Restriction of herpes simplex virus replication by poliovirus: A selective inhibition of viral translation

VIBOLOGY

48, 207-220

(1972)

erpes elective ROMAINE Department

Simplex

Virus

Inhibition

of Viral

E. SAXTON

AND

of Medical I~icrobiology and Immunology, of Medicine, University of California

School

Accepted

Replication

December

Tr~~s~~t~

JACK and Los

G. STEVESS

the Eeed Xeurological Angeles, Caiifornia

Research

Center,

90024

86, 1971

Replication of herpes simplex virus (HSV) in HeLa cells is completely restricted if the cells are superinfected with poliovirus within 5 hr after HSV infection. Poliovirus replicates normally in these dually infected cells nnless HSV is given substantial t,ime and mult,ip!icity advantages. Translation, but not replication, of t,he poliovirus genome appears t,o be required for the restriction of HSV. DNA and RNA synthesis in HSV-infected cells is not significantly altered after poliovirus superinfection, but NW directed protein synthesis is completely inhibited, and poiyribosomes sarrying HSY mRNA are disaggregated within 3 hr after superinfection. These results suggest that the restriction of HSV replication by poliovirus is due to a selective inhibition of HSV mRNh translation. INTRODUCTION

Poliovirus infection of HeLa cells leads to an inhibition of host, cell DNA,l RNA, and protein synthesis (Salzman et al., 1959; Holland and Peterson, 1964). The suppression of HeLa cell protein synthesis is accompanied by a disaggregat,ion of cgtoplasmic polyribosomes, an effect which appears to result from selective inhibition of cellular mRN,4 translation (Willems and Penman, 1966). Infection of mammalian cells with herpes simplex virus leads to a similar inhibition of host cell macromolecular syntheses (Roizman and Roam, 1964; Roizman et al., 1 Abbreviations used in this paper: DNA, deoxyribonucleic acid; HSV, herpes simplex virus; RNA, ribonucleic acid; mRNA, messenger RNA; rRNA, ribosomal RNA; MEM, Eagle’s minimal essential medium; FCS, fetal calf serum; m.o.i., multiplicity of infection; PFU, plaque-forming units; BSS, Hanks’ balanced salts solution; UV, ultraviolet; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane; SDS, sodium dodecyl sulfate; TKM, hypertonic buffer (lo-” ;M M&l2 , 0.25 M KCI, 10p2 M Tris, pH 7.4); DOG, sodium desoxycholate; SSC, standard saline citrate (0.15 fi1 NaCl, 0.015 X sodium citrate, pH 7.0); RSase, ribonuclease; DNase, deoxyribonuclease; epm, counts per minute.

1965 ; Sydiskis and Roieman, 1967). Bn eith pz” system, UV irradiation of the virus prior to infection eliminates the effect. These results suggest that, after infection with either of the viruses, virus specified inhibitors are produced which selectively interfere with the syntheses of cellular macromolecules. Because of the apparently common and drastic effects on host cell macromolecular syntheses following single infection with these otherwise completely dissimilar vi.ruses, we decided t,o investigate their interaction after dual infection. Specifically, we wondered whether these viral inhibitory functions restrict only cellular syn~lieses or whether they also inhibit rnacr~rn~~~~~~~~K synthesis directed by other viruses. In addition, since NW DNA is transcribed in the nucleus, and the viral mRNA is processed, transported to the cytoplasm, and t-csnslated in a manner similar to cell mRNA (Roizman et al., 1970)) dual infection with these viruses might represent a comparatively simp1.e model for studies of euearpotie regukation. 1flATF:RIALS

AXD

METH0193

Cells. HeLa cells x-ere obtained from F!OK Laboratories, Inc., Ingle\%-ood, Geliiornia.

208

SAXTON

AND

The cells were grown at 37” in a 5% COZ humidified atmosphere as monolayer cul‘curesin MEM (Eagle, 1959) containing 10 % newborn calf serum. Viruses. Poliovirus type I was kindly supplied by Dr. John Holland. Stocks were prepared by adsorbing virus at an m.o.i. of 0.1 to nearly confluent cells which were maintained in MEM plus 5 % FCS at 34” for 24 hr. After sedimentation of cells by low speed centrifugation the supernatant fluid was titrated for virus by the method of Holland and McLaren (1959) and usually contained 3 to 5 X log PFU/ml of poliovirus. All virus stocks were stored frozen at -70” until used. The Macro-Plaque strain of HSV was generously provided by Dr. Bernard Roizman. After adsorption of HSV to cells at an m.o.i. of 0.1 the infected cells were maintained in MEM + 5 % FCS at 34” for approximately 35 hr. Cells pelleted by low speed centrifugation were resuspended in l/50 volume of the supernatant fluid, disrupted by 1 min of sonic oscillation (Branson S-75 Sonifier, micro-tip, tap 6) and reconstituted to one-fourth the original volume of MEM. After recentrifugation the cell-free viral suspensionsusually contained 1 to 5 X 10sPFU/ml when assayed in liquid medium containing 0.3 % pooled human y-globulin. This is a standard assay procedure for viruses which passdirectly from cell to cell (Hoggan and Roizman, 1959). Dual Infection. HeLa cell monolayers in 2-0~ French square bottles (2 X lo6 cells/ bottle) were infected for 1 hr at 37” with HSV (adsorbed m.o.i. = 10 PFU/cell) or superinfected with poliovirus (adsorbed m.o.i. = 200 PFU/cell). After viral adsorption, the cells were washed 3 times in BSS and incubated in 2 ml of MEM plus 2% FCS; at various times during the viral growth cycle duplicate monolayers were removed and frozen at -70”. Viruses were released from the dually infected cells by 3 cycles of freezing and thawing. For selective titration of each virus, aliquots of the cellfree supernatant fluids were neutralized for 1 hr at 37” with one-tenth volume of the heterologous antisera. Poliovirus type I rabbit antisera (polio typing antisera, Microbiological Associates, Bethesda, Maryland) or

