Mechanism of interferon action Differential effect of interferon on the synthesis of simian virus 40 and reovirus polypeptides in monkey kidney cells

VIROLOGY

117, 3?9-30

(1982)

Mechanism Differential

of Interferon

Action

Effect of interferon on the Synthesis of Simian Virus 40 and Reovirus Polypeptides in Monkey Kidney Ceils

KATHLEEN Section of Biochemistry

A. DAHER

AND CHARLES

E. SAMUEL’

and Molecular Biobgg, Lkpartment of Biological Sci-, Santa Barbara, California #lo6 Received July 22, 1981; accepted Nmmber

Univemit~ of Cal~mia,

24, 1981

The effect of interferon (IFN) treatment on the synthesis of viral proteins was examined in BSC-1 monkey kidney cells singly and doubly infected with simian virus 40 (SV40) and reovirus. The pattern and amount of SV40 T antigen and reovirus X, cc,and o polypeptide synthesis were comparable in singly and doubly infected cells in the absence of IFN treatment. As determined by Yakobson et aL (Ceu 12: 73-81, 1977) and Kingsman and Samuel (Vi101: 453-465, 1980) SV40-specific RNA synthesis is inhibited by treatment of cells with IFN before infection, but is not inhibited by IFN treatment after infection. IFN treatment initiated before infection inhibited the synthesis of both SV40 T antigen and reovirus polypeptides. However, SV40 T antigen synthesis was not inhibited in SV40 wt or fsA mutant-infected cells when treated with monkey kidney or human leukocyte IFN after infection. By contrast, reovirus polypeptide synthesis was markedly inhibited in BSC-1 cells treated with IFN after SV40 infection and superinfected with reovirus. SV40 infection did not rescue reovirus polypeptide synthesis from the inhibitory action of IFN, and reovirus infection did not alter the resistance of SV40 T antigen synthesis to IFN in cells IFN-treated after SV40 infection. Total cellular protein synthesis was not significantly affected by IFN treatment. These results suggest that the interferoninduced inhibitor(s) of protein synthesis discriminate between SV40 and reovirus mRNAs; the translation of SV40 mRNA, like most cellular mRNAs, is not affected under conditions where the IFN-induced inhibitor(s) significantly affect the translation of reovirus mRNA.

INTRODUCTION

sis of early mRNA, are not significantly affected by IFN (Wiebe and Joklik, 1975; Galster and Lengyel, 1976; Samuel et c& 1980). Two IFN-induced enzymes appear to play important roles in the inhibition of protein synthesis observed in IFNtreated systems, a protein kinase that catalyzes the phosphorylation of the small (a!) subunit of protein synthesis initiation factor eIF-2 (Samuel, 1979) and a 2’,5’-oligoisoadenylate synthetase that catalyzes the synthesis of a family of oligonucleotides of the structure pppA@pEi’A), (Kerr and Brown, 1978) which activate an endoribonuclease (Wreschner et aL, 1981). The primary level of IFN-mediated inhibition of simian virus 40 (SV40) multiplication, in contrast to other viruses such

The multiplication of a wide range of both DNA and RNA animal viruses is inhibited by interferons (Stewart, 1979). Cell culture studies suggest that in many animal virus-host cell systems, including reovirus-infected mouse fibroblast, mouse ascites tumor, and monkey kidney cells (Gupta et a& 1974; Wiebe and Joklik, 1975; Samuel et aL, 1980), the primary level of viral macromolecular synthesis inhibited by interferon (IFN) is the translation of early viral mRNAs into protein. The early steps of reovirus replication, including the conversion of parental reovirions to subviral particles and the subsequent synthe’ To whom reprint

requests should be addressed. 379

0042~6%?2/82/040379-12$02.00/0 Copyright 0 1982 by Academic Pmn, Inc. All righta of reproduction in any form reserved.

