Mechanism of interferon-induced inhibition of early simian virus 40 (SV40) functions

VIROLOGY

68, 58-70 (197%

Mechanism

KIYOSHI Department

of Interferon-Induced Virus 40 (SV40)

YAMAMOTO,

NOBUO

of Tumor Virus Research, Institute

Inhibition Functions

YAMAGUCHI, of Medical

of Early Simian

AND

KINICHIRO

Science, P.O. Takanawa.

ODA

Tokyo 108, Japan

Accepted June 2, 1975 The mechanism of interferon-induced inhibition of early simian virus 40 (SV40) functions was studied in both permissive and nonpermissive cells. The following observations indicate that the step in SV40 replication which is sensitive to the action of interferon is located at a very early stage of infection and suggest that SV40-DNA in the infecting virions cannot be converted to a functional form for transcription by the action of interferon. 1) Synthesis of early viral mRNA as well as T antigen was completely inhibited by interferon when monkey cells were infected at a relatively low multiplicity. This inhibition, however, could be partially overcome by infecting the cells at a high multiplicity. Similar multiplicity-dependent leakiness of the interferon-induced inhibition of viral early functions was observed in nonpermissive mouse cells. 2) Inhibition of SV40-DNA synthesis by interferon was not complemented by the early gene products present in SV40-transformed monkey cells (clone T22). SV40-DNA of the infecting virions was unable to replicate in T22 cells pretreated with interferon. 3) The synthesis of SV40-DNA and -mRNA and the formation of T and V antigens were much less inhibited by interferon when monkey cells were infected with the infectious DNA. The 20-50”; decrease in synthesis of these macromolecules seems to be caused by relatively inefficient penetration of DNA to the nuclei of interferon-treated cells. Under the same conditions. synthesis of these macromolecules was completely inhibited by interferon in the virion-infected cells. INTRODUCTION

by interferon is parallel with the degree of inhibition of SV40-mRNA synthesis in acutely infected cells. In contrast, SV40 T-antigen formation by adeno 7-SV40 hybrid virus, in which 75% of SV40-DNA is integrated into adenovirus type 7 DNA (Kelly and Rose, 1971), is not affected by interferon (Oxman et al., 196713) as much as the formation of T antigen in SV40-transformed cells (Oxman et al., 1967a). This differential effect of interferon on the expression of early SV40 functions provides a good system in which to analyze the mechanism of interferon action. The results of the present study confirmed the inhibitory effect of interferon on the transcription of SV40-DNA in the infected monkey cells. The possible mechanisms of interferon-induced blockage of SV40-DNA transcription are discussed.

The mechanisms of interferon-induced resistance of cells to invading viruses have been studied by many investigators. Most of the studies have shown that the primary site at which an interferon-induced antiviral protein(s) acts is translation of viral mRNA (Joklik and Merigan, 1966; Marcus and Salb, 1966; Levy and Carter, 1968; Kerr, 1971; Friedman et al., 1972; Falcoff et al., 1973). On the other hand, however, several experiments have shown that the inhibition of transcription of viral nucleic acid is the primary site of interferon action (Marcus et al., 1971; Oxman and Levin, 1971; Bialy and Colby, 1972; Manders et al., 1972). In the case of simian virus 40 (SV40), Oxman and Levin (1971) showed that the degree of inhibition of T-antigen formation 58 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction

in any form reserved.

ACTION MATERIALS

AND

OF INTERFERON

METHODS

Cells and viruses. As permissive cells, a secondary culture of African green monkey kidney (AGMK) cells and a passaged AGMK cell culture, G9186 (provided by Dr. T. Kitahara), were cultivated at 37” in Eagle’s medium with 10% calf serum and 10% tryptose phosphate broth. Established cell lines of AGMK, GC7 (Yamaguchi and Kuchino, 1975), Cl4 (Shiroki and Shimojo, 1971), CV-1 and Vero, and a SV40-transformed AGMK cell line, T22 (Shiroki and Shimojo, 1971), were similarly cultivated. As nonpermissive cells, cell lines of mouse fibroblast, L 929 and C3H2K-C4, were cultivated in Eagle’s medium with 10% calf serum. C3H2K-C4 is a cloned cell line of C3H2K which was established by Yoshikura and Hirokawa (1968). SV40 strain 777 was propagated in CV-1 cells and titrated by plaque formation on GC7 cells. The Indiana strain of vesicular stomatitis virus (VSV), supplied by the National Institute of Animal Health, was propagated in GC7 cells and titrated on G9186 cells. The Miyadera strain of Newcastle disease virus (NDV), provided by Dr. M. Toba, was propagated in the allantoic cavity of lo-day-old chick embryos and plaque assayed on primary cultures of chick embryo cells. Preparation and assay of interferon. Monkey and mouse interferons were prepared by NDV infection of G9186 and L 929 cells, respectively, at a multiplicity of lo-20 PFU/cell. At 24-48 hr postinfection (p.i.), the medium was collected, acidified to pH 2 with HCl and kept at 4” for 5 days. The medium was then neutralized with NaOH and concentrated 20-fold by ultrafiltration (Diaflo, UM-10) (Paucker et al., 1970). The sample was dialyzed against 0.1 M sodium phosphate buffer (pH 5.9) and, after centrifugation at 12,000 g for 20 min, the supernatant fluid was applied to a CM-Sephadex (C25) column (15 x 1.5 cm) and eluted with a pH gradient (Merigan et al., 1965). Most of the interferon activity, which eluted at about pH 7, was pooled, dialyzed against Eagle’s medium and filtered through a Millipore filter prior to use.