STEVENS

HSV antisera (prepared in rabbits as described by Stevens and Cook, 1971) were pretested and found to reduce virus titers about lo4 under these conditions. The HSV antiserum neutralized the homologous virus below the level of detection in ,a11dilutions used to assay poliovirus from the dually infected cells. In selective titrations for HSV, poliovirus was first neutralized with the rabbit antisera, and development of poliovirus plaques resulting from the nonneutralized fraction was prevented by the human yglobulin present in the overlay medium. Cell samples singly infected with either virus were incubated with the heterologous sera for 1 hr at 37” prior to titration to correct for any nonspecific inactivation. Ultraviolet irradiation of virions. Five milliliters of stock virus preparations harvested from cells grown in MEM without phenol red were spread over lo-cm sterile petri dishes and exposed to 500 pW/cm2 of UV irradiation from a germicidal lamp at 260 nm for a time predetermined to result in 0.1 % virus survival. DNA, RNA, and protein synthesis. At intervals after infection, cell cultures were washed 3 times with BSS and incubated for 1 hr in MEM plus 2 % dialyzed FCS and 1 #Zi/ml of 3H-leucine (56 Ci/mmole), 3Harginine (20 Ci/mmole), or 0.2 pCi/ml of 14C-lysine (300 mCi/mmole) with 5 % of the normal amount of the corresponding unlabeled amino acid. After washing with chilled BSS, cells were disrupted by 3 cycles of freezing and thawing in distilled water, precipitated overnight with cold 5 % TCA and washed with an excessof TCA on duplicate membrane filters (Millipore, 25 mm, 0.45 pm pore size). After drying, the filters were immersed in a toluene-based Auor and counted in a liquid scintillation spectrometer. RNA and DNA were labeled by the procedure used for proteins except for the direct addition of 5 pCi/ml 3H-uridine (20 Ci/ mmole), 3H-thymidine (15 Ci/mmole), or 1 pCi/ml of 14C-thymidine (45 mCi/mmole) to cell cultures for 1 hr without prewashing. All isotope-labeled nucleotides and amino acids were purchased from Schwarz BioResearch, Inc., Van Nuys, California. Polyacrylamide gel electrophoresis. Cells

RESTRICTION

OF HSV BY POLIOVIRUS

containing labeled polypeptides were scraped off the glass, sedimented by low speed centrifugation, and solubilized for I hr at 37” in I ml of 1O-3 M Tris (pH 8.0) containing 2.5 % SDS, 0.7 M urea, and 1.5 M 2-mercaptoethanol. Dialysis, concentration, electrophoresis, and fractionation of the labeled peptides on 18 cm, 7.5 % polyacrylamideSDS gels were performed as previously described (Stevens et al., 1969). Isolation of polyribosome8. Unlabeled polyribosomes were isolated from duplicate infected HeLa cell monolayers in 2-0x bot’tles by extraction for 15 min at 4” with 0.5 ml of hypert80nic TKM buffer containing 0.5 % DQC and 1% Tween 40. After sedimentation of nuclei and cellular debris by centrifugation at 10,000 g for IO min, half of each pooled duplicate supernatant (0.5 ml) was layered over a 5-ml linear sucrose gradient (4.5 ml of 0.5 &-I.5 n/l- sucrose in hypertonic TKM buffer on a 0.5 ml 2 M sucrose cushion) and sediment,ed by centrifugation at 250,000 g for 2 hr at 4” in a SW 50.1 rotor. Polyribosome sedimentation profiles were automatically recorded for each sample at an absorbancy of 254 nm with an Isco sucrose gradient fraction collector. Polyribosomes labeled with 3H-uridine were isolated from 2 X lo8 normal or infected cells (6 hr after HSV infection) which had been incubated for 2 hr in ME&I containing 20 &i/ml 3H-uridine. In this ease, the polyribosomes were extracted with 3 ml of TKM buffer and detergents, centrifuged at low speed as described, layered over 35ml concave exponential sucrose gradients (30 ml of 0.5-1.5 M sucrose in hypertonic TKM buffer on a S-ml 2 M sucrose cushion) and sedimented by centrifugation for S hr at 4” in a SW 27 rotor at 100,000 g. The fractions containing polyribosomes were collected and pelleted by recentrifugation through hypertonic TKM buffer for 16 hr at 100,000 g. Separation of viral and cellular DNA. Isopycnic CsCl density equilibrium sedimentation was used to separate HSV and HeLa cell D1\TA. Cells labeled with 3H-thymidine were dissolved in I ml of I X SSC containing 3 % SDS, dialyzed overnight in 100 volumes of 0.1 X SSC plus 0.2% SDS, incubated with pronase, and mixed with 14C-HeLa marker DNA in a 4.5 ml of CsCl solution

