Sendai virus replication in Friend erythroleukemia cells. I. Acutely and persistently infected cells become resistant to virus-induced lysis

117

Vtrus Research, 4 (1986) 117-132 Elsevier

VRR 00230

Sendai virus replication in Friend erythroleukemia cells. I. Acutely and persistently infected cells become resistant to virus-induced lysis Arrigo

Benedetto

*, Carla

Amici, Stefania Zaniratti, Maria Pia Camporiondo

Ciuliano

Elia ** and

Summary

Friend leukemia cells (FLC) are susceptible to infection by Sendai virus. a member of the paramyxovirus group. FLC constitute a most suitable model to study virus-host cell interactions, because they grow in suspension (thus avoiding the use of trypsin), and provide an easy way of deriving single-cell clones. When FLC are infected with Sendai virus at high m.o.i., a direct, extensive lysis of the cells ensues, whereas lower doses of virus result in a cytocidal infection whose lethality depends mainly on the virus used, standard or defective interfering egggrown Sendai virus (EGSV), and on the multiplicity of infection (m.o.i.). At later times after infection, FLC become resistant to the Sendai induced lysis (SIL). The SIL resistance can be maintained in single-cell clones that had survived the first infection. The maintenance of the resistant phenotype of the clones requires the serial subcultivation of the cells in the presence of activated EGSV. The mechanisms that presumably regulate the appearance of SIL resistance in Sendai infected FLC are discussed.

Friend

leukemia

cells, Sendai virus, cell lysis

* To whom correspondence should be addressed. ** Permunent uddress: Istituto Medicina Sperimentale

0168-1702/86/$03.50

CNR.

0 1986 Elsevier Science Publishers

Second

University

B.V. (Biomedical

of Rome, Italy.

Division)

118

Introduction The proteolytic cleavage of the viral glycoprotein F is the first step in the process of activation of Sendai virus (a member of the paramyxovirus family). and an important prerequisite for infection to occur (Homma and Ohuchi. 1973; Scheid and Choppin, 1974: Richardson et al., 1980). In the absence of proteolytic cleavage the infectivity of Sendai virus is almost abolished. Viral activation can be achieved in vitro by proteolytic digestion of the virions by trypsin or chymotrypsin (Scheid et al.. 1978; Ishida and Homma, 1978). The replication of fully infectious progeny virus (standard virions) is modulated to some extent by the production of defective interfering (DI) particles which, in turn. require standard virus to replicate (Roux and Holland. 1979). In many cellular systems DI Sendai virus can establish a persistent infection characterized by: (i) the production of defective viral RNA (Kolakofsky. 1976, 1979; Roux and Holland. 1980). (ii) a reduced expression of haemagglutinin (HN) molecules at the cell surface (Roux and Waldvogel, 1983). and (iii) the instability of the viral M protein (Roux and Waldvogel. 1982). Kimura et al. (1979) and Yoshida et al. (1979) have also described a cellular system where persistent infection was sustained by ts mutants of Sendai virus unable to cause cytopathic effect (CPE), and endowed with the ability to cause homologous interference with wild-type Sendai virus. The maintenance of the carrier state established by Sendai virus in cell monolayers, however, may be influenced by the periodic trypsinization of the cultures. a procedure that would mask putative differences in the degree of spontaneous activation of the virus. This appears indeed to be the case, as it has been reported that trypsinization of AGMK cultures persistently infected with Sendai virus resulted in a net increase in the virus production (Benedetto et al., 1979). We decided to study the replication of Sendai virus in Friend leukemia cells (FLC) because of the ability of these cells to grow in suspension (thus avoiding the use of trypsin), their susceptibility to Sendai virus infection, and the ease of selecting individual cell clones. We found that FLC were fully susceptible to Sendai virus infection: they supported the viral growth with subsequent cellular lysis, and conditions were found that resulted in the establishment of a persistent infection of FLC cultures. Furthermore, in this paper we report that FLC are promptly lysed when infected with high doses of Sendai virus. We have studied the cells that have survived a first infection with Sendai virus, and from these populations we have isolated FLC clones that were resistant to Sendai virus induced lysis (SIL). suggesting that the superinfecting virus cannot fuse to the plasma membrane of infected cells. The long-term maintenance of the resistant phenotype requires that the cells be cultivated in medium containing activated Sendai virus.

