the activity of the ATPase was reported to be the same for normal chick embryo cells and cells transformed by Rous sarcoma virus (Bader et al., 1981). However, higher uptake of sodium was apparent in cells transformed by RSV as compared to their untransformed counterparts, though this increase was absent in cells transformed by the Schmidt-Ruppin strain of RSV (Bader et al., 1981). Experiments carried out by Garry et al. (1981) showed that uninfected chick cell cultures incubated in either low or high NaC1 medium express many characteristics of transformed cells. Lowering the NaC1 concentration of the medium causes the cells to appear morphologically similar to cells transformed by the Bryan strain of RSV, whereas, on raising the NaC1 concentration the cells appear similar to cells transformed by the Schmidt-Ruppin strain of RSV. In addition, cells incubated in media with altered NaCI concentration grow to higher saturation densities, synthesise reduced amounts of fibronectin, exhibit increased transport of hexoses, etc. The authors suggest that many, but not all, transformation parameters may be consequences of the altered intracellular monovalent cation concentration. 4.2. EFFECTS ON VIRAL FUNCTIONS If viruses cause modifications in membrane permeability, these changes will clearly have implications for virus development. 4.2.1. Viral Entry As discussed in Section 2, virus entry permeabilizes the cell. The kind of permeabilization induced by each virus varies. For instance, adsorption of Sendai virus produces a generalized increase of permeability as measured using different tests, while entry of picornaviruses (EMC virus, poliovirus) is only found to augment permeability when measured by means of translation inhibitors not permeable to normal cells. No leakage of 86Rb+, nor decrease in the nucleotide pool, is observed. The permeabilization occurs both with viruses that enter the cell by direct translocation and by viropexis, indicating that in both cases membrane permeability is altered (Fernhndez-Puentes and Carrasco, 1980; Carrasco, 1981).

Permeabilization of cells

131

The important question to ask in this section is whether the early virus permeabilization has a physiological significance for the process of virus adsorption. The possibility that viruses increase the permeability of cells by specific mechanism, directed simply at entry, seems logical. We know very little about the mechanisms used by viruses and molecules Io cross biological membranes. Most of our information on this subject comes from the mechanisms used by toxins to get the cytosol. In most cases, the toxin is divided in two moieties. One, the effectomer, has a specific enzymatic activity on a cellular component. The other moiety, called the haptomer, is involved in attaching the toxin to the cell surface and permeabilizing the membrane to the effectomer (Olsnes and Pihl, 1982). The molecular basis of this permeabilization is totally obscure. Curiously enough, a number of virus species can replace the haptomer moiety and permeabilize mammalian cells to effectomers of different microbial and plant toxins (Fern~ndez-Puentes and Carrasco, 1980). A likely hypothesis then is that a virion component acts on the cell in a similar way to the haptomer moiety. This viral component could act by 'pushing' the virion particle through the cellular membrane. Whether the virus penetrates by direct translocation or viropexis is not relevant to this reasoning, since it has to cross a membrane anyway to get to the cytosol. The idea that the virion coat is involved in permeabilizing the membrane to viral nucleic acid is reinforced by the finding that the ionophore, amphotericin B, enhances infectivity of isolated EMC virus RNA up to a hundred-fold (Borden et al., 1979). The entry of bacteriophages also involves the permeabilization of the membrane. Of particular interest is the fact that bacteriophage T4 DNA only enters cells when there exists a membrane potential (Labedan and Goldberg, 1979; Kalauskaite et al., 1980). The energy needed for virus entry can be associated with this protonmotive force, as suggested by the chemiosmotic hypothesis of nucleic acid transport (Kalasauskaite et al., 1980). Animal viruses enter the cytosol efficiently when the proton gradient between the cytoplasm and the culture medium increases. That animal viruses also use this protonmotive force to pass the nucleocapsid through the membrane is an attractive hypothesis, as yet unexplored. 4.2.2. Viral M a c r o m o l e c u l a r Synthesis Modification of intracellular ionic conditions by permeability changes during virus infection is, today, an established fact for a number of animal viruses. Synthesis of viral proteins and viral nucleic acids takes place under these altered ionic conditions, which are ,clearly harmful for cellular macromolecular synthesis. Two observations were important in reaching this conclusion: one was that hypertonic medium selectively blocks cellular protein synthesis in virus-infected cells (Saborio et al., 1974; Nuss et al., 1975); the other was the fact that ionic requirements for optimal translation of cellular and viral mRNAs in cell-free systems were different (Carrasco and Smith, 1976). We suggested that "viruses could be able to create ionic conditions under which host mRNAs are unable to bind to ribosomes, whereas viral mRNAs bind more efficiently under the new conditions. The acquisition of such a property would make the virus more efficient and shorten the time for virus development. On the other hand, the possibility of a cellular response involving protein synthesis which interfered with virus development would be diminished" (Carrasco, 1977). During the last few years, evidence has accumulated that several viral mRNAs are resistant to inhibition by hypertonic shock, and are more efficiently translated in vitro under high ionic conditions (Carrasco et al., 1979). In addition to the well-studied picornaviruses, in which most viral proteins and RNAs are synthesized when the membrane becomes leaky to ions (Lacal and Carrasco, 1982), Semliki Forest virus also offers a good example of viral mRNAs translated late in infection, when shut-off of host protein synthesis takes place, having developed the faculty of being optimally translated under the new ionic conditions (Carrasco et al., 1979). SFV has two known mRNAs. One is the genomic 42S RNA, which is translated early in infection together with all cellular mRNAs. Optimal synthesis of these early viral proteins requires ionic concentrations similar to those found for cellular mRNA translation. By contrast, the subgenomic 26S

132

L. CARRASCOand J. C. LACAL

mRNA requires a higher concentration of monovalent cations for optimal translation (Carrasco et al., 1979). This mRNA is translated in vivo late in infection, when shut-off has taken place and membrane permeability modified. Little is known about the requirements of divalent cations for in vitro synthesis of viral proteins. There are however reasons for believing that the concentration of this cation varies after viral infection (Peterhans et al., 1979). 4.2.3. Virus Assembly The last step in virus development is the assembly of new viral particles. This process occurs in cytolytic viruses, when membrane permeability has been altered and the concentration of cations differs widely from those in a normal cell. In vitro assembly of animal viruses is still an undeveloped field. Assembly of bacteriophages and plant viruses is known to occur under defined pH and calcium concentrations (Klug, 1972; Luria et al., 1978). A higher concentration of calcium ions in the cytoplasm helps the assembly of viral structures, since it keeps different virion macromolecules bound. In fact, chelation of divalent cations in animal virus preparations induces the disassembly of the particle. The basic question is why virus uncoating takes place early during infection whereas assembly of virion structures is seen in the late phase. The answer could lie in the different ionic conditions that exist in both situations. 5. PERMEABILIZATION OF MAMMALIAN CELLS BY OTHER AGENTS Interest in permeabilization of mammalian cells to compounds which do not normally enter cells is twofold: (a) different compounds such as inhibitors, enzymes, metabolite analogs, etc. can be put inside to study their action and (b) we can gain insight into the molecular basis of the factors that govern membrane permeability. Before discussing some of the available methods for permeabilizing mammalian cells, there is one point that needs clarification, viz. what do we understand by the term 'permeabilization'? This term is quite unspecific and refers to any increase in the passage of compounds that normally cross the membrane by passive diffusion, or to the passage of compounds to which cells are thought to be impermeable under normal conditions. We feel that, when referring to permeability changes, the compound to which the membrane becomes permeable should be mentioned. For instance, cells can become permeable to potassium ions and yet retain the nucleotide pool intact. Moreover, some conditions permeabilize cells to the entry of compounds, as measured by the non-permeant inhibitor test, and yet exit of other compounds from the cytosol with similar chemical characteristics is not apparent. In conclusion, to define and characterize a given permeability change induced by a compound under certain conditions, one would have to analyze both the exit and the entry of molecules with different chemical characteristics normally excluded by these cells. Only in some extreme circumstances is a general permeabilization of the cell found. Several methods, which have recently been reviewed (Felix, 1982), have been used to permeabilize mammalian cells to a number of compounds. We will comment on some of them with the aim of gaining more insight into the possible molecular mechanisms that viruses use to permeabilize cells early and late during infection. 5.1. PERMEABILIZATION BY IONOPHORES

Ionophores or complexones are compounds that act on biological membranes and mediate the transport of ions through these membranes (Ovchinnikov et al., 1974; Pressman, 1976; Ovchinnikov, 1979). They are classified in two different groups: those that interact with metal ions acting as ion carriers, and those forming ion-permeable pores or channels through the membranes. Polyene antibiotics belong to the second class of ionophores. They interact with the sterols present in eukaryotic membranes and induce leakage of ions and even macromolecules from cells, leading to cell killing (Hamilton-

Permeabilization of cells

133

CH3

HO

,CH~ "F-'--I

c.

HOOC

o.