330

DAHER

AND

as reovirus, is not resolved. In SV40-infected monkey cells treated with IFN prior to infection, a marked reduction in the accumulation of hybridizable SV40 RNA is well documented (Oxman and Levin, 1971; Yamamoto et a& 1975; Kingsman and Samuel, 1980). The inhibition of early RNA accumulation appears to be due to a reduction in synthesis rather than an enhancement in degradation as the kinetics of SV40 early RNA decay are comparable between untreated and IFN-treated cells as determined both by pulse-chase and hybridization (Kingsman and Samuel, 1980) and by protein synthesis (Daher and Samuel, 1981) methods. The synthesis of SV40 early polypeptides, T and t antigens, is also inhibited in monkey cells treated with IFN prior to infection (Oxman and Levin, 1971; Yakobson et al, 1977; Mozes and Defendi, 1979; Kingsman et aL, 1980) although in SV40-transformed mouse cells neither T nor t antigen synthesis is affected by IFN (Oxman et u& 1967; Kingsman et a& 1980). In contrast to the inhibition in synthesis of SV40 early RNA and protein observed in monkey cells IFN treated before infection with intact SV40 virions, Yamamoto et al, (1975) reported that the synthesis of SV40 mRNA, T antigen, and DNA is not significantly affected by IFN pretreatment when monkey cells are transfected with free SV40 DNA. When IFN treatment is initiated after the time of virus infection, SV40 early RNA accumulation also is not affected in wt and tsA virioninfected monkey cells (S. M. Kingsman and C. E. Samuel, unpublished obseryations, 1979; Yakobson et cd, 1977). However, a marked inhibition of both SV40 T antigen and capsid polypeptide synthesis was reported by Yakobson et al. (1977)in monkey cells treated with IFN after infection. In view of the apparent inconsistency between some of the reported observations concerning the effects of IFN on SV40 multiplication, and the implication that the primary mechanism by which IFN affects SV40 multiplication and reovirus multiplication may be different, we initiated a study to examine the effect of IFN

SAMUEL

on viral protein synthesis in monkey kidney cells singly and doubly infected with SV40 and reovirus virions. The results obtained with BSC-1 cells demonstrate that SV40 protein synthesis is not significantly affected by IFN when treatment is initiated after the time of infection even though the SV40-infected cells possess an antiviral state as demonstrated by the IFN-mediated inhibition of reovirus protein synthesis in doubly infected cells. These results suggest that interferon-induced inhibitors of protein synthesis discriminate between SV40 and reovirus mRNAs; the translation of SV40 mRNA, like most cellular mRNAs, is not affected under conditions where the IFN-induced inhibitors significantly inhibit the translation of reovirus mRNA. MATERIALS

AND

METHODS

Cells and viruses. The BSC-1 line of monkey kidney cells was grown in monolayer culture in Eagle’s minimal essential medium (MEM, GIBCO) containing 5% fetal calf serum (Flow) and supplemented with 1 mM sodium pyruvate (Flow), nonessential amino acids (Flow), 100 units/ml penicillin (GIBCO), and 100 pg/ml streptomycin (GIBCO). Stocks of SV40 temperature-sensitive mutant tsA30 (Tegtmeyer and Ozer, 1971) and its wild-type parent, wt 708 (VA45-54) were prepared at a multiplicity of infection of 0.01 PFU/ cell, and harvested after 75% of the cells showed viral cytopathic effect (Kingsman and Samuel, 1980). Reovirus type 3 (Dearing strain) was grown in suspension cultures of mouse fibroblast L cells and purified as previously described (Samuel and Joklik, 1976). Inteyferom. Monkey interferon induced with Newcastle disease virus was prepared and assayed with vesicular stomatitis virus (VSV) on monkey CV-1 cells as previously described (Samuel and Farris, 1977). Sendai virus-induced human leukocyte interferon (1.9 X lo6 units/mg protein, assayed with VSV on human U cells) was generously provided by Dr. K. Cantell (Central Public Health Laboratory, Helsinki, Finland). A dose of 600 units/ml of