Materials and Methods

FLC (clone 745 A) were routinely

seeded in RPM1 1640 Medium,

supplemented

119 with 10% fetal calf serum and antibiotics at a concentration of ca. 2 X 10’ cells/ml. and were incubated at 37°C in a humidified atmosphere in the presence of 5% CO,. Cell cloning procedures were as previously described (Benedetto et al.. 1982). Viruses Standard stocks of egg-grown Sendai virus (Standard EGSV) were prepared by allantoic inoculation of lo-day-old embryonated eggs. with 0.2 ml of a lOh dilution of infected allantoic fluid. After 48 h at 37°C. allantoic fluid was harvested. clarified by centrifugation (4000 X g, 4”C, 10 min). and stored at -70°C. Egg-grown stocks of defective interfering virus (DI EGSV) were prepared according to Roux and Holland (1979) with the only difference that nine serial undiluted passages in eggs were performed instead of five. Plaque-purified vesicular stomatitis virus (VSV) was obtained from the Laboratory of Virology of the Istituto Superiore di Sanita. Rome. Virus purification The infected allantoic fluid was carefully layered onto a cushion of 50% sucrose in phosphate-buffered saline (PBS). After centrifugation (Spinco rotor SW-27. 22 000 rpm, 5”C, 60 min), the viral band that appeared at the interphase was collected, diluted fourfold in PBS, and centrifuged through a discontinuous density gradient (50, 40. 30 and 20% sucrose in PBS, Spinco rotor SW-27, 25000 rpm, 5°C. 60 min). The viral band was collected with an ISCO density gradient fractionator. Titration Sendai virus was quantified by either plaque assay, or by measuring the amount of haemagglutinin present in the supernatant medium: (a) Monolayers of Madin Darby bovine kidney cells (MDBK. obtained from the Centro Substrati Cellulari, Brescia, Italy) in plastic 3.5-cm Petri dishes were washed twice with serum-free RPMI-1640 medium, and log,,, dilutions of the virus in RPM1 supplemented with 10 mg/ml bovine serum albumin (Sigma, St. Louis, MO, U.S.A.) were added. After adsorption (37”C, 60 min), the inocula (0.5 ml) were removed. and the cells were washed twice as above. The infected monolayers were then overlaid with 3 ml of serum-free RPM1 containing 0.95% Bacto agar and 0.3 pg/ml N-acetyl trypsin (Sigma). Plaques were counted 96 h thereafter without staining, and the counts were confirmed after staining with neutral red (1 : 10000). (b) Haemagglutination tests were performed by standard procedures, using 0.5% human red blood cells (RBC) 0 Rh + suspended in phosphate-buffered saline (PBS). (c) Haemadsorption assays were carried out by measuring the percentage of rosette-forming cells: The infected cultures were washed twice with PBS, and resuspended at 2 x 10h cells/ml. An equal volume of a 0.1% suspension of human red blood cells 0 Rhf was added, the cells were incubated at 4’C for 60 min, and the rosettes were counted in a haemocytometer. Sendai virus-induced cell &sis 2 X lo5 cells were incubated at 37°C 60 min in serum-free RPM1 medium (0.2 ml) containing 20 pCi/ml of [“Crlsodium chromate (Amersham. U.K.). Cells were

120 washed twice with non-radioactive medium, and resuspended in 0.2 ml of fresh medium containing EGSV (32 HAU/ml). After 1 h incubation at 37°C the cells were pelleted, and the radioactivity released into the supernatants was measured in a gamma-counter. The values of radioactivity observed have been considered a measure of the virus-induced cell lysis. 100% potential lysis was defined as the amount of radioactivity released by treatment of “Cr-labelled cells with 2% Triton X-100. The spontaneous release of “Cr was determined by incubating the cells without virus. The percentage of SIL therefore was determined by the formula: Virus-released Triton X-100

radinact.

-

released radioact.

Spontaneously -

released radioact,

Spontaneously

released radioact.

x 100

Complement-induced immunolysis (C”IL) 2 x lo5 cells to be tested for C”IL were labelled with “Cr and washed as described above. The cells were then treated for 60 min at 37°C with diluted 1 : 10 anti-Sendai HN antibody (kindly provided by Prof. R. Calm, Second University of Rome), and 2 units of guinea pig complement. After 45 min incubation the radioactivity released was measured in a gamma-counter. The non-specific release of “Cr was determined by incubating the cells with guinea pig complement in the absence of antibody. The 100% potential cell lysis was determined by incubating the cells with 2% Triton X-100. The percentage of C”IL was calculated according to the formula: Complement

and antibody released radmact. - Radioact.

Triton X-100

released radioact.

- Radioact.

released without antlbody

released wthout

x ,oo

antibody

Anubsis of cellulur und virul proteins Cultures were starved for 30 min in methionine-free medium, and pulse-labelled with [7’S]methionine (10 pCi/ml, 37’C, 30 min). The cells were then pelleted, washed twice with PBS, resuspended in 150 ~1 of sample buffer (62.5 mM Tris-HCI (pH 6.8). 100 mM dithiothreitol, 0.001% bromophenol blue, 2% SDS, 10% glycerol), sonicated to reduce the viscosity, and heated 10 min at 90°C. 100~~1 aliquots were analysed by polyacrylamide gel electrophoresis using 10% gels. Gels were fixed, stained, and prepared for fluorography with PPO/DMSO (20%, w/v) by standard techniques.