~ bH 3 CH 3

CH3

"CH 3

MONENSIN

CH3

CH3 H CO, C,II~ CH3

CH3 CH3 /

CH3

NIGERICIN

FIG. 12. Chemical structure of monensin and nigericin.

Miller, 1973; Martin, 1977). Though polyene antibiotics at lower concentrations do not promote cell killing, they alter membrane permeability to a number of substances. Amphotericin B has been applied successfully to increase the permeability of yeast cells to several inhibitors such as 5-fluorocytosine and rifampicin (Medoffet al., 1972; Shadomy et al., 1975; Battaner and Kumar, 1974). Synthesis of RNA from endogenous nucleoside triphosphates has been possible in yeast cells permeabilized by nystatin (Badaracco and Cassani, 1976). Some of these studies point to the fact that amphotericin B produces pores in the membrane that allow the passage of low molecular weight compounds. However, this interpretation does not seem to be totally correct because an increased uptake of E. coli DNA by HeLa cells and an increased infectivity of EMC virus RNA is induced by amphotericin B at concentrations that do not block cell viability (Kumar et al., 1974; Borden et al., 1979). These results suggest rather that amphotericin B induces cellular permeabilization by a still unknown mechanism. Nigericin and monensim (Fig. 12) are ion-carrier ionophores that do not form pores in biological membranes, and yet increase permeability to some antibiotics in mammalian cells even more efficiently than amphotericin B. Promotion of the entry of translation inhibitors of HeLa cells by nigericin is observed even at concentrations that do not inhibit macromolecular synthesis (Alonso and Carrasco, 1980, 1981a, b, 1982a) (Fig. 13). The conditions of this permeabilization are reversible, and do not affect membrane permeability. Surprisingly enough, nigericin at low concentrations also increases permeability to macromolecules as measured by the entry of the toxic protein alpha-sarcin (Alonso and Carrasco, 198 l b). These results clearly show a similarity between membrane permeability induced by nigericin and that observed during virus infection in the early phase of the replicative cycle. How nigericin permeabilizes mammalian cells is obscure. As shown in Fig. 13, a correlation is found between membrane hyperpolarization and increased permeability to hygromycin B and alpha-sarcin (Alonso and Carrasco, 1982b). These results suggest the following sequence of events when cells are treated with nigericin: the ionophore is inserted in the membrane and translocates potassium ions for protons. In this way, leakage of potassium ions from the cell increases. To maintain the concentration of potassium ions in the cytosol constant, the (Na÷/K ÷) ATPase activity is enhanced, and this leads to a hyperpolarization of the membrane. That membrane hyperpolarization induced by nigericin and monensin could be mediated by the (Na+/K +) ATPase is

134

L. CARRASCO and J. C. LACAL

lOO

20 o

kC u

15

c

/

-r

10

E

2

. . . . . . .

-ll-

1

~4/----o- --oO,_

5

._~ 50

et)

- -,.l..--la

o x

g~

Lo

o.

x

o n.Iz o

.3

E °

u

0 -H0

o

, ,, .I 10 - 6

10 -`5

NIGERICIN

10 - 6

10 -5

(M)

FtG. 13. Effect of nigericin on m e m b r a n e permeability and membrane potential of HeLa cells. Different concentrations o f the ionophore nigericin were added to confluent monolayers of HeLa cells. Two hours after nigericin treatment m e m b r a n e potential was analyzed by the [3H]TPP+ technique in the absence ( O ) or in the presence (C)) of 1 mM ouabain. 86Rb + uptake was determined by 20 min pulses in the absence ( D ) or in the presence (11) of ouabain. In panel B protein synthesis was estimated in control cultures (L,) or in cells treated with 1 mM hygromycin B ( O ) or 10/xg/ml alpha-sarcin (U]).

strengthened by the finding that it is inhibited by ouabain, a known inhibitor of this enzyme activity (Lichstein eta/., 1979). Hyperpolarized cells continue synthesizing proteins at control levels, but their membranes are permeable to a number of compounds (Alonso and Carrasco, 1982a) while ionophores that do not induce hyperpolarization of the membrane do not significantly increase permeability to hygromycin B or alpha-sarcin (Alonso and Carrasco, 1980, 1982a). The idea of hyperpolarization of the membrane by other means should lead to increased permeability if supported by our unpublished experiments in which oligomycin, an ATP synthesis inhibitor not known to act on the cytoplasmic membrane, also hyperpolarizes the cell and increase permeability, though less effectively than nigericin (Otero and Carrasco, unpublished observations). However membrane permeability increases by other mechanisms that do not seem to be linked to modifications in membrane potential since our attempt to find alterations in this membrane parameter during picornaviruses infection have been unsuccesful (Lacal and Carrasco, 1982). Other viruses, such as paramyxovirus, depolarize the membrane early in infection, as occurs with most viruses now studied in the late phase of infection. Membrane depolarization also correlates with increased permeability to ions and other compounds, as observed with amphotericin B (Hamilton-Miller, 1973; Alonso and Carrasco 1982a). 5.2. PERMEABILIZATION BY MODIFICATION OF THE IONIC CONCENTRATIONS

Hypertonicity induces permeability of mammalian cells to small molecules (Castellot et al., 1978). Exposure of cells for several minutes to concentrations of NaC1 above 700 mM permeabilized them to compounds such as cytosin arabinoside triphosphate. The treatment was reversible, though during the permeabilization period macromolecular synthesis was obviously blocked (Castellot et al., 1978). Much milder treatments are now available for rendering cultured cells permeable to inhibitors. For instance, the simple replacement of NaC1 by KCI results in a cell fully permeable to hygromycin B (Fig. 14) (Alonso and Carrasco, 1980). The basis of this permeabilization has not been clarified, but it is perhaps a decrease in membrane potential. Under the conditions described, not only is the treatment reversible, but the cells continue synthesizing proteins at normal levels. To our knowledge, this constitutes the mildest method now available for permeabilizing cells to antibiotics that do not normally cross the cell membrane.

Permeabilization of cells

135

F-

12 ~- A

[KCI] : 5raM

!

o,. -=

[KCI] : 9 0 m M

12

g

L/, 8

.~

x

x

0

,

,

so

6o

---

90

I

120

Iso

0

T" ---Ir--"~-_

,,,, -_-

so

90

60

120

1so

[Na CL] mM FIG. 14. Effect of monovalent ion concentration on the permeability of HeLa cells to hygromycin B. HeLa cells were incubated for three hours in Eagle's medium containing the ionic concentrations indicated in the figure. Protein synthesis was then estimated by a 30min pulse with 0.5 pCi/ml [3SS]methionine. Control culture (A); plus 8 x 10-7M nigericin (O); plus 1 mM hygromycin B ( 0 ) ; plus 8 x 10-7M nigericin and 1 mM hygromycin B ( I ) .

Altering the concentration of divalent cations also influences membrane permeability. For long time a high concentration of calcium ions has been used to introduce nucleic acids into cells (Graham and Van der Erb, 1973; Loyter et al., 1982). Recently, by chance, we found a permeabilization method involving depletion of magnesium ions and addition of low concentrations of calcium ions (Fig. 15). The optimal concentration of calcium ions for permeabilizing HeLa cells is very precise. No entry of hygromycin B is observed when lower or higher concentrations are present. This suggests that a specific activity is triggered dependent on the presence of a defined concentration of calcium and which is inhibited by magnesium. This, still hypothetical, activity increases membrane permeability by a mechanism at present under study in our laboratory.

3O E

D- - f , , . . ~

~

°'"/,L.

,I

?

I

o

\

,b

L

/ t s

x

E

Q. U

10

~ 7d

0

// 0

i

t

1 2

t

I

i

5 I0 20

I

50

I

100

[Ca Ct2]mM FIG. 15. Permeabilization o f HeLa cells to hygromycin B by calcium ions. Confluent monolayers of HeLa cells were incubated in Eagle's medium without magnesium ions and the indicated concentration o f CaC12. Protein synthesis was measured one hour after treatment in the absence ( 0 ) or in the presence (O) of 2mM hygromycin B.