INTERFERON

ACTION IN SV40-INFECTED

the human leukocyte IFN is comparable to a dose of 300 units/ml of the monkey kidney IFN (Samuel and Farris, 1977); a 50% reduction in T antigen synthesis occurs at a dose of 3 units/ml of monkey IFN in cells infected at an m.o.i. of 10 PFU/cell (Kingsman et a& 1930). Virus irQ?m%m and radioactive labeliryl of iqfected cella Confluent monolayer cultures of untreated or IFN-treated BSC-1 cells (about 2 X lo6 cells) in 21-cm2 dishes (LUX) were infected with SV40 (5-10 PFU/cell) and/or reovirus (10 PFU/cell) at 32” unless otherwise indicated. Adsorption was carried out for 2 hr (SV40) or 1 hr (reovirus) in Puck’s saline A modified to contain 20 mlM MgC12 and 1% fetal calf serum. The inoculum was removed after adsorption, and the cultures were refed with supplemented Eagle’s MEM containing 5% fetal calf serum and incubated as indicated under the respective figure legends. At the indicated times, monolayer cells to be labeled were washed with methionine-free MEM; 1.25 ml of methioninefree MEM containing 20-40 &Wml of [?Slmethionine (New England Nuclear, 800-1100 Ci/mmol) was then added to each dish and the cells incubated for 1 hr. Preparation of cell extracts. All extraction and fractionation procedures were carried out at 0” to 4”. Cells were rinsed with phosphate-buffered saline and disrupted with 0.4 ml per dish of lysis buffer [20 mM Tris-HCl, pH 9.0; 137 mM NaCl; 1 mM MgClti 50 pg/ml phenylmethylsulfonyl fluoride; 10% (v/v) glycerol; and 1% (v/v) Nonidet-P40]. After 10 min the solubilized cells were scraped into tubes and centrifuged at 500 g for 10 min; the supernatant fraction was then centrifuged at 25,000 g for 30 min before analysis. Ant&m Anti-SV40 T antigen serum from hamsters bearing SV40-induced tumors was provided by Dr. J. Cole (National Cancer Institute, Bethesda, Md). Antireovirus serum to reovirus capsid polypeptides was prepared in hamsters injected intraperitoneally with 0.5 mg uvinactivated purified virions emulsified in Freund’s complete adjuvant. An identical booster dose was administered 5 weeks later, and 10 days later the animals were

MONKEY

CELLS

381

bled by cardiac puncture. Normal serum was obtained by cardiac puncture from hamsters that had not been immunized with either SV40 or reovirus antigens. Heat-inactivated formalin-fixed Staphylococcus aureua (Cowan I) was provided by Dr. D. Sears and N. Miyamoto (University of California, Santa Barbara). Anal&s of cell &racta Viral proteins were immunoprecipitated from extracts with either antiSV40 T antigen serum or anti-reovirus serum by the indirect S; aureus protein A procedure (Kessler, 1975; Cullen and Schwartz, 1976). All reagents were titered in order to ascertain that the conditions of immunoprecipitation provided a quantitative measure of the levels of viral proteins. Extract (100 ~1) was incubated with normal serum (9 ~1) for 2 hr

T-

FIG. 1. Effect of multiplicity of infection on the sensitivity of SV40 T antigen synthesis to interferonmediated inhibition in cells treated with interferon before infection. WC-1 cells were treated with 600 units/ml of human leukocyte interferon at 37” for 18 hr before infection with wild-type SV40 at input multiplicities of infection of 0, 0.01, 0.1, 1, 10, and 60 PFWcell. Untreated (-) and interferon-pretreated (+) cells were pulse labeled with [S6Sjmethionine for 1 hr at 30 hr postinfection. Extracts were prepared, immunoprecipitated with SV40 anti-T immune serum, and analyzed as described under Materials and Methods.

382

DAHER AND SAMUEL

anti-T 1$40 3

ppt. 14 5

abcde

c

total extract ’ 'z-40 3

’

14 5

c

obcde

T-

4” with 0.5 ml of wash buffer [O.l MTrisHCl, pH 7.4; 0.15 M NaCl], suspended in 40 ~1 of electrophoresis sample buffer, boiled for 10 min, centrifuged, and analyzed by NaDodSOl polyacrylamide gel electrophoresis essentially as described (Laemmli, 1970). Slab gels with a 5% acrylamide stacking layer and a 11.5% acrylamide running layer were routinely used. For detection of radioactive bands, gels were impregnated with EN’HANCE (New England Nuclear), dried onto filter paper under vacuum with steam heat, and used to expose Kodak XRP-1 film at -80”. RESULTS

SK@ Tumor Antigen Synthesis in BSC-1 CeuSTreated with Inteyferon before and q&r Infection