Results

Fusion of FLC by stundud und Dkontuining Sendui virus preparations Complement-induced immunolysis (C”IL) is a very sensitive procedure to reveal Sendai virions fused to the host cell plasma membrane (Fan and Sefton, 1978). Standard or DI-containing Sendai virus preparations (3.8 X 10h pfu/HAU or 1.1 X lo5 pfu/HAU, respectively) were equally able to fuse FLC plasma membrane independently of their different specific infectivity. This is seen in Fig. 1: for cells infected with either DI or standard EGSV virus stocks, C”IL was complete in the range of 4-0.5 HAU/lOh cells, and declined rapidly under 0.25 HAU/lO” cells.

121

J 1

2

3

4

5

Vrrus 64 32 16

7

6

dilution

8

4

2

8

icolog,)

1

Fig. 1. Sensitivity of FLC. complement.

-I

.5 .25 32 06 .03

HAU/lO’

and

9101112

1

FIX.

infected

with

scalar

doses of standard

0) Sendai virus. were assayed for complement-induced t*and Methods. Fig. 2. Kinetics of “Cr HAU/lO’

(O--

immun~~lysis as described

release from FLC treated with high doses of Sendai virus. “Cr-labelled RPM1

cells of purified

1640. either normal (O----

standard

EGSV

were collected and SIL was appreciated

3

Time (hours)

cells

newly infected with various doses of Sendai virus. to anti-Sendai

“Cr-labelled

suspended in serum-free

2

and incubated

antibodies 0)

or

DI

m Materiais

FLC‘ were

0) or Ca”-free (O---O). containing 32 at 37°C. AI indicated time intervals. cultures

as described in Materrais

and Methods.

Infection of FLC with doses of virus higher than 4 HAU/lO” cells resulted in the direct lysis of the cultures. Since P-induced immunolysis can detect the presence of a few viral polypeptides fused to the cell plasma membrane, it is reasonable to assume that the cells which escaped immunolysis induced by anti-HN antibodies represent those cells free of viral HN protein on their surface. The minimal dose of input virus (measured as HAU) that gave C”IL of 100% of cells was similar for both virus stocks: 0.25 HAU/lO” cells.

High doses of both standard and DI-containing EGSV (from 128 to 8 HAU/lO” cells) exerted a rapid, direct cytolytic effect on FLC without any cell agglutination or cell fusion (Fig. 1, asterisk). The Sendai-induced lysis (SIL) was indeed a virus-dependent phenomenon, since heat-inactivation of the input virus (%*C. 30 min) abolished completely such an effect. Moreover. a minimum of 8 HAU/lO” cells were required to secure the lysis of 100% of the cells. Values of Sit obtained by counting the amount of “Cr released as described in Materials and Methods. were confirmed by direct counting of the viable cells. SIL seems to be temperature dependent. with

122 an optimum at 37°C. and reached its maximum value 1 h after the addition virus to the cells (Fig. 2). Ca’ ’ ions can be dispensed with (Fig. 2).

of the

Virus retdicutim it1 FLC’ infected with difjCerent doses of DI or standurd EGS V As a first approach to investigate Sendai virus-host cell interactions. we looked at the effect of m.o.i. (defined as pfu/cell) on the production of virus, and on host-cell metabolism. FLC were infected with either standard (3.8 X 10” pfu/HAU) or DI-containing (1.1 x 10’ pfu/HAU) stocks of EGSV at HAU/lO“ cell ratios of 0.25, 1 and 2. These values corresponded to m.o.i.‘s of - 1. - 4 and - 8 pfu/cell. and - 0.03. - 0.1 and - 0.2 pfu/cell for standard and DI-contatning EGSV, respectively. At daily intervals thereafter. the viable cells were counted, the number of rosette-forming cells was determined. and the amount of viral HAU released into the media was quantified (Fig. 3). Some remarkable features emerged from these experiments: (a) In cells initially infected with Standard EGSV. the production of virus (as

100

150

mo1=4

80

120

60

90

60

120

9lJ

60

30

1

2

3

4

1 after

Time Fig. 3. Effect of standard

and DI

growth rate of FLC. Time-course (A: m.o.i. = X. 2 HAU/lO”

EGSV

.\tock\. 0 -0.

4

( days)

~nfectwn at different

m.o.i.‘s (i.e.. pfu/crll)

n)

cells) and

0.

F: m.o.i. = 0.03. 0.25 HAU/lO’ HAU released into the medium.

by Trypan

Blue exclusion dye method. each

cells (9::); Omeawred

and

with standard

c: m.o.l. - 1. 0.25 HAU/IO”

E: m.o.l. = 0.1. I HAU/IO”cells:

rosette-forming

I\ indicated (m----

m.o.i.‘s on expression of infectwn

infected at different

cells: B: m.o.~. = 4. 1 HAU/lOhcells:

In the insets. the number of viable cells ( x IOh). da) after Infection.