136

L. CARRASCO and J. C. LACAL

5.3. LYSOLECITHIN

Lysolecithin is a hemolysin derived from lecithin by enzymatic cleavage. A number of animal cell lines are permeabilized to exogenous molecules by this compound (Miller et al., 1978, 1979; Wydro et al., 1980). At low lysolecithin concentrations, the treatment is reversible, and permeabilization to small molecules occurs. Permeabilization to macromolecules is achieved at higher concentrations, but in this case it is not reversible (Castellot, 1980). 5.4. SELECTIVE PERMEABILIZATION OF TRANSFORMED CELLS BY EXOGENOUS A T P

It would be useful to find a selective permeability of transformed cells to certain inhibitors. However, all comparative studies on selected permeability to drugs of normal and transformed cells do not show any differences with respect to this question. Rozengurt and Heppel (1975) found that incubation of transformed cells in a particular medium in the presence of external ATP induced a rapid permeabilization, as measured by the efflux ofmetabolites (Fig. 16). Maximal effect was found at 0.5 mM ATP (Rozengurt et al., 1977;

A CELLULAR,,.,~ MEMBRANE~: ~p_~ pH>8"~

OUT (

IAntl~ycln

~ r ~

iN

D

B

pH>8

FCCP

9~

C

q

ii

pH~
-,,D IN

OUT( ~ ~

ProteinKinale

PROTEINA ~ . ~ p

(Normal permeability)~f~

®L -

i

IN ATPInhlbltors

PROTEIN-(~

(Increasedpermeability) Na F

Phosphatose FIG. 16. Schematic representation of the permeabilization of transformed cells by ATP. The presence of ATP in the incubation medium at pH 8 induces the permeabilization of the cells (panel B). This effect increases when the cells are treated with uncoupling agents such as FCCP or antimycin A (panel C). Permeability is also attained at neutral pH provided that a phosphate inhibitor such as NaF is present.

Permeabilization of cells

137

Rozengurt and Heppel, 1979). A much smaller permeabilization is observed with resting or growing untransformed cell lines and with secondary cultures of embryo fibroblasts. This permeabilization effect is highly specific and is not induced by GTP, CTP, UTP or ADP (Rozengurt et al., 1977). A synergistic effect is observed in the presence of either energy transfer inhibitors or inhibitors of respiration (Rozengurt and Heppel, 1979). Rapid resealing of the membrane takes place when cells are replaced in normal medium, and they grow normally afterwards. Maximal permeability by these methods is observed with high external and a low intraceUular ATP concentration. The molecular basis for increased permeability perhaps lies in the phosphorylation of a membrane protein that could play a part in the control of passive permeability (Rozengurt and Heppel, 1979; Makan, 1979, 1981). The basic pH required for the permeabilization to occur, seems to be involved in the inhibition of phosphatases, since the presence of sodium fluoride together with external ATP allows permeabilization to occur at neutral pH (Makan, 1978). This is consistent with the idea that there is a fluoride inhibitable protein phosphatase involved in the control of membrane permeability in transformed cells. Do virus increase permeability because of phosphorylation of membrane proteins? Many animal viruses code for protein kinases, and in some cases the virion contains a proton kinase activity. But it is not yet known if permeabilization by naked viruses, such as picornaviruses or adenoviruses, has something to do with the phosphorylation of membrane proteins. Carrasco (1980a) and Kitagawa (1980) found, independently, that selective killing of transformed cells is achieved by means of the ATP permeabilization technique. The rationale behind these experiments is similar to that used to block selectively macromolecular synthesis in virus-infected cells (Carrasco, 1978). Since permeabilization by this technique is specific for transformed cells, the presence of non-permeant inhibitors such as GppCH 2p, hygromycin B or araCTP during the permeabilization treatment would lead to the selective inhibition of transformed cells (Carrasco, 1980; Kitagawa, 1980). As permeabilization in this case occurs by selective phosphorylation of a membrane protein, and this is only found in transformed cells, is of interest to recall that the gene involved in cellular transformation by some retroviruses codes for a protein kinase activity that is located in the membrane (Willingham et al., 1979, 1980; Krueger et al., 1980). 5.5. THIONINS AND OTHER PROTEINS

A number of proteins that interact with the membrane, such as thionins or mellitin, increase permeability in mammalian cells considerably (Carrasco et al., 1981a; Boone and Skalka, 1981). They might act in a way similar to detergents. Once inserted in the membrane, they distort the phospholipid bilayer and, at high concentrations lyse the cells. These proteins could also serve as models to compare whether some virus proteins that interact with cellular membranes produce similar effects and hence he could think that viruses would lyse cells in a similar way. 5.6. OTHER METHODS There are many more methods used to permeabilize cells. The reader is referred to a review by Felix (1982). Many of them use detergents, organic solvents or physical treatments. We consider that though they are useful for permeabilizing cells they are less relevant for understanding the molecular mechanisms used by viruses to modify membrane permeability. 6. F U T U R E PROSPECTS Much work has been done in the last decade in an effort to understand the molecular biology of animal virus development. The progress observed in the characterization of the molecular modifications of cellular membranes induced by animal virus infection is not only an important issue per se, but also because several alterations of cellular metabolism

138

L. CARRASCO and J. C. LACAL

are a consequence of them and are better explained if the changes in membrane permeability are known. The elucidation of the mechanisms used by viruses to enter a host cell is a subject under intense research at present. Although the entry of enveloped viruses by fusion or viropexis is a fairly well understood process, models for entry of naked viruses are less developed. Progress in this subject is clearly connected with the understanding of an important problem in biology: how macromolecules cross cell membranes. Studies on the alteration in membrane permeability early in infection by naked or enveloped viruses are already under way. Except for Sendai virus, where the work done has characterized most of the membrane alterations induced early during infection, for most animal viruses we do not know the viral component involved in permeabilization and its molecular mechanism of action on the membrane. With regard to the changes observed late in infection in the membrane, we consider that the work in this area should be extended to other virus species. The use of mutants can be very profitable in characterizing the viral component responsible for these alterations. Several laboratories have measured modifications in the content of monovalent cations throughout viral infection. We believe that future progress could benefit by the use of more powerful microprobe techniques, such as X-ray diffraction, combined with electron microscopy (Goldstein et al., 1981) and also by the analysis of possible modifications of divalent cations, such as calcium, that so profoundly affect cellular processes (Schackmann and Shapiro, 1982; Epel, 1982; Takai et al., 1982). More knowledge of permeabilization methods for mammalian cells will not only help to introduce different molecules into the cell and study their effects, but also shed more light on the processes that govern membrane permeability. In addition, results obtained with the use of these methods are relevant to an understanding of the modifications in permeability induced by viral infection. Last, but not least, knowledge of the changes in the membrane have already proved to be profitable in designing specific inhibitors of virus-infected cells (Carrasco, 1978; Carrasco et al., 1981b; Carrasco and Vfizquez, 1983). The antiviral effects of some non-permeant translation inhibitors are evident in culture cells (Lacal and Carrasco, unpublished results). The development of new inhibitors, to which virus-infected cells are more permeable, has obvious practical applications. Moreover, the selectivity of some already-known antiviral compounds can be improved, taking advantage of the increased permeability of the cells observed during virus replication. Acknowledgements--J. C. Lacal is the holder of a Caja de Ahorros fellowship. We thank CAICYT for financial support.

REFERENCES AGGELER, J., KAPP, L. N., SHEFFER, C. G., TSENG and WERB,Z. (1982) Regulation of protein secretion in Chinese hamster ovary cells by cell cycle position and cell density. Expl Cell Res. 139: 275-283. AGOL, V. I. (1980) Structure, translation and replication of picornaviral genomes. Prog. reed. ViroL 26:119-157. AHARONOV, A., VLODAVSKY,I., PRUSS, R. M., FOX, C. F. and HERSCHMAN, H. R. (1978) Epidermal growth factor induced membrane changes in 3T3 cells. J. cell. Physiol. 95: 195-202. ALONSO M. A. and CARRASCO, L. (1980) Action of membrane active compounds on mammalian cells. Permeabilization of human cells by ionophores to inhibitors of translation and transcription. Eur. J. Biochem. 109: 535-540. ALONSO, M. A. and CARRASCO,L. (1981a) Relationship between membrane permeability and the translation capacity of human HeLa cells studied by means of the ionophore nigericin. Eur. J. Biochem. 118: 289-294, ALONSO M. A. and CARRASCO,L. (1981b) Permeabilization of mammalian cells to proteins by the ionophore nigericin. FEBS Letts 127:ll2-114. ALONSO M. A. and CARRASCO, L. (1981c) Selective inhibition of cellular protein synthesis by amphotericin B in EMC virus-infected cells. Virology 114: 247-251. ALONSO M. A. and CARRASCO,L. (1981d) Reversion by hypotonic medium of the shut-off of protein synthesis induced by encephalomyocarditis virus. J. Virol. 37: 535-540. ALONSO, M. A. and CARRASCO,L. (1982a) Molecular basis of the permeabilization of mammalian cells by ionophores. Eur. J. Biochem. 127: 567-569. ALONSO M. A. and CARRASCO,t . (1982b) Translation of capped viral mRNAs in poliovirus-infected HeLa cells. The EMBO Journal 1: 913-917.