FIG. 2. Effect of interferon treatment initiated either before infection or at increasing times after infection on the synthesis of SV40 T antigen and cellular proteins in w&infected cells. BSC-1 cells were treated at 37’ with 600 units/ml of human leukocyte interferon at (b) 40 hr before infection, (c) 5 hr after infection, or (d) 14 hr after infection with wt SV40. Interferon-treated (b-d) and untreated (a, e) cultures were pulse labeled with [86S]methionine for 1 hr at 36 hr postinfection. Extracts were prepared and analyzed as described under Materials and Methods. (Left) Aliquots, 100 pl, immunoprecipitated with SV40 anti-T immune serum for measurement of T antigen synthesis; (right) 4-~1 aliquota analyzed directly without immune precipitation for measurement of overall cellular protein synthesis.

at 4”; 100 ~1 of heat-inactivated, formalinfixed S. aureus (10% w/v) was then added, and incubation was continued for 30 min at 4’. The precipitate obtained was removed by centrifugation at 15,000 g for 3 min. The resulting supernatant fraction was incubated with immune serum, either 6 ~1 of SV40 anti-T or 6 ~1 of anti-reovirus, for 16 to 24 hr at 4”; 100 ~1 of S. aureus was then added, and incubation was continued for 30 min at 4”. The immunoprecipitate was collected by centrifugation at 15,000 g for 3 min, washed three times at

The synthesis of SV40 T antigen in BSC1 cells was inhibited by treatment with interferon 18 hr before infection (Fig. 1). This is a well-established observation (Oxman and Levin, 1971; Yabobson et aL, 1977; Kingsman et d, 1980). However, as shown in Fig. 1, the degree of inhibition of T antigen synthesis depended upon the multiplicity of infection, which was varied from 0.01 to 50 PFU/cell. At an m.o.i. of 1 PFU/cell T antigen synthesis was inhibited about loo-fold, at 10 PFU/cell about 25-fold, and at 50 PFWcell about 3 to 4fold as quantitated by densitometric scanning of autoradiograms. In contrast to the significant inhibition of T antigen synthesis observed in wildtype SV40-infected BSC-1 cells treated with IFN before infection (Figs. 1 and 2 left, track b), T antigen synthesis measured in a 1-hr pS]methionine pulse at 36 hr postinfection was not significantly reduced when cells were treated continuously with IFN beginning at either 5 or 14 hr qfter infection (Fig. 2 left, tracks c and d). Interferon treatment initiated either before or at increasing times after SV40 infection did not significantly affect the synthesis of cellular proteins present in extracts analyzed before immunoprecipitation with anti-T immune serum (Fig. 2, right).

INTERFERON

ACTION IN SV40-INFECTED

SV40 tsA mutants overproduce early RNA and T antigen at 41”, and to a lesser extent at 32”, because of the inability of the mutant T antigen to regulate early transcription (Tegtmeyer et uL, 1975; Alwine et aL, 1977; Khoury and May, 1977). The overproduction of early mRNA coding for T antigen by tsA mutants facilitates certain studies on the mechanism of interferon action in SV40-infected cells because of the increased amounts of early RNA, and of protein when measured in short pulses, available for analysis. In the experiment shown in Fig. 3, BSC-1 cells infected with SV40 tsA30 at 32” were treated with IFN before infection, at infection, or 18 or 44 hr after infection. After a 1-hr $S]methionine pulse at 16, 24, or 60 hr postinfection, the infected cultures were harvested and analyzed for T antigen synthesis in the IFN-treated as compared to untreated control cells. In cells IFN treated for 16 hr beginning at 16 hr before infection, T antigen synthesis was greatly reduced when measured at both 24 and 60 hr postinfection. However, when IFN treatment for 16 hr was initiated at the time of infection or 18 or 44 hr after infection, the synthesis of T antigen was not significantly reduced when measured at either 24 or 60 hr postinfection. The sensitivity of SV40 capsid polypeptide VP-l synthesis to the inhibitory action of IFN followed a pattern similar to that observed for T-antigen in both wtand ha-infected cells. IFN treatment before infection reduced significantly the amount of de nova synthesized VP1 present in the nuclear fraction of BSC-1 cells, whereas IFN treatment after infection did not significantly alter the amount of VP1 synthesized at late times (results not shown). Viral