3

infection

study of FLC,

Dl (D: m.o.~. = 0.2. 2 HAU/IO”cells; cells) EGSV

2

123 revealed by the number of rosette-forming cells, and the amount of HAU in the medium) was directly proportional to the initial m.o.i. (Fig. 3A-C). Moreover. a large proportion of cells infected at m.o.i. = - 1 failed to give rosettes. (b) Concomitantly, the overall rate of cell survival (inserts) declined steadily with increasing m.o.i.‘s and, apparently, no cell could survive 48 h after infection at m.0.i. = 8; (c) At lower m.o.i.‘s, after an initial lag of 24-48 h. Sendai virus infected cells not only survived, but were even able to multiply almost normally (Fig. 3B. C. insert). (d) As expected, FLC infected with DI-containing EGSV produced significantly lower amounts of virus. This was accompanied by either the mere survival of the culture (Fig. 3D insert). or an almost normal cell replication rate (Fig. 3E, F. inserts) at low m.o.i.‘s. (e) The formation of polykaryocytes as a result of the infection was never observed. We have previously reported that in Sendai-infected AGMK cells the virus protein synthesis increased as a function of the m.o.i., even though the overall rate of protein synthesis remained constant (Benedetto et al.. 1980). The question then arose as to whether the observed inhibition of cellular replication and the virus-induced cell lethality were due to the virus-induced inhibition of cell protein synthesis (shut off). Accordingly, we labelled with [“Slmethionine FLC cultures infected with standard or DI-containing EGSV at various m.o.i.‘s. Fig. 4A shows the protein pattern of control, uninfected 745 A cells. Fig. 4B presents the pattern shown bv 745 A cells infected with standard EGSV at m.o.i. = - 8 in which almost only viral bands are recognizable. DI EGSV-infected cultures (m.o.i. = - 0.2 pfu/cell. Fig. 4C) present a picture which resembles that of standard EGSV infected ones. even though the host cell protein synthesis is less compromised. Sendai virus directed peptides (notably NP and M) are also observed in FLC infected with standard EGSV at a m.0.i. of - 1 (Fig. 4D), but under these conditions the synthesis of high molecular weight proteins of cellular origin appeared to persist to a certain extent (Fig. 4D and corresponding densitometry). These findings raise the question as to whether the cells that survived the first infection had been really infected. Accordingly. for each m.o.i. used, the percentage of cells that would escape infection was estimated on the basis of the Poisson distribution of probabilities, and these theoretical ,:aiues were compared with the experimental ones. As shown in Table 1, the percentage of rosette-forming cells (i.e.. the cells actually engaged in viral protein synthesis 24 h p.i.) in FLC infected with standard EGSV was in good agreement with the predicted values. whereas in cultures infected with DI-containing stocks the percentage of uninfected cells was significantly lower than expected. a situation that the intrinsic heterogeneitv of the viral population of any DI-containing stock (infectious and defectivle particles) may be accounted for. Since a defined portion of the cell population survived the first infection. it was tempting to speculate that those are the cells that, on the basis of a random Poisson distribution of the infective particles, did never get infected at all. That this is not the case can be inferred from the fact that at all the HAU/cell ratios used 100”; of

124

P ~HN -

NP

43K-

31 K-

A

B

C

D

E

Ftg. 4. Effect of standard or DI EGSV infection at different m.o.i.‘s on protein synthesis of FLC. (A) Control uninfected 745 A cells: (B) standard EGSV infected 745 A cells. m.o.i. = 8: (C) DI EGSV Infected 745 A cells. m.o.1. - 0.2: (D) standard EGSV infected 745 A cells, m.o.i. = 1: (E) 745 A cells. infected at m.o.i. = 4 with standard EGSV and labelled 5 days p.i. On the right-hand side of the figure are reported the densitometric scans corresponding to each autoradiogram. Standard proteins: phosphorylase h (M, = 94K): bovine serum albumin (M, = 68K): ovalbumin (M, = 43K): carbonic anhydrase (M, = 3lK).

TABLE

I

EFFECTIVENESS

OF INFECTION

OF FLC BY STANDARD

AND

DI SENDAI

VIRUS

Virus stock

Inuculum size (HAU/lOh cells)

m.0.i. (pfu/cell)

Rosette-forming cells (%;) ”

Predicted number of infected cells (‘%)h

Standard

2 1 0.25

7.600 3.800 0.950

100 7s 50

100 97.76 61.32

DI

2 1 0.25

0.220 0.110 0.027

40 24 10

19.74 10.41 2.66

I’ Measured h According

at 24 h pi.: mean values of triplicate experiments. to Poisson’5 distribution. The following formula was applied:

s = (1 -em ““‘~L”‘).lOO.