Permeabilization of cells

139

ALLISON, A. C. and DAVIES, P. (1974) Mechanisms of endocytosis and exocytosis. Symp. Soc. exp. Biol. 28: 419-446. APRILETTI, J. W. and PENHOET, E. E. (1974) Recovery of DNA-dependent RNA polymerase activities from L cells after mengovirus infection. Virology 61: 597-601. BABLANIAN,R. (1975) Structural and functional alterations in cultured cells infected with cytocidal viruses. Prog. Med. Virol. 19: 40-83. BADARACCO,G. and CASSANI,G. (1976) Ribonucleic acid synthesis dependent on exogenous triphosphates in nystatin-treated cells of Kluyveromyces lactis. Antimicrobial Ag. Chemother. 9: 748-753. BADER,J. P., OKAZAKI,T. and BROWN, N. R. (1981) Sodium and Rubidium uptake in cells transformed by Rous sarcoma virus. J. cell. Physiol. 106: 235-243. BADER, J. P., SEGE,R. and BROWN, N. R. (1978) Sodium concentrations affect metabolite uptake and cellular metabolism. J. cell. Physiol. 95: 179-188. )3ANERJEE, S. P., BOSMANN, H. B. and MORGAN, H. R. (1977) Oncogenic transformation of chick-embryo fibroblasts by Rous sarcoma virus alters rubidium uptake and ouabain binding. Expl Cell. Res. 104: l 11-117. ~ARROS, F., DE LA PEI~A, P., GASCON, S., RAMOS, S. and LAZO, P. S. (1981) Cholera toxin induces changes in the ion permeability of intestinal brush border membranes. Biochim. Biophys. Acta 644: 143-146. BATTANER, E. and KUMAR, V. (1974) Rifampin: Inhibition of ribonucleic acid synthesis after potentiation by amphotericin B in Saccharomyces cerevisiae. Antimicro. Ag. Chemother. 5: 371-376. BAXT, B. and BABLANIAN, R. (1976) Mechanism of vesicular stomatitis virus-induced cytopathic effects. II. Inhibition of macromolecular synthesis induced by infectious and DI particles. Virology 72: 383-392. BEATRICE, M. C., PALMER, J. W. and PFEIFGER, D. R. (1980) The relationship between mitochondrial membrane permeability, membrane potential and the retention of Ca 2+ by mitochondria. J. biol. Chem. 255: 86638671. BENEDETTO, A., ROSSl, G. B., AMICI, C., BELARDELLI, F., CIOE, L., CARRUBA, G. and CARRASCO, L. (1980) Inhibition of animal virus production by means of translation inhibitors unable to penetrate normal cells. Virology 106: 123-132. BIENZ, K., EGGER, D., RASSER, Y. and LOEFFLER, H. (1978) Differential inhibition of host cell RNA synthesis in several picornavirus-infected cell lines. Intervirology 10: 209-220. BISHOP, J. M. and VARMUS, H. (1982) Functions and origins of retroviral transforming genes. In RNA tumor viruses, pp 999-1108, WEISS, TEICH, VARMUS and COFFIN (Eds). Cold Spring Harbor, New York. BOONE, L. R. and SKALKA,A. M. (1981) Viral DNA synthesized in vitro of avian retrovirus particles permeabilized with melittin. Kinetics of synthesis and size of minus and plus-strand transcripts. J. Virol. 37: 117-126. BORDEN, E. D., BOOTH, B. W. and MCBAIN, J. A. (1979) Enhancement of infectivity of encephalomyocarditis virus RNA by amphotericin B methyl ester. J. gen. Virol. 42: 297-303. BORSA, J., MORASH, B. O., SARGENT, M. D., CoPPS, T. P., LIEVAART,P. A. and SZEKELY,J. G. (1979) Two modes of entry of reovirus particles into L cells. J. gen ViroL 45: 161-170. BRENDLER, T., GODEEROY-COLBURN,T., CARLILL, R. D. and THACH, R. E. (1981) The role of mRNA competition in regulating translation II Development of a quantitative in vitro assay. J. biol. Chem. 256: 11747-11754. BRODSKY, W. A., SADOFF, J. C., DURHAM, J. H., EHRENSPECK, G., SCHACHNER, M. and IGLEWSKI, B. H. (1979) Effects of pseudomonas toxin A, diphtheria toxin and cholera toxin on electrical characteristics of turtle bladder. Proc. natn. Acad. Sci. U.S.A. 76: 3562-3566. CANTOR,H. and BOYSE,E. A. (1977) Lymphocytes as models for the study of mammalian cellular differentiation. Immunological Rev. 33: 105-124. CARAEOLI,E. (1982) Membrane Transport of Calcium. Academic Press, London. CARRASCO, L. (1981) Modification of membrane permeability induced by animal viruses early in infection. Virology 113: 623-629. CARRASCO,L. and ESTEBAN, M. (1982) Modification of membrane permeability in vaccinia virus-infected cells. Virology 117: 62-69. CARRASCO,L. (1979) The development of new antiviral agents based on virus-mediated cell modification. In Antiviral Mechanisms for the Control of Neoplasma, pp 623-63 l, CHANDRA,P. (Ed.). Plenum Press, New York. CARRASCO,L. (1980a) Selective inhibition of translation in transformed cells. FEBS Lefts 110: 341-343. CARRASCO, L. (1980b) Action of nucleotide derivatives on translation in encephalomyocarditis virus-infected mouse cells. J. gen. Virol. 54: 125-134. CARRASCO, L., V.~ZQUEZ,D., HERN.~NDEZ-LUCAS,C., CARBONERO,P. and GARCiA-OLMEDO,F. (1981a) Thionins: plant peptides that modify membrane permeability in cultured mammalian cells. Eur. J. Biochem. 116: 185-189. CARRASCO,L., FERNANDEZ-PUENTES, C., ALONSO, M. A., LACAL, J. C., Mu/qoz, A., FERNANDEZ-SOUSA,J. M. and V/~ZQUEZ,D. (1981b) New approaches to antiviral agents. In Medicinal chemistry advances, pp 211-223, DE LAS HERAS, F. and VEGA, S. (Eds). Pergamon Press. CARRASCO,L. and SMITH, A. E. (1976) Sodium ions and the shut-off of host protein synthesis by picornaviruses. Nature 264: 807-809. CARRASCO,L. (1978) Membrane leakiness after viral infection and a new approach to the development of antiviral agents. Nature 272: 694-699. CARRASCO,L., HARVEY, R., BLANCHARD,C. and SMITH, A. E. (1979) Regulation of translation of eukaryotic virus mRNAs. In: Modern Trends in Human Leukemia Ill, pp 189-192, NETH, R., HOFSCHENEIDER, P. and MANNWILER, K. (Eds). Springer-Verlag, New York. CARRASCO,L. (1977) The inhibition of cell functions after viral infection. A proposed general mechanism. FEBS Letts 76: 11-15. CARRASCO, L. and VAZQUEZ, D. (1983) Viral translation inhibitors. In Antibiotics, Vol. VL HANH, F. (Ed.). Springer-Verlag. CARRASCO,L. and SMITH, A. E. (1980) Molecular biology of animal virus infection. Pharmac. Ther. 9:311-355.

140

L. CARRASCO and J. C. LACAL

CASTELLOT,J. J., MILLER,M. R. and PARDEE,A. B. (1978) Animal cells reversibly permeable to small molecules. Proc. natn. Acad. Sci. U.S.A. 75: 351-355.