Protein Synthesis in Ini?e@krmTreated BSGl CeUa Sin&y and Doubly Infected with SV.‘. and Remirus

Although no significant reduction in SV40 protein synthesis was observed when BSC-1 cells were treated with IFN after infection, the duration of IFN treatment postinfection should have been more than

pulse t

IFN t

MONKEY

CELLS

383

16 hr 24 hr 60hr ‘E ‘c -16 0% -16 0 18°C -16 0 18 445’ obcdefghijklm

T-

FIG. 3. Effect of interferon treatment initiated either before infection or at increasing times after infection on the synthesis of SV40 T antigen in fsAinfected cells. BSC-1 cells were treated with 606 units/ml of human leukocyte interferon at 16 hr before infection (b, e, i), at the time of infection (c, f, j), or at 18 hr (g, k) or 44 hr (1) after infection with fsA SV40. Interferon was present in the culture medium of interferon-treated ceils for a total of 16 hr, except for the experiment represented by track g, in which interferon treatment was for 6 hr only. Interferon-treated (b, c, e-g, i-l) and untreated (a, d, h. m) cultures were pulse labeled with [8SSknethionine for 1 hr at 16 (a-c), 24 (d-g), or 60 hr (h-m) postinfection. Extracts were prepared, immunoprecipitated with SV40 anti-T immune serum, and analyzed as described under Materials and Methods.

sufficient to allow for the maximal induction of the two enzymes that are believed to play a role in the inhibition of viral protein synthesis in IFN-treated cells, a 2’,5’-oligoadenylate synthetase and a protein kinase (Samuel et al, 1977; Baglioni et aL, 1979; Kimchi et aL, 1979). Reovirus replication is sensitive to IFN in monkey cells, and the inhibition of synthesis of reovirus polypeptides is the apparent level of IFN action in monkey and mouse cells (Gupta et al, 1974; Wiebe and Joklik, 1975; Samuel et aL, 1980). Double infections with SV40 and reovirus were therefore carried out to determine whether viral protein synthesis, in the case of a translationally

DAHER AND SAMUEL

334

virus serum

R S S/R RSRSRS abcdef

uninf

A{ TPC

u 1

FIG. 4. Synthesis of SV40 and reovirus proteins in infected and superinfected cells. Single (a, h) and double (c, d) infections of BSC-1 cells with SV40 fsA and reovirus were at 32”. Cultures were pulse labeled with pS]methionine for 1 hr and extracts were prepared, immunoprecipitated with either SV40 anti-T immune serum (b, d, f) or anti-reovirus immune serum (a, c, e), and analyzed as described under Materials and Methods. (a) Reovirus infected, labeled at 33 hr postinfection; (h) SV40 infected, labeled at 60 hr postinfection; (c, d) SV40 infected and then superinfected with reovirus at 38 hr post-SV40 infection, labeled at 60 hr post-SV40 infection; (e, f) uninfected, labeled 1 hr. R, reovirus; S, SV40. The arrows indicate the positions of reovirus polypeptides (X, p, u) and SV40 T antigen (T).

sensitive virus such as reovirus, sitive to the inhibitory action

was sen-

of IFN in SV40-infected cells under conditions where SV40 early and late protein synthesis was resistant to the inhibitory action of IFN. As shown in Fig. 4, SV40 and reovirus proteins are synthesized in comparable amounts in singly and doubly infected BSC-1 cells in the absence of IFN treatment. Thus, heterologous interference does not appear to occur between SV40 and reovirus when examined at the level of viral polypeptide synthesis. The effect of noncontinuous IFN treatment

initiated

either

before

or after

SV40

infection on the synthesis of viral proteins in SV40-infected, reovirus-superinfected cells is shown in Fig. 5. BSC-1 cells were