125 the cells were susceptible to C”-induced immunolysis, consistent with the notion that all the cells had fused at least one viral particle (Fig. 1). If this apparent discrepancy is explained as a result of the presence of altered particles (virions able to fuse the cell membrane but unable to undergo a productive infectious cycle) one would expect that as the cells divide the percentage of cells free of viral protein in the plasma membrane would increase steadily. Here again this appears not to be the case: Cells which have been infected at HAU/lOh cell ratios of 1 and 0.25 either with standard or DI EGSV, at the 4th day p.i. showed almost regular cell doubling rate. All these cells, however, were infected, as they underwent C”-induced immunolysis (not shown). Furthermore, PAGE analysis of the proteins synthesized by the cells infected with Standard EGSV at an m.o.i. - 4 and labelled 5 days thereafter showed that viral polypeptides were produced, even though the host cell protein synthesis was largely maintained (Fig. 4E). These cultures, therefore, appeared to satisfy the main criterion established by Walker (1964) to define a persistent infection: production of virus with conserved growing ability of the host cells. Reinfection of Friend cells that survived the first infection with Sendui virus Cultures persistently infected with Sendai virus become resistant to superinfection with the homologous virus (Roux and Holland, 1979; Benedetto et al., 1979). To check whether FLC persistently infected with Sendai virus behaved similarly, the following experiment was performed: 5 days after the initial infection at a HAU/lOh cells ratio = 1 with either DI or standard EGSV. these cultures were re-infected at an m.o.i. - 8 (2 HAU/106 cells) with Standard virus. In contrast to the large cytocidal effect following the first infection of FLC at an m.o.i. - 8 (Fig. 2A) the reinfected cultures remained viable, and maintained a normal growth rate. These persistently infected FLC cultures, however, were fully susceptible to infection by VSV (10 pfu/cell), indicating that the resistance to the superinfection by the homologous virus was independent on the production of interferon. In these conditions it was important to check whether the resistance to superinfection observed in persistently infected FLC cultures implied also a state of resistance to SIL, induced by high amounts of Sendai virus. Accordingly, we measured the degree of SIL, after the addition of 32, 64 or 128 HAU/lOh cells of standard EGSV to these persistently infected FLC. Table 2 shows that the FLC persistently infected with either standard or DI EGSV were equally resistant to SIL, whereas the mock-infected controls were fully susceptible to Sendai induced lysis. These findings suggest that the attachment and/or the fusion of the superinfectant homologous virus to the cell surface are seriously impaired in persistently infected FLC. Possible mechanisms regulating the resistance to the superinfection of FLC The kinetics of appearance of the resistance to SIL after infection of FLC cultures with standard EGSV at an m.o.i. - 4 was then investigated. Beginning 1 h after virus infection through the 3rd hour, the cells were fully susceptible to SIL (Fig. 5). Then the sensitivity to SIL decreased steadily, and by lo-12 h reached a plateau value of ca. 20%. It is worth noting at this point that the maximal resistance to SIL was observed concomitantly with the beginning of the release (and successive accumula-

126 TABLE

2

SENDAI-INDUCED COVERED FLC Cell culture5

CYTOLYSIS

(SIL)

UNINFECTED

AND

FIRST

INFECTION

RF.-

Percent of cells lysed by various doses of EGSV 32 HAU/lOhcells

Uninfected Standard DI

OF

FLC

64 HAU/lOhcells

12X HAU,‘lO”cells

a

b

c

a

b

5

a

h

c

95 1x 17

90 21 15

x2 25 20

97 23 I9

100 I6 21

91 18 I6

92 21 20

89 15 1X

100

a. b and c indicate sets of values obtained in three distinct cultures recovered from first infectlon with 1 HAU/lOhcells

29 25

experiments. Standard or DI represent FLC of standard or DI EGSV. respectively.

tion) of cell-grown Sendai virus (CGSV). The question then arose as to whether the CGSV released into the medium played a role in this phenomenon: conceivably. the viral particles produced in the infected cells and released in the medium can prevent the SIL either by digesting the receptor moieties of the cellular plasma membrane via their neuraminidase activity, or by binding more stably to the receptor. Such a stable binding may result from the reduced ability of CGSV to fuse the cells. In fact, in the absence of proteolytic activation. CGSV would just be able to adsorb and occupy the cellular receptor. A third possibility to be taken into account is that the insertion of viral proteins into the cell plasma membrane. and their accumulation at later times after infection would modify the cell surface in such a way as to hinder the fusion process of the superinfecting Sendai virions. That the latter is indeed the case appears from the following experiment: FLC cultures were infected with standard EGSV at an m.o.i. - 4, and incubated for 12 h thereafter. The cultures were washed and transferred to fresh medium containing: (a) 3% fetuin (Sigma, St. Louis, MO): (b) rabbit anti-Sendai antisera diluted 1 : 10: (c) mouse IFN (1000 IU/ml, kindly

3

6

9 12 15 18 21 24

Fig. 5. SIL of FLC cultures at different time mtervals during first infection. FLC cultures were infected ;Lt m.o.i. - 4 with standard EGSV and incubated. At indicated time intervals. cultweb were labelled with “Cr and assayed for SIL as described in Materials and Methods.