CASTELLOT,J. J. (1980) Lysolecithin-permeabilized animal cells as a tool for studying cell growth and metabolism. In: Introduction o f Macromolecules into Viable Mammalian Cells, pp 297-324, LIss, A. R. (Ed.). (Wister Inst. Symp. Ser 1). CHOY, P. C,, PADDON, n . B. and VANCE, D. E. (1980) An increase in cytoplasmic CTP accelerates the reaction catalyzed by CTP: Phosphocholine cytidyltransferase in poliovirus-infected HeLa cells. J. biol. Chem. 255: 1070-1073. CHOPPIN, P. W. and SHEID, A. (1980) The role of glycoproteins in adsorption, penetration and pathogenicity of viruses. Rev. infect. Dis. 2: 40-61. COHEN, P. (1980) The role of calcium ions, calmodulin and troponin in the regulation of phosphorylase kinase from rabbit skeletal muscle. Fur. J. Biochem. 111: 563-574. COLLINS, F. D. and ROBERTS, W. R. (1972) Mechanism of mengovirus-induced cell injury in L cells: Use of inhibitor of protein synthesis to dissociate virus-specific events. J. Virol. 10: 969-978. COMPANS, R. W. and KEMP, M. C. (1978) Membrane gycoproteins of enveloped viruses. In: Current Topics in Membranes and Transport, 1Iol. II, pp. 233-277, BRONNER, F. and KLEINZELLER, A. (Eds). Academic Press, New York. CONE, C. D. and CONE, C. M. (1976) Induction of mitosis in mature neurons in central nervous system by sustained depolarization. Science 192: 155-156. CONE, C. O. (1971) Unified theory on the basic mechanisms of normal mitotic control and oncogenesis. J. theor. Biol. 30: 151-181. CONTRERAS,A. and CARRASCO,L. (1979) Selective inhibition of protein synthesis in virus-infected mammalian cells. J. ViroL 29: 114-122. COOMBS, K., MANN, E., EDWARDS, J. and BROWN, D. T. (1981) Effects of chloroquine and cytochalasin B on the infection of cells by Sindbis virus and vesicular stomatitis virus. J. Virol. 37: 1060-1065. Cox, D. C. and CLINKSCALES,C. W. (1976) Infectious reovirus subviral particles: virus replication, cellular cytopathology and DNA synthesis. Virology 74: 259-261. DALES, S. (1973) Early events in cell-animal virus interactions. Bact. Rev. 37: 103-135. DE Lg PE/qA,P., BARROS,F., GASCON, S., LAZO, P. S. and RAMOS, S. (1981) Effect of yeast killer toxin on sensitive cells of Saccharomyces. J. biol. Chem. 256: 10420-10425. DIMMOCK, N. J. (1982) Initial stages in infection with animal viruses. J. gen. Viron. 59: 1-22. DORI~E, M., MOREAU, M. and GUERRIER, P. (1978) Hormonal control of meiosis. In vitro induced release of calcium ions from the plasma membrane in starfish oocytes. Expl Cell. Res. 115: 251-260. DUNCAN, J. L. and BUCKINGHAM, L. (1977) Effects of streptolysin O on transport of amino acids, nucleosides and glucose analogs in mammalian cells. Infect. Immun. 18: 688-693. DUNNEBACKE, T. H., LEVINTHAL,J. D. and WILLIAMS,R. C. (1969) Entry and release of poliovirus as observed by electron microscopy of cultured cells. J. ViroL 4: 505-513. EGBERTS, E., HACKETT, P. B. and TRAUB, P. (1977) Alteration of the intracellular energetic and ionic conditions by mengovirus infection of Ehrlich ascites tumor cells and its influence on protein synthesis in the midphase of infection. J. Virol. 22: 591-597. EGGLETON, K. H. and NORKIN, L. C. (1981) Cell killing by simian virus 40: The sequence of ultrastructural alterations leading to cellular degeneration and death. Virology 110: 73-86. EPEL, D. (1982) The physiology and chemistry of calcium during the fertilization of eggs. In: Calcium and Cell Function vol. II, pp 356-383, CrtEUNG, W. Y. (Ed.). Academic Press, New York. PAGAN, J. B. and RACKER, E. (1978) Determinants of glycolytic rate in normal and transformed chick embryo fibroblasts. Cancer Res. 38: 749-758. FALLON, R. J., COX, R. P. and GHOSH, N. K. (1979) Induction of human Choriogonadotropin and follitropin in HeLa cell cultures by hyperosmolality. J. cell. Physiol. 98: 613-618. FAN, D. P. and SEFTON, B. M. (1978) The entry into host cells of Sindbis virus, vesicular stomatitis and Sendal virus. Cell 15: 985-992. FARNHAM, A. E. and EPSTEIN, W. (1963) Influence of encephalomyocarditis (EMC) virus infection on potassium transport in L cells. Virology 21: 436-447. FELIX, H. (1982) Permeabilized cells. Ann. Biochem. 120: 211-234. FERNANDEZ-PUENXES, C. and CARRASCO, L. (1980) Viral infection permeabilizes mammalian cells to protein toxins. Cell 20: 769-775. FOSTER, D. O. and PARDEE, A. B. (1969) Transport of amino acids by confluent and nonconfluent 3T3 and Polyoma virus-transformed 3T3 cells growing on glass cover slips. J. biol. Chem. 244: 2675-2681. FRANKLIN, R. M. and BALTIMORE,D. (1962) Patterns of macromolecular synthesis in normal and virus-infected mammalian cells. Cold Spring Harbor Symp. Quant. Biol. 27: 175-198. FRANCOER, A. M. and STANNERS,C. P. (1978) Evidence against the role o f K + in the shut-off of protein synthesis by vesicular stomatitis virus. J. gen. Virol. 39: 551-554. FUCHS, P. and GmERMAN, E. (1973) Enhancement of potassium influx in baby hamster kidney cells and chicken erythrocytes during adsorption of parainfluenza I (Sendal) virus. FEBS Letts 31: 127-130. FucHs, P., GRUDER, E., GITELMAN,J. and KOHN, A. (1980) Nature of permeability changes in membrane of HeLa cells adsorbing Sendal virus. J. cell. Physiol. 103:271 278. FUCHS, P., SPIEGELSTEIN,M., CHA1MSON,M., GITELMAN,J. and KOHN, A. (1978) Early changes in the membrane of cell absorbing Sendal virus under conditions of fusion. J. cell. Physiol. 95: 223-234. GARRY, R. F., BISHOP, J. M., PARKER, S., WESTBROOK,K., LEWIS, G. and WAITE, M. R. F. (1979) Na ÷ and K + concentrations and the regulation of protein synthesis in Sindbis virus-infected chick cells. Virology 96: 108-120. GARRY, R. F., MOYER, M. P., BISHOP, J. M., MOVER, R. C. and WAITE, M. R. F. (1981) Transformation parameters induced in chick cells by incubation in media of altered NaCl concentration. Virology 111: 427-439.

Permeabilization of cells

141

GARRY,R. F. and WAITE,M. R. F. (1979) Na + and K + concentrations and the regulation of the interferon system in chick cells. Virology 96: 121-128. GAZITT, Y. (1979) Early decrease of 2-Deoxy glucose and y-amino isobutyric acid transport are among the first events in differentiating synchronized murine erythroleukemia cells. J. cell. Physiol. 99: 407-416. GENTY, N. (1975) Analysis of uridine incorporation in chicken embryo cells infected by vesicular stomatitis virus and its temperature-sensitive mutants: uridine transport. J. Virol. 15: 8-15. GILBERT (1963) Enzyme release from tissue cultures as an indicator of cellular injury by viruses. Virology 21: 609-616. GOLDSTEIN, J. E., NEWBURY, D. E., ECHLIN, P., JOY, D. C., FIORI, C. and LIFSHIN, E. (1981) Scanning Electron Microscopy and X-ray Microanalysis. Plenum Press, New York. GOLDSTEIN, J. L., ANDERSON, R. G. W. and BROWN, M. S. (1979) Coated pits, coated vesicles and receptor mediated endocytosis. Nature 279: 679-685. GOLINI, F., THACH, S. S., BIRGE,C. H., SAFER, B., MERR1CK, W. C. and THACH, R. E. (1976) Competition between cellular and viral mRNAs in vitro is regulated by a messenger discriminatory initiation factor. Proc. HatH. Acad. Sci. U.S.A. 73: 3040-3044. GRAHAM, F. L. and VAN DER ERB, A. J. (1973) A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52: 456-467. GROLLMAN, E. F., LEE, G., AMBESI-IMPIOMBATO,F. S., MELDOLESI, M. F., ALOJ, S. M., COON, H. G., KABACK, H. R. and KOHN, L. D. (1977) Effects of thyrotropin on the thyroid cell membrane: hyperpolarization induced by hormone-receptor interaction. Proc. HatH. Acad. Sci. U.S.A. 74: 2352-2356. IHACKSTADT, T. and MALLAVlC, L. P. (1982) Sodium and potassium transport in herpes simplex virus-infected cells. J. gen. Virol. 60:199 207. HAMAGUCHI, Y. and HIRAMOTO, Y. (1981) Activation of sea urchin eggs by microinjection of calcium buffers. Expl Cell Res. 134: 171-179. HAMILTON-MILLER, J. M. T. (1973) Chemistry and biology of the polyene macrolide antibiotics. Bact. Rev. 37: 166--196. HAMMARSTROM,L. and SMITH, C. I. E. (1979) Reversible inhibition of lymphocyte proliferation by rubidium ions. Expl Cell Res. 119: 343-348. HANSEN, J. and EHRENFELD, E. (1981) Presence of the capbinding protein in initiation factor preparation from poliovirus-infected HeLa cells. J. Virol. 38: 438-445. HAROLD, F. M. (1977) Ion currents and physiological functions in microorganisms. Ann. Rev. Microbiol. 31: 181-203.