209

(1.700 g/cm”) as descr previously (Stevens and Cook, 1971). r eentrifuga-tion to equilibrium at 25” for 44 hr in a SW’ 50.1 rotor at 115,000 g, j-drop fractions were collected on 2.3 cm Whatman No. 3MM cellulose filter pads. The pads were dried and assayed for both isotopes in a liquid s&nil-lation spectrometer adjusted for dual labei counting. RNA-DNA hybridizatzon. Unlabeled DP;A from normal or KSV-infected cells was extracted from cells as described and separated by CsCl isopycnic density sedimentation, Infected cell DIVA was collected in 5 fract~iions, and the tubes contaking DNA (1.720-1.730 g/cm3) were pooled. DNA from normal cells was isolated by the same procedure, and fractions between the densities of I.690 and 1.720 were pooled. After alkaline hydrolysis of residual Rr\iA and extensive dialysis against 0.1 X SSC, the DfGTA samples were fragmented by son-. ication, denatured by heating to HOO” for 10 min, diluted into cold 4 X SSC, and bound to membrane filters (cf. Baluda and Kayak, 1970). ?II-Uridine-labeled RNA from peileted polyribosomes of normal and HSVinfected cells were extracted 3 times with cold phenol, incubated with DNase, reextracted with phenol! fragmented by sonicaCon, and annealed with DNA immobilized on filters by the method of Gillespie and Spiegelman (1965). After hybridization, the fibers were washed extensively, reheated at 70” in 4 X SSC, and treated with ElNase as described by Baluda and Nayak (4970) prior to counting for radioactivity and DNA assay by standard methods. RESULTS Replication of NSV and Poliovirus Infected HeLa Cells.

in DuaiIg

To determine whether interference occurred between HSV and poliovirus the replication of b0t.h viruses was followed in dually infected HeLa cells. Nearly confiuent HeLa cell monolayer cultures were infected with HSV (m.o.i. = 10) and 1, 5, 20, or 15 hr later the cells were superinfected with poliovirus (m.o.i. = ZOO). These viral multiplicities ensured dual infection of greater than 99 % of the cells.

210

SAXTON

AND

HSV growth cycles (Fig. 1A) were obtained by assaying the infected cells for HSV after selective neutralization of poliovirus. During HSV single infection, a one-step growth cycle wit,h lo-hr latent and rise periods leading to nearly a lOOO-fold increase in virus within 30 hr is observed. Figure 1A also shows the growth cycles of HSV in HeLa cells superinfected with poliovirus at 1 or 5 hr. The replication of HSV is completely inhibited after poliovirus superinfection at these times. However, in the cells superinfected with poliovirus at 10 l-u, the HSV growth cycle is only partially rest,ricted. The HSV replication cycle after poliovirus superinfection at 15 hr is nearly identical to that following single infection, indicating that by this time replication of HSV has progressed to a point insusceptible to interference. By 8 hr after HSV infection, the entire HeLa monolayer was observed to undergo rapid polykaryocyte formation. Despite the 107

I

I

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I

STEVENS

extensive alterations in the cytoplasmic membrane which accompany cell fusion there was no measureable loss of poliovirus receptor sites since poliovirus adsorbed as efficiently to HSV-induced HeLa cell polykaryocytes as to normal cells (our unpublished results). Thus, the inability of poliovirus to completely inhibit HSV replication in the later superinfections was not due to an adsorption deficiency. Figure 1B shows the growth of poliovirus assayed from the same dually infected cells. HSV infected cells superinfected with poliovirus at 1 or 5 hr support normal poliovirus replication. Later poliovirus superinfection resulted in the marked reduction in poliovirus replication shown in the lo-hr and 15hr superinfection growth cycles. The results presented in Fig. 1 indicate that poliovirus and HSV can induce reciprocal, although unequal, interference in dually infected cells. It appears that until

I

106

p

IO5

8 *g 2 a

104

103

102

HOURS

POST

INFECTION

FIG. 1. HSV and poliovirus growth cycles in dually infected HeLa cells. Cells were infected with HSV at zero time (H) and superinfected with poliovirus at the indicated times (PI , Ps , PIO , PIS) after HSV infection. (A) HSV was assayed at 5-hr intervals in cells singly infected with HSV (O-0) and in cells superinfected with poliovirus at 1 hr (a-q ), 5 hr (n---n), 10 hr (A--A), or 15 hr (O--O) after HSV infection. (B) The poliovirus growth cycle in singly infected cells (a- @) and in cells superinfected with poliovirus at I hr (O--O); 5 hr (A--A), 10 hr (m--E), and 15 hr (n--O) after HSV infection are shown. Further details concerning met,hods are given in the text.