127 TABLE

3

INFLUENCE OF FETUIN. INTERFERON AND ANTI-SENDAI SIL SENSITIVITY OF INFECTED AND UNINFECTED FLC Treatment

TREATMENT

ON

SIL(%) Uninfected

None Fetuin Interferon Anti-Sendai

ANTIBODY

antibody

Infected

cells

a

b

a

95 100 90 97

97 91 97 91

18 20 55 19

a and b indicate sets of values obtained percentage of rosette-forming cells.

in two different

experiments.

cells b

(97) (95) (45) (92)

21 23 60 25

(93) (99) (51) (95)

Values in parentheses

indicate

the

provided by Dr. F. Belardelli, 1st. Sup. Sanita, Rome); or (d) no addition. At 24 h p.i., SIL was assayed by adding standard EGSV to the cultures (32 HAU/lO’ cells). Early experiments had shown that addition of 3% fetuin prevented completely the desialation of FLC by 2.5 IU/ml of Vibrio choleru neuraminidase, as determined by the method of thiobarbituric acid assay (Warren, 1959). Table 3 shows that fetuin and anti-Sendai serum did not modify the susceptibility of the infected culture to SIL, while IFN treatment (at doses that halved the percentage of rosette-forming cells; Table 3, figures in parentheses) restored the SIL to a significant extent. We interpreted these results as an indication that CGSV released into the medium does not play a major role in the acquisition of resistance to SIL either by means of the neuraminidase activity (which is blocked by the high doses of fetuin; Kimura et al., 1980) nor by occupying stably the cellular receptors. In fact, by neutralizing the ability of the virions to bind to the competent receptors with specific antibody, the SIL resistance remained unchanged. It was rather the inhibition of the viral replication resulting from the IFN treatment the condition that prevented the appearance of the resistance to SIL, consistent with the notion that the membrane of persistently infected FLC were modified from within. Stability of the SIL-resistant phenotype Since FLC persistently infected with Sendai virus grew normally, it was important to determine how long the SIL-resistant character persisted upon long-term cultivation of the cells. Accordingly, one culture which had survived the first infection with standard EGSV was divided in two and maintained in the presence or absence of EGSV in the culture medium for more than 30 generations. EGSV (2 HAU/ml) was added to the cultures at every cell subculturing, i.e. every fifth day. The addition of virus to the cell population surviving the first infection slowed down the growth rate slightly but, as the cellular generation number increased, the slowing effect exerted on the cell growth by the new virus was less evident until it was completely suppressed. After about 15 cell generations each culture was cloned in soft agar, in the presence or absence of EGSV in the agar medium. The cloning efficiency of the two cultures was practically the same. Eight single cell derived

128 TABLE

4

HN EXPRESSION AND SIL RESISTANCE SURVIVING SENDAI INFECTION Original

population

Clones

OF CLONES

DERIVED

HN on the plasma membrane

FROM

SILh ”

0 2 5 0 1 3 2 0

95 88 92 90 X6 95 90 97

80 95 90 85 94 100 92 91

21 15 5 12 21 17 8 12

Maintained in the absence of virus

1 2 3 4 5 6 7 8

Maintained in the presence of virus

IV 2v 3v 4v 5v 6v 7v uv

.’ Percentage * Percentage

HN molecule on their surface as appreciated SIL (see Materials and Methods).

of cells expressing of cells undergoing

CELL POPULATIONS

by C”IL

clones from each culture were expanded, and cultivated in the presence or absence of the virus. The clones were studied for the expression of Sendai-virus infection as revealed by: (i) the presence of HN antigens on the cellular plasma membrane (C”-induced immunolysis), and (ii) the susceptibility to SIL. The data reported in Table 4 suggest that the cultivation of first-infection surviving cells in the absence of virus selects cell clones which do not express HN on the plasma membrane. Since they are unable to form rosettes, and cannot be stained with FITC-labelled anti-Sendai antibodies (not shown), they seem to be completely recovered from the first infection. All these clones are sensitive to SIL. On the other hand, cellular clones cultivated in the constant presence of EGSV are resistant to SIL, and carry HN antigens in their membranes, suggesting that their resistance to SIL is induced by the infectious process, and that its maintenance requires the continuous addition of active (egg-grown) virus to the culture. To confirm that this was indeed the case, we studied the SIL and rosette-forming cells in one clone, cultivated for several generations either in the presence or absence of standard or DI EGSV (Fig. 6). The long term cultivation of the clone in the presence of standard or DI EGSV maintained substantially unchanged the SIL-resistant phenotype, while the percentage of rosette-forming cells tended to vary in the range of 20-40s (standard EGSV), and lo-30% (DI EGSV) (Fig. 6A,B). In the absence of EGSV the SIL-resistant character of the clone was progressively lost after ca. 20 passages. Concomitantly, the expression of Sendai virus infection, as revealed by the percentage of rosette-forming cells, declined steadily (Fig. 6C).

129

4

8

12 16 20 24 28 32 36 40 Generation

Fig. 6. Stability added.

SIL

populations

number

of SIL-resistant

sensitivity (Cl-lv),

(o--

phenotype 0)

and

long term cultivated

during cell cultivation frequency

of

in presence or absence of new virus

rosette-forming

in presence of standard EGSV

cells (0 -

0)

in clonal

(A), in presence of DI EGSV

(B)

or in absence of virus (C).