HARRISON, P. R. (1982) Stem cell regulation in erythropoiesis. Nature 295: 454-455. HATANAKA, M. and HANAFUSA, H. (1970) Analysis of a functional change in membrane in the process of cell transformation by Rous sarcoma virus: Alteration in the characteristics of sugar transport. Virology 46: 470~79. HEINZ, E. (1981) Electrical potentials in biological membrane transport. Mol. Biol. Biochern. Biophys. Vol 33. Springer-Verlag. HELENIUS, A., MARSH, M. and WHITE, J. (1980) The entry of viruses into animal cells. Trends Biochem. Sci. 5: 104-106. HIRATA, F. and AXELROD, J. (1978) Enzymatic methylation of phosphatidyl-ethanolamine increase erythrocyte membrane fluidity. Nature 275: 219-220. HOSAKA, Y. and SHIMIZU, J. K. (1972) Cell fusion by Sendai virus. Cell Surf. Rev. 2: 129-155. Hsu, M. C., SCHEID, A. and CHOPPIN (1979) Reconstitution of membranes with individual paramyxovirus glycoproteins and phospholipid in cholate solution Virology 95: 476-491. HUANG, A. S. and WAGNER, R. R. (1965) Inhibition of cellular RNA synthesis by non-replicating vesicular stomatitis virus. Proc. Natn. Acad. Sci. U.S.A. 54: 1579-1584. {MPRAIN, C. C., FOSTER, K. A., MICKLEM, K. J. and PASTERNAK,C. A. (1980) Nature of virally mediated changes in membrane permeability to small molecules. Biochem. J. 186:847 860. .IOHNSON, M. A. and WEBER, M. J. (1979) Potassium fluxes and ouabain binding in growing, density-inhibited and Rous sarcoma virus-transformed chicken embryo cells. J. cell. Physiol. 101: 89-100. .IULIANO,R. L. and GAGALANE,E. 0979) The effect of membrane-fluidizing agents on the adhesium of CHO cells. J. cell. Physiol. 98: 483-490. KACHAR, B. and REESE, T. S. (1982) Evidence for the lipidic nature of tight junction strands. Nature 296: 464. KALASAUSKAITE, E., GRINIUS, L., KADISAITE, D. and JASA1TIS, A. (1980) Electrochemical H + gradient but not phosphate potential is required for Escherichia coli infection by phage T4. FEBS Letts 117: 232-236. KAPLAN, J. G. (1979) Activation of cation transport during lymphocyte stimulation: the molecular theology of spinning metabolic wheels. Trends Biochem. Sci. 4:147 149. KIEFER, H., BLUME, A. I. and KABACK, H. R. (1980) Membrane potential changes during mitogenic stimulation of mouse spleen lymphocytes. Proc. HatH. Acad. Sci. U.S.A. 77: 2200-2204. I(IMELBERG,H. K. (1977) The influence of membrane fluidity on the activity of membrane-bound enzymes. Cell. surf. Rev. 3: 205-293. KITAGAWA, T. (1980) Selective inhibition of macromolecular synthesis in transformed mouse cells by means of ATP-treatment. Biochem. biophys Res. Commun. 94: 167-173. KLEMPERER, H. G. (1960) Hemolysis and the release of potassium from cells by Newcastle disease virus (NDV). Virology 12: 540~552. KLENK, H. D. and ROTT, R. (1970) Cotranslational and post-translational processing of viral glycoproteins. Curr. Topics Microbiol. Immunol. 90: 19-48. KLUG, A. (1972) Assembly of tobacco mosaic virus. Fedn Proc. 31: 3042. KOCH, K. S. and LEFFERT, H. L. (1979) Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation. Cell 18: 153-163. KOHN, A. (1979) Early interactions of viruses with cellular membranes. Adv. Virus. Res. 24: 223-276.

142

L. CARRASCO and J. C. LACAL

KOREN, R. and SHOHAMI,E. (1979) Magnesium ions and serum activation of uridine uptake by quiescent hamster fibroblasts. FEBS Letts 106: 149-152. KRUEGER, J. G., WANG, E. and GOLBERG, A. R. (1980) Evidence that the src gene product of Rous sarcoma virus is membrane associated. Virology 101: 25-40. KUMAR, B. V., MEDOFF, G., KOBAYASHI,G. and SCHLESSINGER,D. (1974) Uptake of Escherichia coli DNA into HeLa cells enhanced by amphotericin B. Nature 250: 323-325. LABEDAN, B. and GOLBERG, E. B. (1979) Requirement for membrane potential in injection of phage T4 DNA. Proc. natn. Acad. Sci. U.S.A. 76: 4669-4673. LACAL, J. C. and CARRASCO,L. (1982) Relationship between membrane integrity and the inhibition of host translation in virus-infected mammalian cells. Comparative studies between encephalomyocarditis virus and poliovirus. Eur. J. Biochem. 127: 359-366. LACAL, J. C., V.~ZQUEZ,D., FERNANDEZ-SOUSA,J. M. and CARRASCO,L. (1980) Antibiotics that specifically block translation in virus-infected cells. J. Antibiotics 33: 441-447. LAGE-DAVILA, A , HOFMAN-CLERC, F., TORPIER, G. and MONTAGNIER, L. (1979) Glucose-regulated membrane properties of untransformed and virus-transformed BHK-21 cells. Expl Cell Res. 120: 181-189. LANG, D. R. and WEBER,M. J. (1978) Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus. J. cell. Physiol. 94:315-320. LAWRENCE, C. and THACH, R. E. (1974) Encephalomyocarditis virus infection of mouse plasmocytoma cells. I. Inhibition of cellular protein synthesis. J. Virol. 14: 598-610. LAZO, P. S., BARROS,F., PEI~A,P. and RAMOS,S. (1981) Ion gradients as candidates for transmembrane signalling. Trends Biochem. Sci. 6: 83-85. LEDBET'rER, M. L. and LUBIN, M. (1977) Control of protein synthesis in human fibroblasts by intracellular potassium. Expl Cell Res. 105: 223-236. LENARD, J. and MILLER, D. K. (1982) Uncoating of enveloped viruses. Cell 28: 5-6. LEVANON, A., KOHN, A. and INBAR, M. (1977) Increase in lipid fluidity of cellular membranes induced by absorption of RNA and DNA virions. J. ViroL 22: 353-360. LEVENSON, R., MACARA, I. G., SMITH, R. L., CANTLEY,L. and HOUSMAN, D. (1982) Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation. Cell 28: 855-863. LEV1NSON,A. D., COURTNEIDGE,S. A. and BISHOP, J. M. (1981) Structural and functional domains of the Rous sarcoma virus transforming protein (pp 60st0. Proc. natn. Acad. Sci. U.S.A. 78: 1624-1628. LICHSTEIN, D., KABACK, H. R. and BLUME,A. J. (1979) Use of a lipophylic cation of determination of membrane potential in neuroblastoma-glioma hybrid cell suspension. Proc. natn. Acad. Sci. U.S.A. 76: 650654. LOYTER, A., SCANGOS, G. A. and RUDDLE, F. H. (1982) Mechanisms of DNA uptake by mammalian cells: Fate of exogenously added DNA monitored by the use of fluorescent dyes. Proc. natn. Acad. Sci. U.S.A. 79: 422~,26. LUBIN, M. (1967) Intracellular Potassium and macromolecular synthesis in mammalian cells. Nature 213: 451-453. LURIA, S. E., DARNELL, J. J. E., BALTIMORE,D. and CAMPBELL, A. (1978) General Virology, John Wiley, New York. MAEDA, T., ASANO, A., OKADA, Y. and OHNISHI, S. I. (1977) Transmembrane phospholipid motions induced by F glycoprotein in Haemagglutining virus of Japan. J. lHroL 21: 232-241. MAKAN, N. R. (1978) Induction of permeability change and restoration of membrane permeability barrier in transformed cell cultures. Expl Cell Res. 114: 417-427. MAKAN, N. R. (1979) Role of cytoplasmic ATP in the restoration and maintenance of a membrane permeability barrier in transformed mammalian cells. J. cell. Physiol. 101: 481-492. MAKAN, N. R. 0981) Passive membrane permeability to small molecules and ions in transformed mammalian cells: Probable role of surface phosphorylation. J. cell. Physiol. 106: 49-61. MARKS, P. A. and RIFKIND, R. (1978) Erythroleukemic differentiation. A. Rev. Biochem. 47: 419-448. MARTIN, J. F. (1977) Biosynthesis of polyene macrolide antibiotics. A. Rev. Biochem. 31: 13-38. MCGRAW, C. F., NACHSHEN, D. A. and BLAUSTE1N, M. P. (1982) Calcium movement and regulation in presynaptic nerve terminals. In Calcium and Cell Function, CHEUNG,W. Y. (Ed.). Academic Press, New York. MCSHARRY, J. J. and CHOPPING, P. W. (1978) Biological properties of the VSV glycoprotein I. Effects of the isolated glycoprotein on host macromolecular synthesis. Virology 84: 172-182. MEDOEF, G., KOBAYASHI, G. S., KWON, C. N., SCHLESSINGER, D. and VENKOV, P. (1972) Potentiation of rifampicin and 5-fluorocytosine as antifungal antibiotics by amphotericin B. Proc. natn. Acad. Sci. U.S.A. 69: 196-199. MENDOZA, S. A., WIGGLESWORTH,N. M., POH.IANPELTO,P. and ROZENGURT, E. (1980) Na + entry and Na+-K + pump activity in murine, hamster and human cells--Effect of monensin, serum, platelet extract and viral transformation. J. cell. Physiol. 103: 17-27. MILLER, M. R., CASTELLOT,J. J. and PARDEE,A. B. (1978) A permeable animal cell preparation for studying macromolecular synthesis DNA synthesis and the role of deoxyribonucleotides in S phase initiation. Biochemistry 17: 1073-1080. MILLER, M. R., CASTELLOT,J. J. and PAROLE, A. B. (1979) A general method for permeabilizing monolayer and suspension cultured animal cells. Expl Cell Res. 120: 421. MILLER, H. I. and PENHOET, E. E. (1972) Differential inhibition of nuclear RNA polymerases in L cells infected with mengovirus. Proc. Soc. exp. Biol. Med. 140: 435-438. MONDOVI, B., STROM, R., FINAZZIAGRO, A., CAIAEA,P. DE SOLE, P., BOZZI, A., ROTILLO, G. and RossI FANELLI, A. (1971) Effect of polyenic antibiotics. Cancer Res. 31: 505-509. MOOLENAAR, W. H., MUMMERY,C. L., VAN DER SAAG, P. T. and DE LAAT, S. W. (1981) Rapid ionic events and the initiation of growth in serum-stimulated neurobalstoma cells. Cell 23:789 798.