treated with IFN at 32”, infected with tsA30 SV40, and then superinfected with reovirus at 42 hr post-SV40 infection as outlined in the schematic diagram (Fig. 5). Viral protein synthesis was measured in a 1-hr pulse with pS]methionine at 65 hr post-SV40 infection. SV40 T antigen was detected by immunoprecipitation of extracts prepared from doubly infected cells with anti-SV40 T antigen serum (Fig. 5A) and reovirus X, I.C,and (Tproteins were detected by immunoprecipitation with antireovirus serum (Fig. 5B). When cells were IFN treated for 24 hr before SV40 infection and then maintained in medium in the absence of IFN for 65 hr before pulse labeling with [?S]methionine, both SV40 and reovirus polypeptide syntheses were inhibited as compared to untreated cells (Fig. 5, tracks b and c). IFN treatment at either 3 or 12 hr after SV40 infection did not affect the synthesis of SV40 T antigen (Fig. 5A, tracks d and e); by contrast, the synthesis of reovirus polypeptides was significantly inhibited in the superinfected cells IFN treated at 3 or 12 hr after SV40 infection (Fig. 5B, tracks d and e). These results indicate that IFN-induced inhibitors of viral protein synthesis are present and functional in IFN-treated BSC-1 cells, and suggest that the translation of SV40 early mRNA is not significantly affected in intact cells under conditions where the translation of reovirus mRNA is markedly inhibited by IFN. The degree of inhibition of reovirus protein synthesis was not maximal either in the case of noncontinuous IFN treatment before SV40 infection (Fig. 5B, track c) or in the case of IFN treatment for 6 hr before reovirus infection (track f) when compared to the levels of reovirus protein synthesis in untreated cells (track b) and cells treated for longer periods of time with IFN (tracks d and e). The apparent modulation in the degree of inhibition of reovirus protein synthesis dependent upon the time of IFN treatment is consistent with the observed kinetics of induction and decay of the IFN-induced antiviral state and PJeIF-2cu protein kinase. Relevant to the results shown in Figs. 5-7, 6 hr of IFN treatment is sufficient time to

INTERFERON

ACTION IN SV40-INFECTED

MONKEY

CELLS

385

(4) uninfected UJ) infected, no IFN (c-f) infected, IFN-treoted

PW$ Reovirus infection 1

sv40

infection 1

-24-

(clIFN

0-3-12

36-42-

(cl) I-------------c tell---------

(A) anti-W40

T

obcdef

Harvest 1 t

65-66

(f)+-

(8) anti-MO obc

def

T-

FIG. 5. Effect of noncontinuous interferon treatment initiated either before or after SV40 infection on the synthesis of viral proteins in SV40-infected, reovirus-superinfected cells. BSC-1 cells were treated with 300 units/ml of monkey kidney CV-1 interferon at 32” as outlined in the schematic diagram shown below. Cultures were infected with tsA SV40 and then superinfected with reovirus at 42 hr post-SV40 infection. Infected cells were pulse labeled with [8SSjnethionine for 1 hr at 65 hr post-SV40 infection, extracts were prepared and immunoprecipitated with either (A) anti-W40 T immune serum or (B) anti-reovirus immune serum, and analyzed as described under Materials and Methods.

partially induce both an antiviral state and protein kinase activity but full induction is not observed until after 12 to 16 hr of IFN treatment (Samuel et c& 19’77; Kimchi et d, 1979). Both the IFN-induced antiviral state and protein kinase activity decay significantly by 2 to 3 days after the removal of IFN and within 4 to 5 days have returned to levels comparable to those observed in untreated cells (C. E. Samuel and G. S. Knutson, manuscript in preparation).

The amounts of reovirus polypeptides synthesized in singly infected BSC-1 cells and cells doubly infected with SV40 and reovirus were comparable (Figs. 4 and 6), and reovirus superinfection of SV40-infected cells did not render SV40 T antigen sensitive to the inhibitory action of IFN (Fig. 5). As shown in Fig. 6, prior infection of BSC-1 cells with SV40 also did not rescue reovirus protein synthesis from the inhibitory action of IFN. The sensitivity of reovirus polypeptide synthesis was

DAHER

386 (a) (b) (c-f)

AND SAMUEL

uninfected infected, no IFN infected, IFN-treated (A)+ Iej- SV40 infection t -24 -o-4--13 (d) 1 (e) 1

IFN (A)

SV40,.REO

abcdef

Reovirus infection t 3741 -6566

Horvesi 1 t c c *

(f) (Bl -,

REO

bcdef’