Taken together, our data suggest that infection of FLC lines can be maintained only by t-e-infecting the culture: without new active virus added, the cloned cells tend to recover from their infection, reacquiring in this process their original sensitivity to SIL.

Discussion In the present paper we have studied Sendai virus replication in Friend erythroleukemia cells grown in suspension, and we have investigated the conditions that maintain persistent infection in clones that had survived the first infection. Roux and Holland (1979) and Koiakofsky (1976) have extensively studied the mechanism of generation and the biological properties of DI-containing preparations of Sendai virus. The amount of DI particles generated at each cycle of viral replication appears to depend on the DI/standard virions ratio in the inoculum. Since the production of the former seems to be favored during replication, we cannot exclude the possibility

130 that the preparations of standard virus used in these studies might contain also some DI contaminants. However, the high specific infectivity of the standard Sendai virus (about 40 times that of DI-containing stocks) tends to exclude this explanation. Several remarkable features of Sendai virus infection of FLC emerge from these studies: When FLC are infected with high doses of Sendai virus, a rapid cytolysis occurs. This is not unexpected, since it is well known that Sendai virus (Okada et al., 1966; Toyama et al., 1977) and other paramyxoviruses (Polos and Gallaher, 1981) induce cell lysis and fusion mediated by the F glycoprotein. Ca” ions appear to play a critical role in this respect: in the presence of Ca’+. the activity of the F protein results in fusion rather than lysis of the infected cells. The Sendai-induced lysis of FLC, on the contrary, seems to be independent to a considerable extent of the presence of Ca’ + ions, and dependent just on the m.o.i. Moreover, Sendai virus appears to be unable to induce celllcell fusion in FLC. While the mechanism responsible for SIL of FLC is still poorly understood, it seems reasonable to hypothesize that the FLC plasma membrane becomes particularly fragile when fused to a critical amount of viral particles. This would be hardly surprising, as other cellular systems have been described. where Sendai virus induces modifications of the properties of the cell surface such as alterations of the ion barrier (Okada et al., 1975) and increased plasma membrane fluidity (Maeda et al., 1977). Doses of Sendai virus of 4 HAU/lOh cells or lower are unable to lyse FLC directly: in these conditions, cells become infected, and produce virus. The survival of infected cells depends largely on the choice of DI or standard EGSV for the infection and, for each viral stock, on the m.o.i. used. At lower m.o.i. the infected cells maintained their growth potential. At the same HAU/cell ratio the infection with standard EGSV has a more cytotoxic effect than DI virus. In the experimental conditions described the progressive inhibition of the host cell protein synthesis may be accounted for the increased rate of lethality of the infected cells. The infected cells and the cell population that survived the first infection have lost their susceptibility to SIL. Such resistance was not mediated by the neuraminidase activity of the cell-grown virus released in the medium: in fact, the addition of 3% fetuin (a concentration of competitor substrate sufficient to prevent the effects of added enzyme on the sialic acid residues of the cell membrane) failed to restore their susceptibility to SIL. The state of resistance to SIL, instead, was reduced only by blocking the viral replication with IFN, suggesting that the acquisition of the resistance to SIL requires some later step of the viral replication. In this respect, a possibility to be considered is that this phenomenon involves the insertion of the viral proteins in the cell plasma membrane, such as to modify its physiology through a modification of its microviscosity (Kohn, 1979). The critical importance of the cell surface in Sendai virus replication is also underlined by the inhibitory effect exerted by plasma membrane active compounds as the prostaglandins of the A series on the production of Sendai virus in AGMK cells (Santoro et al., 1981) and on the ability of these hormones to prevent the establishment of a carrier state (Santoro et al., 1980). The last prominent feature emerging from our study concerns the persistence of

131 the infection in populations of Friend cells that survived the first infection with Sendai virus. The cloning experiments indicate that the long-term maintenance of the infectious state and of the SIL resistance of Friend cells requires the continuous re-infection of the culture with EGSV: in the absence of fresh activated virus added, the growth of the infected cells leads to a progressive loss of the infected state.

Acknowledgements

The authors are indebted to Prof. Milton W. Taylor (Bloomington. IN, U.S.A.) and Prof. R. Perez-Bercoff (Rome, Italy) for most useful comments and criticisms. This work was supported in part by Grant x3.00627.52 (P.F. Controllo Malattie da Infezione) from the Italian National Research Council (CNR).