Permeabilization of cells

143

MORGAN, C., ROSENKRANZ, H. S. and MEDNIS, B. (1969) Structure and development of virus as observed in the electron microscope X. Entry and uncoating of adenovirus. J. Virol. 4: 777-799. MORONEY, S., SMITH, A., THOEI, L. D. and WENNER, C. E. 0978) Stimulation of 86Rb+ and 32pi movements in 3T3 cells by prostaglandins and phorbol esters. J. cell. Physiol. 95: 287-294. MOSSER, A. G., CALIQUIRI,L. A. and TAMM, I. (1972) Incorporation of lipid precursors into cytoplasmic membranes of poliovirus-infected HeLa cells. Virology 47: 3947. MUI~oz, A. and CARRASCO,L. (1981) Protein synthesis and membrane integrity in interferon-treated HeLa cells infected with encephalomyocarditis virus. J. gen. Virol. 56:153 162. NAIR, C. N. (1981) Monovalent cation metabolism and cytopathic effects of poliovirus-infected HeLa cells. J. Virol. 37: 268-273. NAIR, C. N., STOWERS, J. W. and SINGFIELD, I. (1979) Guanidine-sensitive Na + accumulation by poliovirusinfected HeLa cells. J. Virol. 31: 184-189. NALECZ, M. J., ZBOROWSKI, J., FAMULSKI, K. S. and WOJTCZAK,L. (1980) Effect of phospholipid composition on the surface potential of liposomes and the activity of enzymes incorporated into liposomes. Eur. J. Biochem. 112: 75-80. NEUMAYER,E., HOFF, R. and HOFFMAN, C. (1965) Antiviral activity of amantadine hydrochloride in tissue culture and in vivo. Proc. Soc. exp. Biol. Med. 119: 393-396. NISHIOKA, D. and MAGAGNA, L. S. (1981) Increased uptake of thymidine in the activation of sea urchin eggs. Expl Cell Res. 133: 363-372. NORKIN, L. C. (1977) Cell killing by simian virus 40: Impairment of membrane formation and function. J. Virol. 21: 872-879. NORRIE, D. H., WOLSTENHOLME,J., HOWCROFT, H. and STEPHEN,J. (1982) Vaceinia virus-induced changes in Na + and K + in HeLa cells. J. gen. Virol. 62: 127-136. Nuss, D. L. and KOCH, G. (1976) Differential inhibition of vesicular stomatitis virus polypeptide synthesis by hypertonic initiation block. J. Virol. 17: 283-286. NUSS, O. L., OPPERMANN,H. and KOCH, G. (1975) Selective blockage of initiation of host protein synthesis in RNA virns-infected cells. Proc. HatH. Acad. Sci. U.S.A. 72: 1258-1262. OKADA, Y., KASEKI, I., KIM, J., MAEDA, Y., HASHIMOTO, T., KANNO, Y. and MATSUI, Y. (1975) Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendal virus). Expl Cell Res. 93: 368-378. OLSNES, S. and PIHL, A. (1982) Toxic lectins and related proteins. In: Molecular Action o f Toxins and Viruses, pp 51-105, COHEN and VAN HEYNINGEN (Eds). Elsevier Biomedical Press. OPPENHEIMER,S. B. (1977) Current Topics in Developmental Biology, Vol 1, p 1, MOSCONA, A. A. and MONROY A. (Ed.). Academic Press, New York. ORR, C. W., YOSHIKAWA-FUKADA, M. and EVERT, J. D. (1972) Potassium: effect on DNA synthesis and multiplication of Baby-Hamster Kidney cells. Proc. HatH. Acad. Sci. U.S.A. 69: 243-247. OVCHINNIKOV, Y. A., IVANOV,V. T. and SHKROB, A. M. (1974) Membrane Active Complexomes. Elsevier. Amsterdam. OVCmNNIKOV, Y. A. (1979) Physico-chemical basis of ion transport through biological membranes: Ionophores and ion channels. Eur. J. Biochem. 94: 321-336. OXENDER, D. L., LEE, M. and CECCHINI, G. (1977) Regulation of amino acid transport activity and growth rate of animal cells in culture. J. biol. Chem. 252: 2680-2683. PASTERNAK, C. A. and MICKLEM, K. J. (1973) Permeability changes during cell fusion. J. Membrane. Biol. 14: 293-303. PASTERNAK, C. A. and MICKLEM, K. J. (1974) The biochemistry of virus-induced cell fusion. Biochem. J. 140: 405~, 11. PASTERNAK, C. A. and MICKLEM, K. J. (1981) Virally induced alterations in cellular permeability: A basis of cellular and physiological damage? Bioscience Rep. 1: 431~148. PATTERSON, S., OXFORD, J. S. and DOURMASHKIN,R. R. (1979) Studies on the mechanism of influenza virus entry into cells. J. gen. Virol. 43: 223-229. PEARSE, B. (1980) Coated vesicles. Trends Biochem. Sci. 5: 131-134. PENMAN, S. (1965) Stimulation of the incorporation of choline in poliovirus-infected cells. Virology 25:148-152. PETERHANS, E., HAENGGELI, E., WILD, P. and WYLER, R. (1979) Mitochondrial calcium uptake during infection of chicken embryo cells with Semliki Forest virus. J. Virol. 29: 143-152. PEEFFERKORN, E. R. and SHAPIRO, D. (1974) Reproduction of togaviruses. In: Comprehensive Virology, Vol. 2, pp. 171-230, FRAENKEL-CONRAT, H. and WAGNER, R. R. (Eds). Plenum Press, New York. POSTE, G. and PASTERNAK, C. A. (1978) Virus-induced cell fusion. Cell S u r f Revs 5: 306-349. POTTER, J. D. and JOHNSON, J. D. (1982) Troponin. In: Calcium and Cell Function, CHEUNG, W. Y. (Ed.). Academic Press, New York. PRESSMAN, B. C. (1976) Biological applications of ionophores. A. Rev. Biochem. 45: 501-530. ROTnSTEIN, A. (1978) The cell membranes a short historical perspective. Curr. Topics Membranes trans. 11: 1-13. ROZENGURT, E. and HEPPEL, L. A. (1975) A specific effect of external ATP on the permeability of transformed 3T3 ceils. Biochem. biophys. Res. Commun. 67: 1581-1588. ROZENGURT, E. and HEPPEL, L. A. (1979)Reciprocal control of membrane permeability of transformed cultures of mouse cell lines by external and internal ATP. J. biol. Chem. 254: 708-714. ROZENGURT, E., HEPPEL, L. A. and FRIEDBERG,I. (1977) Effect of exogenous ATP on the permeability properties of transformed cultures of mouse cell lines. J. biol. Chem. 252: 458~4590. ROZENGURT, E., LEGG, A. and PET~CAN, P. (1979) Vasopresin stimulation of mouse 3T3 cell growth. Proc. natn. Acad. Sci. U.S.A. 76: 1284-1287. ROSE, J. K., TRACHSEL, n., LEONG, K. and BALTIMORE, D. (1978) Inhibition of translation by poliovirusinactivation of a specific initiation factor. Proc. natn. Acad. Sci. U.S.A. 76: 2732-2736. RUmN, H. (1981) Growth regulation, reverse transformation, and adaptability of 3T3 cells in decreased Mg 2+ concentration. Proc. natn. Acad. Sci. U.S.A. 78: 328-332.