FIG. 6. Effect of continuous interferon treatment initiated either before or after SV40 infection on the synthesis of reovirus polypeptides in SV40-infected, reovirus-superinfected cells. BSC-1 cells were treated with 600 units/ml of human leukocyte interferon at 32” as outlined in the schematic diagram shown below. Cultures were (A) infected with tsA SV40 and then superinfected with reovirus at 41 hr post-SV40 infection, or (B) not infected with SV40 but infected with reovirus as for (A). Infected cultures were pulse labeled with PSJmethionine for 1 hr at 65 hr post-SV40 infection, and extracts were prepared, immunoprecipitated with anti-reovirus immune serum, and analyzed as described under Materials and Methods.

comparable in the presence (Fig. 6A) and absence (Fig. 6B) of SV40 infection of BSC1 cells treated continuously with 600 units/ ml of IFN. By contrast, continuous treatment of doubly infected BSC-1 cells with doses of either 600 units/ml (results not shown) or 3000 units/ml (Fig. 7) of IFN did not significantly inhibit SV40 T-antigen synthesis when the IFN treatment was initiated Qi%r infection (Fig. ‘7, tracks d-f) but markedly inhibited T antigen synthesis when treatment was initiated before infection (Fig. 7, track c). DISCUSSION

The results reported here demonstrate that the synthesis of SV40 and reovirus

proteins in intact monkey kidney cells is differentially affected by interferon treatment. The translation of SV40 early mRNA into T antigen was not significantly inhibited by IFN treatment after infection. The insensitivity of T antigen synthesis to IFN treatment initiated after SV40 infection is not due to a lack of expression of IFNmediated translational inhibitors because the synthesis of reovirus polypeptides was markedly inhibited in BSC-1 cells doubly infected with SV40 and reovirus. An analogous situation concerning the effect of IFN on SV40 early polypeptide synthesis exists in SV40-transformed mouse cells; the synthesis of T antigen in SV40-transformed cells is completely resistent to the

INTERFERON

ACTION IN SV40-INFECTED

inhibitory action of IFN even though the replication of vesicular stomatitis virus is extensively inhibited in IFN-treated SV40transformed cells (Oxman et d, 1967; Mozes and Defendi, 1979; Kingsman et aL, 1980; Samuel et aL, 1980). Recently, SV40 early RNAs from lytically infected and transformed cells were analyzed and found to be similar to each other with respect to the genomic localization of their 5’ and 3’ termini, their major splicing patterns, and their 5’ cap and methylation patterns (Reddy et ah, 1979; Haegeman and Fiers, 1980). Other recent studies indicate that, by the criterion of peptide mapping, the major form of T antigen found in 3T3 cells transformed with SV40 mutants possessing characteristic T antigen tryptic peptides was indistinguishable from T antigen found in monkey cells lytically infected with the respective mutants (Denhardt and Crawford, 1980). It thus appears that in both lytically infected cells and in transformed cells the major forms of SV40 early mRNA and T antigen protein product are structurally similar at the level of primary sequence, and that the SV40 early mRNA coding for T antigen is not recognized as “viral” by the interferon system. By contrast, reovirus mRNA is sensitive to IFN-induced translational inhibition in viva in cell culture systems and in vitro in cell-free systems (Gupta et cd, 1974; Samuel and Joklik, 1974; Wiebe and Joklik, 1975; Miyamoto and Samuel, 1980; Samuel et cd, 1980). Yakobson et al, (1977) reported a marked inhibition in SV40 T antigen and VP-l synthesis in BSC-1 cells treated with IFN at 24 hr after infection with wt strain 777. We, however, have not observed a significant inhibition of SV40 T antigen synthesis in cells treated and pulse labeled at various times after infection in studies utilizing both monkey and human IFNs, CV-1 and BSC-1 cells, and wt 708 and tsA30 virus strains. The reason for this contradiction is unclear but may be related to the virus strains employed. However, the results of several independent studies of IFN action in SV40-infected and SV40transformed cells can be best explained by an IFN-induced reduction in the forma-