References Benedetto. A., Zaniratti. S.. Meo. P. and Rossi. G.B. (1979) Modulation by cell trypsinization of Sendai virus expression in AGMK cells: first infection and establishment of a carrier state. J. Gen. Virol. 45. 407.-421. Benedetto, A., Rossi. G.B., Amici. C.. Belardelli, F.. Cioe’, L.. Carruba. G. and Carrasco, L. (1980) Inhibition of animal virus production by means of translational inhibitors unable to penetrate normal cells. Virology 106, 123-132. Benedetto. A.. Amici, C., Dyaczenko, W.. Zaniratti. S. and Elia, G. (1982) Acquisition of sensitivity to exogenous fibronectin by Friend leukemia cells correlates with reduction of their tumorigenic potential. Int. J. Cancer 30. 663-667. Fan. D.P. and Sefton. B.M. (1978) The entry into the host cells of Sindbis virus, vesicular stomatitis virus. and Sendai virus. Cell 15. 985-992. Homma, M. and Ohuchi. M. (1973) Trypsin action on the growth of Sendai virus in tissue culture cells. 111: Structural differences of Sendai viruses grown in eggs and in tissue culture. J. Virol. 12. 1457-1465. Ishlda. N. and Homma. M. (1978) Sendai virus. Adv. Virus Res. 23. 349-383. Kimura. J., Orvell. C. and Norrby. E. (1979) Characterization of the polypeptides synthesized in cells infected with a temperature sensitive mutant derived from HVJ (Sendai virus) carrier culture. Arch. Virol. 61, 23-33. Kimura. Y.. Aoki, H.. Shimokata. K.. Ito, Y.. Takano. M. and Maeno. K. (1980) Characterization of altered BHK cells resistant to HVJ (Sendai virus) infection. J. Gen. Virol. 47. 193-197. Kahn. A. (1979) Early interactions of viruses with cellular membranes. Adv. Virus Res. 24. 223-276. Kolakofsky, D. (1976) Isolation and characterization of Sendai virus DI RNAs. Cell 8, 547-555. Kolakofaky, D. (1979) Studies on the generation and amplification of Sendai virus defectwe-interfering genomes. Virology 93. 581-593. Maeda, Y.. Kim. J.. Koseki. I.. Mekada. E., Shiokada. Y. and Okada. Y. (1977) Modification of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus). Exp. Cell Res. 108, 95-,106. Okada. Y. and Murayama, F. (1966) Requirement of calcium ions for the cell fusion reaction of animal cell by HVJ. Exp. Cell Res. 44. 527-551. Okada, Y.. Koseki, I., Kim, J.. Maeda. Y.. Hashimoto, T., Kanno, Y. and Matsui. Y. (1975) Modifications of cell membranes with viral envelopes during fusion of cells with HVJ (Sendai virus). I. Interactions between cell membranes and virus in the early stage. Exp. Cell Res. 93. 368-378. Poloa, P.G. and Gallaher. W.R. (1981) A quantitative assay for cytolysis Induced by Newcastle disease virus. J. Gen. Viral. 52. 259-265.

132 Richardson. C.D., Scheid. A. and Choppin. P.W. (1980) Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F, or HA, viral polypeptides. Virology 105. 205-222. Roux. L. and Holland. J.J. (1979) Role of defective interfering particles of Sendai virus in persistent infection. Virology 93, 91-103. Roux, L. and Holland. J.J. (1980) Viral genome synthesis m BHK-21 cells persistently infected with Sendai virus. Virology 100. 53-64. Roux, L. and Waldvogel. F. (1982) Instability of the viral M protein in BHK-21 cells persistently infected with Sendai virus. Cell 28, 2933302. Roux, L. and Waldvogel, F. (1983) Defective interfering particles of Sendai virus modulate HN expression at the surface of infected BHK cells. Virology 130. 91-104. Santoro. M.G.. Benedetto, A.. Carruba, G.. Garaci. E. and Jaffe, B.M. (1980) Prostaglandin A compounds as antiviral agents. Science 208. 1032-1034. Santoro. M.G., Carruba. G.. Garaci. E.. Jaffe, B.M. and Benedetto, A. (1981) Prostaglandins of the series A inhibit Sendai virus replication in cultured cells. J. Gen. Viral. 53, 75-83. Scheid. E. and Choppin, P.W. (1974) Identification of the 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. Scheid, E.. Graves, M.C., Silver, S.M. and Choppin, P.W. (1978) Studies on the structure and functions of paramyxovirus glycoproteins. In: Negative Strand Viruses and the Host Cell (Mahy, B.W.J. and Barry, R.D.. eds.), pp. 181-193. Academic Press, London. Sugamura. K., Tozawa, H.. Homma. M. and Ishida. N. (1974) Factors influencing the establishment of persistent infection of HVJ (Sendai virus) in L cells. Jpn. J. Microbial. 18. 3499355. Toyama. S.. Toyama, S. and Uetake. H. (1977) Altered cell fusion capaci.y in lines of KB cells resistant to Sendai virus-induced cytolysis. Virology 76. 503-515. Walker, D.L. (1964) The viral carrier state in animal cell cultures. Progr. Med. Viral. 6. 111-148. Warren, L. (1959) The thiobarbiturtc acid assay of sialic acids. J. Biol. Chem. 234. 1971-1975. Yoshida, T., Nogai, Y., Maeno, K.. Jimma. M.. Hamaguchi. M., Matsumoto. T.. Nagayoshi. S. and Hoshino, M. (1979) Studies on the role of M protein in virus assembly using a Ts mutant of HVJ (Sendai virus). Virology 92. 1399154. (Manuscript

received 26 July 1985)