144

L. CARRASCO and J. C. LACAL

RUBIN, H., MIDAIR, C. and SANOl, H. (1981) Restoration of normal appearance, growth behavior, and calcium content to transformed 3T3 cells by magnesium deprivation. Proc. natn. Acad. Sci. U.S.A. 78: 2350-2354. SABORIO, J. L., PONG, S. S. and KOCH, G. (1974) Selective and reversible inhibition of initiation of protein synthesis in mammalian cells. 3. tool. Biol. 85:195-211. SANDVIG, L., OLSNES, S. and P1HL, A. (1979) Inhibitory effect of ammonium chloride and chloroquine on the entry of the toxic lectin modeccin into HeLa cells. Biochem. biophys. Res. Commun. 90: 648-655. SCHACKMANN,R. W. and SHAPIRO, B. M. (1982) Calcium and the metabolic activation of spermatozoa. In: Calcium and Cell Function vol. II, pp 339-353, CHEUNG,W. V. (Ed.). Academic Press, New York. SCHADT,M. and HAEUSLER,G. (1974) Permeability of lipid bilayer membranes to biogenic amines and cations: changes induced by ionophores and correlations with biological activities. J. Membrane Biol. 18: 277-294. SCHAEFER, A., K/3HNE, J., ZIBIRRE, R. and KOCH, G. (1982) Poliovirus-induced alterations in HeLa cell membrane functions. J. Virol. 44: 444-449. SCHANNE, F. A. X., KANE, A. B., YOUNG, E. E. and FARBER, J. L. (1979) Calcium dependence of toxic cell death: A final common pathway. Science 206: 700-702. SCHEID, A. and CHOPPIN, P. W. (1974) Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57: 475-490. SCHWARTZ, L. B., LAWRENCE, C., THACH, R. E. and ROEDER, R. G. (1974) Encephalomyocarditis virus infection of mouse plasmacytoma cells II. Effect and host RNA synthesis and RNA polymerases. J. Virol. 14:611-619. SCHLEGEL, R., DICKSON, R. B., WILLINGHAM,M. C. and PASTAN, I. R. (1982) Amantadine and dansylcadaverine inhibit vesicular stomatitis virus uptake and receptor-mediated endocytosis of 2-macroglobulin. Proc. HatH. Acad. Sci. U.S.A. 79: 2291-2295. SCHLEHOFER, J. R., HABERMEHL, K. O., DIEFENTHAL, W. and HAMPL, H. (1979) Reduction of 5ICE-permeability of tissue culture cells by infection with herpes simplex virus type 1. Intervirology 11: 158-166. SCHUPERLI,D., PETERHANC,E. and WYLER,R. (1978) Permeability changes of plasma and lysosomal membranes in HeLa cells infected with rabbit poxvirus. Arch. Virol. 58: 203-212. SEGEL, G. B. and LICHTMAN, M. A. (1978) The effect of method on the measurement of K + concentration in PHA-treated human blood lymphocytes. Expl Cell Res. 112: 95-102. SHADOMY, S., WAGNER, G., SPINEL-INGROFF, A. and DAVIS, B. A. (1975) In vitro studies with combinations of 5-fluorocytosine and amphotericin B. Antimicro. Ag. Chemother. 8:117-121. SIMILI, M., DE FERRA, F. and BAGLIONI,C. (1980) Inhibition of RNA and protein synthesis in inteferon-treated HeLa cells infected with vesicular stomatitis virus. J. gen. Virol. 47: 373-384. SINGER,S. J. (1974) The molecular organization of membranes. A. Rev. Biochem. 43: 805-833. SKEI-IEL, J. J., HAY, A. J. and ARMSTRONG, J. A. (1977) On the mechanism of inhibition of influenza virus replication by amantadine hydrochloride. J. gen. Virol. 38:97-110. SMETS, L. A., ENNINGA, I. C. and VAN ROODY, H. (1982) Cell communication reduced by changes in cell surface carbohydrates. Expl Cell Res. 139: 181-189. SMITH, N. R., SPARKS, R. L., POOL, T. B. and COMERON,I. L. (1978) Differences in the intracellular concentration of elements in normal and cancerous liver cells as determined by X-ray microanalysis. Cancer Res. 38: 1952-1959. SPENDLOVE, R. S. and SCHAEFER, F. L. (1965) Enzymatic enhancement of infectivity of reovirus. J. Bact. 89: 597-602. STORGESS, J., MOSCARELLO, M. and SCHACHrER, H. (1978) The structure and biosynthesis of membrane glycoproteins. In: Current Topics in Membranes and Transport, Vol. 11, pp 15-105, BRONNER, E. and KLEINZELLER, A. (Eds). Academic Press, New York. TAKAI, Y., KISHIMOTO, A. and NISHIZUKA, Y. (1982) Calcium and phospholipid turnover as transmembrane signaling for protein phosphorylation. In: Calcium and Cell Function, Vol. II, pp 386-412, CHEUNG, W. Y. (Ed.). Academic Press, New York. TOLMACH, L. J. (1959) Attachment and penetration of cells by viruses. Adv. Virus Res. 4: 63-110. TOOZE, J. (1981) DNA Tumor Viruses. Cold Spring Harbor, New York. TRACHSEL, H., SONEMBERG, N., SHATKIN, A. J., ROSE, K., LEONG, K., BERGMANN, J. E., GORDON, J. and BALTIMORE, D. (1980) Purification of a factor that restores translation of vesicular stomatitis virus mRNA in extracts from poliovirus-infected HeLa cells. Proc. natn. Acad. Sci. U.S.A. 77: 770-774. TRUMP, B. F., CROKER, B. P. and MERGNER, H. J. (1971) The role of energy metabolism, ion and water shifts in the pathogenesis of cell injury. In: Cell membranes, Biological and Pathological Aspects, pp 84-128, RICHER, G. W. and SCARPELLI, D. G. (Eds). Williams & Wilkins, Baltimore. TSIEN, R. Y., POZZAN, T. and RINK, T. J. (1982) T-cell mitogens free Ca 2+ and membrane potential in lymphocytes. Nature 295: 68-71. VANCE, D. E., TRIP, E. M. and PADDON, H. B. (1980) Poliovirus increases phosphatidylcholine biosynthesis in HeLa cells by stimulation of the rate-limiting reaction catalyzed by CTP: Phosphocholine cytidylyltransferase. J. biol. Chem. 255: 1064-1069. VAZQUEZ, D. (1979) Inhibitors of proteinbiosynthesis. In: Molecular Biology Biochemistry and Biophysics, Vol. 30. KL~NZELLER, A., SPRINGER, G. F. and WITTMANN, H. G. (Eds). Springer-Verlag, Berlin. WALOEN, W. E., GODEFROY-COLBURN, T. and THACH, R. E. (1981) The role of mRNA competition in regulating translation I. Demonstration of competition in vivo. J. biol. Chem. 256:11739-11746. WEISS, R., TE1CH, N., VARMUS,H. and COFFIN, J. (1982) R N A Tumor Viruses. Cold Spring Harbor Laboratory. New York. WERTZ, G. W. and YOUNGNER, J. S. (1972) Inhibition of protein synthesis in L cells infected with vesicular stomatitis virus. J. Virol. 9: 85-87. WHITE, J., MATL1N, K. and HELENIUS, A. (1981) Cell fusion by SFV, influenza and VS viruses. J. cell. Biol. 89: 674-679. WHITTINGHAM,D. G. and SIRACUSA, G. (1978) The involvement of calcium in the activation of mammalian oocytes. Expl Cell Res. 113: 311-317.

Permeabilization of cells

145

WILLIAMS,R. J. P. (1982) Free manganese (II) and ion (II) cations can act as intracellular cell controls. FEBS Letts 140:3 10. WILLINGHAM, M. C., JAY, G. and PASTAN, I. (1979) Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell 18: 125-134. WILLINGHAM,M. C., PASTAN,I., Snm, T. Y. and SCOLNlCK, E. M. (1980) Localization of the src gene product of the Harvey strain of MSV to the plasma membrane of transformed cells by electron microscopy immunocytochemistry. Cell 19: 1005-1014. WOJTCZAK, L. and NALECZ,N. J. (1979) Surface charge of biological membranes as a possible regulator or membrane-bound enzymes. Eur. J. Biochem. 94: 99-107. WYDRO, R., BROT, N. and WEISSBACrt,H. (1980) RNA synthesis in permeable HeLa cells. Arch. Biochem. Biophys. 201: 73-80. '~NYKE, A. M., IMPRAIN, C. C., KNUTTON, S. and PASTERNAK, C. A. (1980) Components involved in vitally mediated membrane fusion and permeability changes. Biochem. J. 190: 625-638. YAMAIZUMI,M., UCH1DA,T. and OKADA,Y. (1979) Macromolecules can penetrate the host cell membrane during the early period of incubation with HSJ (Sendai virus). Virology 95:218 221. YAMAZAKI,S. and WAGNER,R. R. (1970) Action of interferon: Kinetics and differential effects on viral functions. J. Virol. 6: 421~,29. YAOI, Y., MITSUI, H. and AMANO, M. (1970) Effect of u.v.-irradiated vesicular stomatitis virus on nucleic acid synthesis in chick embryo cells, J. gen. Virol. 8: 165-172. YOSHIOKA, T. and INOUE, H. (1981) Activation of sea urchin eggs by a channel-forming ionophore amphotericin B. Expl Cell Res. 132: 461-464.

JPT 23/I--J