MONKEY

IFN t

CELLS

387

C-24 3 12 36 b

cdef

T-

FIG. 7. Effect of continuous interferon treatment initiated either before or after SV40 infection on the synthesis of SV40 T antigen in SV40-infected, reovirus-superinfected cells. BSC-1 cells were treated with 3000 units/ml of human leukocyte interferon at 32”, infected with &.A SV40, and superinfected with reovirus. The timing of the experiment was as summarized by the schematic diagram shown under Fig. 5 except that interferon dishes were refed with medium containing interferon at 0 (c) and at 43 hr (cf). Extracts were prepared, immunoprecipitated with anti-SV40 T immune serum, and analyzed as described under Materials and Methods. (b) Infected, no IFN. (c-f) Infected and IFN treated at: (c) -24 to 65; (d) 3 to 65; (e) 12 to 65; and (f) 36 to 65 hr.

tion of active early transcriptional complexes in permissive monkey cells treated with IFN before infection rather than by IFN-mediated effects on the translation perse of SV40 RNA. First, attachment and penetration of SV40 virions does not appear to be affected by IFN treatment initiated before infection because the amount of input SV40 DNA genome present in untreated and IFN-treated cells is comparable at early times postinfection (Metz et uL, 1976; W-q. Zeng and C. E. Samuel, unpublished observations). Second, the

388

DAHER

AND

accumulation of SV40 early RNA is markedly reduced in monkey cells treated with IFN before infection with virions (Oxman and Levin, 19’71; Yamamoto et aZ., 1975; Metz et al, 1976; Yakobson et cd, 1977; Kingsman and Samuel, 1980). However, the accumulation of early RNA is not reduced in monkey cells when treated with IFN either after infection with SV40 virions (Yakobson et aL, 1977; S. M. Kingsman and C. E. Samuel, unpublished observations) or before transfection with free SV40 DNA of wt strain 777 (Yamamoto et aL, 1975). Third, the kinetics of SV40 early RNA decay are comparable between untreated and IFN-treated monkey cells as determined by both pulsechase and hybridization (Kingsman and Samuel, 1980) and protein synthesis (Daher and Samuel, 1981) techniques. Fourth, the translation of SV40 early mRNA coding for T antigen is not significantly inhibited in monkey cells IFN treated after infection with virions (Figs. 2, 3, 5, 7) or before transfection with naked DNA (Yamamoto et & 1975). SV40 early protein synthesis is also not inhibited by IFN in IFN-sensitive transformed cells (Oxman et al, 1967; Mozes and Defendi, 1979; Kingsman et a!., 1980). Fifth, the reduction in T antigen synthesis in monkey cells treated with IFN before infection with SV40 wt strain 777 is comparable to the reduction in amount of early RNA synthesized (Oxman and Levin, 1971; Metz et cd, 1976). Sixth, the yield of progeny SV40 in single cycle growth determinations with wildtype 830 and deletion mutant 894 is not significantly decreased when IFN treatment is initiated 24 or 36 hr after infection but is markedly decreased when IFN treatment is initiated 24 hr before infection (S. M. Kingsman and C. E. Samuel, unpublished observation). The results summarized above are all consistent with an effect of IFN in monkey cells at a very early stage of SV40 infection following virion penetration but prior to early transcription. SV40 temperature sensitive group D mutants and deletion mutants such as d11261 and d11262 which map within the genes for virion proteins VP2 and VP3

SAMUEL

behave as though they contain a tightly binding repressor of transcription (Avila et al, 1976; Llopis and Stark, 1981). Such mutants are affected at a very early stage of infection. The tsD mutants have an unusual property in that the first cycle of virus replication is not temperature sensitive when infection is carried out with free viral DNA rather than with virions; tsD mutants also fail to complement SV40 ts mutants of any other class (Robb and Martin, 1972; Chou and Martin, 1975a, b). The tsD mutants are defective at the restrictive temperature at a step after virion attachment and penetration but prior to viral DNA synthesis (Lebowitz and Weissman, 1979). Indeed, SV40 tsD mutants possess some properties in untreated cells characteristic of wild-type virus and tsA mutants in interferon-treated cells. Conceivably IFN pretreatment of monkey kidney cells leads to subtle changes in the uncoating process of SV40 virions which result in alterations in VP2 and/or VP3 associated with SV40 DNA such that expression of early RNA is repressed. ACKNOWLEDGMENTS

This work was supported in part by research grants from the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service, and the American Cancer Society. C.E.S. is a Career Development Awardee of the National Institute of Allergy and Infectious Diseases, U. S. Public Health Service. REFERENCES

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