RESTRICTION

OF

late in the HSV growth cycle poliovirus can completely restrict HSV replication. These temporal patterns of interference were observed consistently if the viral multiplicities were carefully controlled. However, if cells were dually infected under condit’ions designed to give HSV the multiplicity advantage, HSV was found to interfere partially with poliovirus replication even after poliovirus superinfect’ion at 5 hr. Figure 2 illust’rates the result’s of this reversed multiplicity experiment,. Poliovirus replication after superinfection at 5 hr is restkcted to 1% of the growth seen in control cells singly infected with poliovirus. This int,erference wit,h poliovirus growth is nearly abolished in cells preinfected with SV after UV irradiation of PEW to 0.1% survival. Therefore, it appears that the expression of an intact HSV genome 108r

I 10 81

4

6

8

10

12

14

BSV

BY

POLIOVIRUS

2B’i

is necessary for inhibition of poliovirus rep lication. Since HSV induces only a partial inhibition of poliovirus growth in dually infected cells even when given this substantial time and multiplicity advantage, we did not investigate the phenomenon in detail. Requirements gcGTPoZio~vh~~ Interference WSB Replication

wiir’

To determine whether replication of the poliovirus RNA was necessary for the inhibition of HEW, cells were infected with (m.o.i. = IO) and superinfected with poliovirus (m.0.i. = 200) under conditions identical to those in Fig. 1 except for add&km of 1.5 mM guanidine t,o the medium. Guanidine at this concentration has been shown to restrict poliovirus growth by blocking replieation but not translation of the viral RNA (Penman and Summers, 1965). In our system 1.5 mM guanidine also completely inhibits the replication of poliovirus in cells (our unpublished observations). BSTi growth cycles presented in Fig. 3A show &at’ HSV replicates normally after addition of guanidine but that pohovirus completely blocks HSV replication after xuperinfe&ion at 1r or 5 h.r in the presence of guamdine, Thus, replication of the poliovirus RKA does not appear to be a prerequisite for thy: inhibition of HSV repiication. If expression of a functional poliovirus genomeis required for interference, UV irradiation of the superinfecting virus shdd. abolish the effect (cf. Penman and Smnmers, 1965). To test this possibility, the experiment was repeated using pohovirus aftw LTV irradiation to 0.1% survival. As shown in Fig. 3B, interference with completely eliminated in cells superinfe~t~ed. with UV Irradiated poliovirus. Therefore, i:, appears t’hat, translation of poliovirus RNA. is necessary for inhibition.

hours postinfection Frc. 2. Poliovirus replication cycles in HeLa cells infected with HSV (N) at a high multiplicity (m.o.i. = 130) and superinfected 5 hr later with poliovirus (Psi at a low multiplicity (m.0.i. = 5). Poliovirus replication after single infection of cells (O---O); poliovirus replication in dually infected cells (Op.-. 0); poliovirus replication in dually infect,ed cells after UV irradiation of HEX

infected Cells The inhibition of HSQ replication by poliovirus could the result of a direct interference with ‘si macromolecular syntheses, similar to the inhibition of HeLa cell syntheses a,fter single infection with po!iovirus (Holland and Peterson, 1964). Therefore, the synthesis of DNA, RKA, and pro-

SAXTON

5

10

15

20

25

AND STEVENS

30

L

0

HOURS POST INFECTION FIG. 3. HSV replication in HeLa cells superinfected with nonreplicating poliovirus. (A) HSV growth cycles in the presence of guanidine in HSV singly infected cells (a---@) and after poliovirus superinfection at 1 hr (m--E), 5 hr (A--A), 10 hr (U----O), or 15 hr (A--A) were determined. (B) HSV replication after addition of guanidine in singly infected cells (O-.-a 0), and after superinfection at 5 hr with either viable (O---O) or UV-irradiated poliovirus (@- - 0).

tein was examined in dually infected cells. In all experiments, guanidine was added to block poliovirus replication, and viral multiplicities similar to those presented in Fig. 1 were used.

and 20 PFU/cell. As shown in Fig. 4A poliovirus superinfections at 5, LO, or 15 hr lead to a rapid inhibition of this protein synthesis. Thus, poliovirus can efficiently interfere with protein synthesis in HSV-infected cells even when poliovirus RNA replication is blocked Protein Synthesis with guanidine. The rate of protein synthesis was measIn the same experiment, equal numbers of ured as TCA-insoluble radioactivity after HSV infected and 5 hr poliovirus superinincubation of cell cultures with 1 &X/ml of fected cells (10’ cells/culture) were incubated 3H-leucine for 1 hr at various times after for 2 hr with 1 MCi/ml 3H-leucine at a time virus infection. After HSV infection, the rate when protein synthesis appeared to be preof incorporation of 3H-leucine into acid- dominantly HSV specified (8-10 hr, as indiprecipitable material decreasesinitially and cated by the dotted line in Fig. 4A). Proteins then increasesto a maximum between 4 and were extracted from these cells and separated 6 hr (Fig. 4A). These results suggest that by SDS-polyacrylamide gel electrophoresis. HSV rapidly inhibits synthesis of cellular Figure 4B shows the peptide profiles obpolypeptides and that by 6 hr after infection tained from these infected cells. The labeled viral protein synthesis predominates in these proteins from cells singly infected with HSV cells. However, the time of maximum HSV appear to be typical HSV-induced peptides, protein synthesis varies significantly de- since the electrophoretic profile is similar to pending on the viral multiplicity between 5 that obtained by Olshevsky and Becker