ON SV40

59

Mock interferon was similarly prepared from the medium of G9186 and L929 cell cultures. Monkey interferon thus prepared usually contained approximately 1600 units/ml with a specific activity of 6.5 x lo3 unitslmg of protein. Both monkey and mouse interferons had no inhibitory effect on cell growth when added to AGMK and C3H2K-C4 cell cultures, respectively. The activity of interferon was assayed by plaque reduction of VSV on the interferontreated cells. Cells in 60-mm petri dishes were exposed for 20 hr to twofold serial dilutions of the interferon preparation and then challenged with about 50 PFU of VSV. One unit of interferon was defined as the reciprocal of the dilution that gave a 50% plaque reduction. Preparation of SV40. SV40 was prepared from the infected CV-1 cells as previously described (Iida and Oda, 1975). In brief, the cell extract prepared by sonication was treated with 1% sodium deoxycholate (Difco) at 37” for 30 min. The extract was centrifuged at 10,000 g for 20 min, and the supernatant fluid was layered on 10 ml of saturated KBr solution and centrifuged at 24,000 rpm for 3.5 hr in a Spinco SW 25.1 rotor, according to Yoshiike and Axelrod (personal communication). The SV40 band formed in the KBr solution was collected, dialyzed against 0.01 M Tris-HCl (pH 8.0), 5 mM EDTA and rebanded in CsCl (p = 1.34) by centrifugation at 38,000 rpm for 24 hr in a Spinco 40 rotor. The band was collected and similarly dialyzed. When [3H]thymidine-labeled SV40 was prepared, CV-1 cells maintained in Eagle’s medium without serum and tryptose phosphate broth for 2 days were infected at a multiplicity of 1 PFU/cell, and 2 pCi/ml of [3H]thymidine (15 Ci/mmol) was added to the culture at 30-40 hr after infection. Isolation of SV40-DNA. The purified SV40 virions were incubated in 0.01 M Tris-HCl (pH 8.0), 0.01 M EDTA, 0.3 M 2-mercaptoethanol and 250 pg/ml of papain (Sigma) at 37” for 5 min. DNA was extracted once with water-saturated phenol (pH 8.0) and twice with chloroformisoamyl alcohol (24:l) after addition of l/10

60

JAMAMOTO.

YAMAGUCHI

volume of 5 M NaClO,. After dialysis against l/10 x SSC (NaCl, Na citrate) containing 0.01 M EDTA, it was sedimented through a 5-20% sucrose gradient in 0.02 M Tris-HCl (pH 8.0), 0.1 M NaCl, 0.01 M EDTA by centrifugation in a Spinco SW 25.1 rotor at 24,000 rpm for 18 hr at 4”. Component I was collected, dialyzed against l/10 x SSC containing 2 mM EDTA and stored at -80”. For preparation of infectious DNA, component I was precipitated by the addition of 2.5 volumes of ethanol and kept at -20” overnight. The DNA was collected by centrifugation and dissolved in a minimal volume of Tris-buffered BME (one volume of 0.2 M Tris-HCl (pH 7.4) and three volumes of Eagle’s medium without antibiotics). Infection of cells with SV40-DNA. Subconfluent cultures of GC7 and G9186 cells treated with interferon were washed once with prewarmed phosphate-buffered saline (PBS) without Caz+ and Mg’+. SV40-DNA and DEAE-dextran (MW, 2 x 10’) at a final concentration of 500 pg/ml were layered over the cell monolayer at 37” for 30 min (McCutchan and Pagano, 1968). The cells were washed twice with prewarmed Eagle’s medium and incubated in medium with 4% calf serum and 5% tryptose phosphate broth. Anti-SV40 rabbit serum (64 units/ml) was added at 4 hr p.i. Measurement of the uptake of labeled SV40-DNA. The cells infected with either 3H-labeled SV40 virions or 3H-labeled SV40 DNA were washed seven times with prewarmed Eagle’s medium after adsorption for 2 and 0.5 hr respectively. The cells were incubated in Eagle’s medium with 5%’ calf serum and 5% tryptose phosphate broth at 37”. At the times indicated, the cells (ca. 2 x 106) were harvested and suspended in isotonic buffer (0.14 M NaCl, 0.01 M Tris-HCl (pH 7.4), 1.5 mM MgCl,) containing 0.5% Nonidet P-40 at 0” for 10 min (Borun et al., 1967). The nuclei, deposited by centrifugation, were washed once with isotonic buffer and suspended in 2 ml of RSB (0.01 M NaCl, 0.01 M TrisI-ICI (pH 7.4), 1.5 mM MgCl,). To the nuclear suspension, 0.3 ml of a detergent mixture made of one part of 10% (W/W)