RESTRICTION

OF

HSV

BY

21.3

POLIOVIRUS

tide after gel e~e6tro~hores~sof proteins ted from uninfected cells. Many peptides can be seen. Unlike the peptide heterogeneity seen in normal cell fected cell electropherogram (Fig. tains about 12 we!1 separated pepti correspond almost exactly in size and position with previously reported values for the olypeptides (OlshavFrom this peptide profile it appears few, if any, normal tides are still being synthesized

FIG. 4. Protein synthesis in HSV-infected and poliovirus-superinfected HeLa cells. (A) Incorpora,tion of 3H-leucine during 1 hr intervals into TCA-precipitable material in HSV singly infected cells (@--a) and cells superinfected with poliovirus (A-A) at 5, 10, or 15 hr after HSV infection. H, Pj , PI0 , and PUS indicate the time at which cells were infected with HSV or superinfected with poliovirus. (B) Polyacrylamide gel electropherograms of 3H-leucine labeled polypeptides extracted from HSV singly infected (upper profile) and poliovirus superinfected (lower profile) HeLa ~41s:

cytosine content of herpesvirus DNA (GC = 70%) codes for proteins relatively rich. ifi arginine and d.eficient in lysine. As sho-vvnrby Kaplan et a!. (1970), the change in the amino acid composition of peptides synthesized after infection can be used as a very ser,sitive method ol identifying herpesvirus induced Deotides, Peptides from normal HeLa ceils &~d~cells8 hr after HSV infection were labeled with 3H.. arginine and 14C-lysine, The electropherogram in Fig. 6A showsthat norma! eia cdl peptides contain uniform ~ro~o~t~ons of the labeled amjno acids in nearly aP1proteins with an overall 3H: l*C ratio of I : 2. In con:zpoo1

--

(1970). After poliovirus superinfection, incorporation of 3H-leucine into these polypeptides is greatly reduced (lower electrophoretie profile of Fig. 4B) indicating that HSV peptide synthesis is uniformly inhibited by poliovirus. Identi$cation

oj NSV

Proteins

in Infected

Cells

To show more rigorously that these peptides were HSV induced, patterns of migration in polyacrylamide gels and the amino acid composition of proteins from uninfected and NSV infected cells were compared. Monolayers of normal HeLa cells and cells infected with BSV for 8 hr (10’ cells/culture) were incubated with 5 &IX/ml 3H-leucine for an additional 2 hr. Figure 5A showsthe pep-

3-

20

40

60

80

‘3

GEL FRCCT OUS

FIG. 5. Electropherograms of 3H-leucine polypeptides from normal HeLa cells similar cells 8-10 hr after WSV infection

labeled (A) and (B).

214

SAXTON AND STEVENS 1600

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800 600 Im XZ u”

200

400 200

“0 T; 0 ? E f3 aJ 1000 .E .c F Q 800

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= 1000

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800

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,5 600

600 -++ 400

200

IOC1

GEL

FRACTIONS

FIG. 6. Electropherogramsof 3H-arginineand W?-lysinelabeled polypeptides from normal HeLa cells (A) and cellslabeled8-10hr after HSV infection (B).

trast, the electropherogram presented in Fig. 6B shows that the relatively few peptides synthesized in the HSV infected cells contain a 3H-arginine: 14C-lysineratio of about 3: 1. Thus, compared to uninfected cells, there is .a 6-fold increase in the arginine:lysine ratio in all peptides synthesized by 8 hr after HSV infection. These results strongly support the conclusion that at this time, protein synthesis is almost completely virus specified in HSV-infected cells. However, to determine whether this change in peptide labeling truly reflects a replacement of cellular peptides with arginine-rich and lysine-depleted HSVinduced peptides, rather than representing merely a virus-induced change in the specific activity of the intracellular amino acid pools, it was necessary to estimate the uptake and turnover rates for these essential amino acids in normal and infected cells (cf. Kaplan et al., 1970). To estimate the turnover rate of these amino acids, the release of acid soluble 3H-

arginine and 14C-lysinefrom prelabeled proteins of normal and infected cells was measured. As shown in Table 1, normal and infected cells have a similar turnover rate for these amino acids. About 3 % of the arginine and 1.5 % of the lysine is released per hour from the prelabeled proteins in both normal and infected cells. Figure 7 shows that, the uptake of 3H-arginine and 14C-lysine into proteins of normal cells (Fig. 7A) and HSVinfected cells (Fig. 7B) becomes linear soon after addition of label to the medium. In addition, a comparison of the ordinates in Fig. 7A and 7B reveals the expected 6-fold change in incorporation rate of arginine relative to lysine into proteins of HSV-infected cells. If these cells are washed twice at 20 min and reincubated in unlabeled MEM, a rapid decrease occurs in the incorporation of both labeled amino acids into proteins. These results show that the arginine and lysine pools equilibrate rapidly with the medium in both normal and infected cells. Thus, infec-

RESTRICTION TABLE PROTZIN

'TURNOVER

TCA-soluble Time after labeling” (hr)

UNINFECTED

HELA

P1,200/9,780 22,800/19,970 25,lQO/22,300 30,900/28,000

cells

AND

CELLS

radioactivity/2 (cpm 3H-arginine/ cpm 14C-lysine)

Uninfected 2 4 6 8

1 IN

HSV-IKFECTED

OF

Infected

X 106 cells

cells

10,100/8,0!0 19,800/17,850 22,000/23,400 26,300/26,600

a Cells were labeled for 1 hr with 2.5 &i/ml “H-arginine and 0.5 $Zi/ml ‘Glysine in MEM with ZyO dialyzed FCS and 5% of the normal amount of these unlabeled amino acids. After washing, the cells were infected with HSV for 1 hr (m.o.i. = 10) or mock-infected, rewashed, and incubated in complete MEM plus 2% dialyzed FCS. -4t 2.hr intervals cell samples were precipitated with cold TCA, and total TCA-soluble and precipitable radioactivity was determined. After 1 hr of labeling, 2 X 106 cells contained about 90,000 cpm 3H-arginine and 200,000 cpm W-lysine in proteins as total acid-insoIuble radioactivity.

EISV

2 3 .bj

BY

tion does not s~gni~~a~tl~ alter the ce813iar uptake or protein turnover of these esential amino acids. Therefore, a sea! shift from synthesis of cellular pept’ides to arginine rich 8-v is viral peptidex after in:ection with indicated,

RNA Synthesis in

ually Injected Ceils

The interference with HSV proteisl syn-curs after polisvirus super-be due to an inhibition of transcription of the HSV genome. To examine this possibility the rate of RNA synthesis was measured in MSW-infected cells and eel1 cultures superinfected wit,h poiiovirus at 5 hr. As shoa-n in Fig. 8A, incorporaCon of 3 -uridine in-to TCA-precipitable material increased initially in HSV-infec;F:d cells, reaching a peak at 3-4 hr! and then gradually declined. These resultx ma27 in& cate that prior to infection the monolayers were supporting only a minimal rate of RXA synthesis. Superinfection wihh poliovirus did not suppress RNA synthesis belou- th4: lever

minutes

7.

Uptake of %I-arginine and

‘Glysine into proteins of uninfected HeLa cells (A) and “Gls 8 hr after HSV infection (B). Incorporation of radioactivity int,o TCA-precipitable material during incubation of cells in MEM with 5 &i/ml 3H-arginine and 1 wCi/ml l&C-lysine plus 5% of ;he norma’k amount of the corresponding unlabeled amino acids (A - -A). Since the arginine and lysine -uptake were found to be proportional at each IO-min interval, the results were plotted as single points representing both amino acids. At 20 min, selected cell samples were washed rapidly with complete medium free of label and reincubated in this unlabeled MEM until assayed for t.otal TCA precipitable radioaetivit,y (O--O). Fro.

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800

0

4

8

HOURS

10

POST

0

3

6

3

12

INFECTION

F c. 8. Comparison of RNA and protein synthesis during 1-hr intervals in HSV- and poliovirus-infected HeLa cells. (A) Incorporation of 3H-uridine into TCA-precipitable material of HSV singly infected cells (e---a), poliovirus singly infected cells (A---A), or cells superinfected with poliovirus 5 hr after HSV infection (A--A). (B) I ncorporation of 3H-arginine into TCA precipitable material of HSV singly infected cells (0 - - 0) and cells superinfected with poliovirus 5 hr after HSV infection

(A--A). seen in the HSV singly infected cells. This is in contrast to the 50 % reduction seen in 3Huridine incorporation in the normal HeLa cells singly infected with poliovirus at the same time. Therefore, RNA synthesis in HSV-infected cells appears to be insusceptible to any measurable poliovirus induced inhibition. It is possible that the inability of poliovirus to restrict RNA synthesis in superinfected cells is due to a previous HSV induced inhibition of rRNA synthesis (Wagner and Roizman, 1969) which poliovirus rapidly inhibits during single infections (Darnell, 1968). Protein synthesis measured during the same experiment using identical viral multiplicities in parallel cell cultures was subject to the expected rapid inhibition after superinfection (Fig. SB) . DNA Synthesis in Dually Infected Cells

HeLa cells infected with poliovirus exhibit a slow decline in DNA synthesis (Holland and Peterson, 1964). Herpesvirus DNA is replicated in the nucleus in a semiconservative manner (Kaplan and Ben-Porat, 1963) and may be susceptible to inhibition by poliovirus.

To determine the effect of poIiovirus superinfect.ion on HSV DNA synthesis, cells were infected with HSV and after 5, 10, and 16 hr were labeled with 3H-thymidine for 1 hr. Infected cells were also superinfected with poliovirus at 10 hr when HSV DNA synthesis was maximal, and after 6 additional hours were also labeled for 1 hr with 3H-thymidine. DNA was extracted from an equal number of cells of each infected culture and centrifuged to equilibrium in CsCl gradients. As shown in Figs. 9A and 9B, incorporation of 3H-thymidine into HeLa cell DNA (p = 1.705 g/cm3) decreases by 10 hr after HSV infection, as incorporation into viral DNA (p = 1.725 g/cm3) is increased. These results, in agreement with those of Roizman and Roane (1964), indicate that HSV inhibits cellular DNA synthesis. A comparison of the gradients containing the 16-hr HSVinfected and poliovirus-superinfected DNA samples (Fig. 9C and 9D) sbows that the incorporation of 3H-thymidine into HSV and HeLa DNA is almost the same in single and superinfected cells. Thus, neither HSV nor HeLa DNA synthesis appears to be markedly inhibited within 6 hr after poliovirus

RESTRICTION

OF

HSV

drops

BP

POLIOVIRUS

217

&Cl

FIG. 9. C&l density gradient profiles of aH-thymidine-labeled DNA from HSV infected cells (heavy line). **C-Thymidine-labeled DIVA from normal HeLa cells (fine line) was added as marker. The dotied lines represent densities determined for each gradient. DNA was extracted from HSV infected cells labeled for 1 hr with Yi-thymidine at 5 hr (A), 10 hr (B), or 16 hr (C), and cells superinfected with poliovirus at 10 hr and labeled at 16 hr (D) after HSV infection.

superinfection. This clearly demonstrates SV DNA synthesis is not susceptible to any detectable inhibition by poliovirus at a time when herpesvirus protein synthesis can be completely inhibited (Fig. 4A). Polyribosonae

Disaggregation

after

;5

hE.2.

Poliovirus

From the experiments presented above, poliovirus interference with HSV replicaCon appears to be limited to a direct inhibition of protein synthesis. This translation inhibition could result from interference with the binding or movement of ribosomes along the mRNA. If binding were inhibited, polyribosomes would rapidly disaggregate as ribosomerunoff occurred. If, on the other hand, ribosome movement were retarded, polyribosomes might be expected to persist. To determine which of these phenomena occur, polyribosomes were extracted at 2-hr intervals from equal numbers of HSV-infected cells and cells superinfected at 5 hr with poliovirus. After velocity sedimentation, the UV optical density recordings of the polyribosomal sucrose gradient elution profiles were compared (Fig. IO). After HSV infection, polyribosomes appear to be rapidly

/ I I !

2-h

J---J

I

GRADIENT FRACTtONS

l__i_-.

FIG. 10. Pdyribosomal absorbaney profiles after velocity sedimentation of cytoplasmie extracts through 5-ml linear sucrose gradients. Polyribosornes appearing in the left half of each absorbancy profile are shown for urrinfeeted cells (HeLa) and for oells processed at successive 2 hr intervals after HSV infection (II2 ) I34 , XI;, I& and Nlo). Profiles from HSV-infected cells superinfected with poliovirus at 5 hr are shown at similar 2-hr intervals (P5 , Pa , and $10).

218

SAXTON

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degraded and then increase to a maximum between 6 and S hr, presumably after the ribosomes reform on viral mRNA. Within 1 hr after poliovirus superinfection, extensive degradation of these polyribosomes is observed. Therefore, the mechanism of poliovirus inhibition of HSV protein synthesis involves polyribosome disaggregation and could result from a selective alteration in the binding of ribosomes to HSV mRNA. Hybridization

of Polyribosomal

RNA

To establish that polyribosomes carry HSV mRNA by 8 hr after HSV infection, polyribosomal RNA derived from infected and normal cells was hybridized with HSV and HeLa cell DNA immobilized on membrane filters. As shown in Table 2, labeled polyribosomal RNA from HSV-infected cells binds over 10 times more efficiently to viral DNA than to cellular DNA. Addition of a IO-fold excess of unlabeled cellular RNA reduces the binding of HSV polyribosomal RNA to viral DNA by only one-third, indicating that the majority of the labeled polyribosomal RNA contains unique HSV specified sequences and is insusceptible to competition by normal HeLa cell RNA. The competed fraction probably represents either nonspecific binding or residual labeled HeLa RNA present in the infected cell polyribosomes. This appears likely since an excess of unlabeled cellular RNA virtually eliminates binding of polyribosomal RNA derived from infected cells to HeLa cell DNA. In the reciprocal hybridization assay, normal HeLa cell polyribosomal RNA anneals to viral DNA only about 10% as efficiently as HSV-infected cell polyribosomal RNA. The relatively inefficient hybridization between HeLa cell DNA and normal cellular polyribosomal RNA probably represents rRNA annealing since the reassociation rate between mammalian mRNA and DNA is usually slow (cf. McCarthy and Church, 1970), These hybridization experiments show that, by 8 hr after HSV infection, cellular polyribosomes contain viral mRNA. This HSV mRNA apparently is unable to form an initiation complex with ribosomes after poliovirus superinfection.

STEVENS TABLE

2

HYBRIDIZATKON BETWEEN POLYRIBOSOMAL RNA AND DNA OBTAINED FROM HSVINFECTED AND NONINFECTED HELM

CELLS

RNase-resistant bound Source of RNAa

Source of DNA”

HSV HSV HeLa HeLa

HSV HeLa HSV HeLa

radioactivityC Uncompeted @pm) 850 68 102 164

Competed (w4 558 13 -

a 3H-Uridine-labeled RNA was extracted from polyribosomes of infected cells (HSV) or uninfected cells (HeLa). Each hybridization vial contained 100,000 cpm of TCA-precipitable RNA. The specific activity of the HSV RNA was about 5 X lo5 cpm/mg and the HeLa RNA was 2 X lo6 cpm/mg. b Four micrograms of purified viral DNA (HSV) or of cellular DNA (HeLa) was immobilized on each membrane filter. After annealing with RNA, the amount of DNA remaining bound to each filter was determined, and no measurable loss was detected. The small amount of DNA from uninfected cells banding at the viral density (1.720-1.730 g/cm3) did not anneal significantly with labeled viral or cellular DNA. c Average cpm bound to DNA on each of 3 filters corrected for nonspecific binding (about 75 cpm) to blank filter after annealing for 16 hr at 70” in 1 ml of 4 X SSC plus 0.2% SDS. The “uncompeted” reactions contained only labeled polyribosomal RNA and the DNA filters. In addition to the labeled RNA, about 2 mg of unlabeled RNA from normal HeLa cells was added in the “competed” hybridization reactions. This unlabeled RNA eliminated all nonspecific binding to the blank filters. DISCUSSION

Our results show that poliovirus completely restricts the replication of HSV in dually infected HeLa cells provided that poliovirus superinfection occurs within the first 5 hr after HSV infection. However, poliovirus replicates normally after early superinfection in these cells. Translation, but not replication, of a functional poliovirus genome appears to be required for this interference with HSV, since inhibition is observed after poliovirus RNA synthesis is

RESTRICTION

OF

blocked with guanidine, but is completely abolished after irradiation of t’he poliovirus. An interference which has also been suggested to require genomic expression by the lntcrfering virus has been reported by Marcus and Carver (1967). There, inhibition of Newcastle disease virus replication was demonstrated in cells preinfected with poliovirus, rubella virus, or several arboviruses. However, interference between viruses does not always require expression of the viral genome since Viiagines and McAuslan (1970) have described an interference with vaccinia virus by frogvirus 3 which appears to be mediat’ed by a st8ructural component of the frogvirus virion. In one other dual infection system reported recemly, involving mutual interference between vaccinia and mengovirus (Freda and Buck, 1971) the role of viral genomes was not defined. We find that HSV can partially interfere with poliovirus replication in dually infected cells if HSV is given substantial time and multiplicity advantages. This inhibition appears to require a functional viral genome since UV irradiation of &ions eliminat,es the effect. The incomplete in’cerference may be due to poliovirus partially outstripping an HSV specified translation inhibitor. Alternatively, nonspecific HSV-induced cellular destruction could explain the partial inhibition of poliovirus replication. We did not investigate the molecular basis of this interference. e mechanism by which poliovirus interfe with HSV replication was examined in detail. EKSV directed protein synthesis was uniformly inhibited within 3 hr after superinfection, but neither RKA nor DNA synthesis was significant’ly altered within 6 hr after e&ion. Polyribosomes carKA were rapidly disaggregated after poliovirus superinfection. From this, we conclude that the interference with HSV replication can be attributed to a poliovirus-specified inhibitory function which selectively restricts translation of HSV mRNA while allowing normal translation of the poliovirus messenger. We have consistently observed an accumulation of 80 S ribosomes rather than subunits after poliovirus disaggregation of pol yribo-

HSV

BY

POLIOVIRUS

219

somes from normal eLa cells or HSV in;fected cells. It seem likely that this polyribosomal disaggregation results from either a poliovirus specific alteration in these ribosomes, or initiation factors which decrease the affinity bet,ween the ribosoma;il subunits and the subunit binding sites of al? mRNA moiecules lacking a unique poliovirus RNA base sequence. Willems and Penman (1966) have shown that the mechanism of host cell protein synthesis restriction by poliovirus involves a disaggregation of po4p ribosomes. This probably results from an inability of cellular mRNA to form an initiation complex with ribosomes. This me&nism of selective messenger recognition is not absolute, slnee other picornaviruses are insensitive to inhibition by poliovir~us (,cf. McCormick and Penman, 1968). An alteration in translation initiation specificity which appears to be analogous to that described here has been found in ~s~~~~~~~~~ coli after mfection with the DXA pbage T, (Hsu and Weiss, X969). The ribosomai binding affinity for E. coli mRNA or the R5.A of several RNA phages is decreased by novel initiation factors which appear on ribosomes after T4 infection (Dube and land, 1970). In euearyotic cells an apparently similar interference with vacciniia replication occurs in a~enQ~rus-~re~~fe~~e HeLa cells. In t&s system, the int’erfere~~~e with vaccinia has been traced to an -inaMity of vaccinia virus mRNA to associate with ribosomes in these doubly infected cells, (Giorno and Rates, 1971). From these results, it is clear that seiective alteration of the cellular translation machinery is a control mechanism used by a number of different viruses &Ming Tg, aden~virus, and pohovirus. It, may be significant 44~1% this regulation is manifested at the level zsf translation, the only step in ma~rorno~e~~la~ synthesis common to replication of all DXA viruses, RNA viruses, and normal celis

This work was supported by ginited States Public Health Service grmts Al-06246 and Al00249. We are indebted to the laboratories of M. A. Baluda and F. 0. Wettstein for their assistance and advice during the course of these studies

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