AND ODA

sodium deoxycholate and two parts of 10% (w/w) Tween 40 was added (Penman, 1966). The nuclei, deposited by centrifugation, were washed with RSB and the trichloracetic acid (TCA)-insoluble radioactivity retained on the glass-fiber filter (Toyo-Roshi, Type GB 60) was counted. Immunofluorescence tests. SV40infected cells on a coverslip were stained with either fluorescein-conjugated antiSV40 T-antigen serum from hamsters bearing virus-free, SV40-induced tumors or fluorescein-conjugated anti-SV40 V-antigen rabbit serum. The number of T- or V-antigen-positive cells was counted under a fluorescent microscope. DNA-DNA and DNA-RNA hybridization. Hybridization was carried out with DNA immobilized on Millipore membrane filters (Gillespie and Spiegelman, 1965). For DNA-DNA hybridization, the filters, previously soaked in 3 x SSC containing 0.02% each of Ficoll, polyvinylpyrrolidone and bovine albumin (Denhardt, 1966), were incubated in 6 x SSC with the denatured labeled DNA at 66” for 16 hr. The filters were then washed on both sides with 3 mM Tris-HCl (pH 9.4), dried and counted. DNA was labeled with [3H]thymidine in the presence of 5 x 10m6 M thymidine, 5 x 10e6 M uridine and 2 x lo-’ M fluorodeoxyuridine. Labeled DNA was denatured and fragmented in 0.5 N NaOH by heating at 100” for 5-7 min just before hybridization. DNA-RNA hybridization was carried out in 1 ml of 6 x SSC containing 0.1% sodium dodecyl sulfate (SDS) at 66” for 20 hr. RNA was labeled with [3H]uridine together with lo-’ M thymidine. RESULTS

Interferon Sensitivity Lines

of the AGMK

Cell

Sensitivity of the established cell lines of AGMK to interferon was assayed by reduction of VSV plaque formation. The cells, treated with various concentrations of interferon, were challenged with VSV, and the number of plaques formed at each interferon concentration was plotted as percent of that formed in untreated control

ACTION

OF INTERFERON

cells. As shown in Fig. 1, the sensitivity was quite different among these cell lines although the efficiency of plaque formation by VSV in these various cell lines was not significantly different. G9186 cells were most sensitive and pretreatment of the cells with 4 units/ml of interferon completely inhibited VSV plaque formation. At this concentration, however, the efficiency of plaque formation in CV-1 and Vero cells was unaffected. The sensitivity of cells to interferon inhibitions of VSV plaque formation was as follows: G9186 > AGMK > Cl4 > GC7 > T22 > CV-1 > Vero. Sensitivity of the cells to interferon was similarly assayed in terms of the efficiency of T-antigen formation in cells infected with SV40. Since the sensitivity of these cell lines to SV40 infection is different, secondary cultures of AGMK, GC7, C14, and CV-1 cells, treated with various concentrations of interferon, were infected with SV40 at a multiplicity of 5, 5, 15 and 75 PFU/cell, respectively, so that more than 90% of the cells in untreated culture exhibit T antigen. As shown in Fig. 2, at a concentration of 100 units/ml, the number of T-antigen-forming cells detected by immunofluorescence was reduced to less than 10% of the control in secondary cultures of

Interferoni

units

ml

FIG. 1. Interferon sensitivity of the AGMK cell lines assayed by VW plaque reduction. The confluent monolayers of the cells, treated with the indicated concentration of monkey interferon; were challenged with VSV and the plaques were counted as described in Materials and Methods. -A-, G9186; -O-, secondary culture of AGMK; --O--, C14; -0--, GC7; -W-, T22; -A-, CV-1; -*-, Vero.

61

ON SV40 1

FIG. 2. Interferon sensitivity of the AGMK cell lines assayed by SV40 T-antigen formation. Secondary cultures of AGMK (01, GC7 (01, Cl4 (0) and CV-1 (A) cells on coverslips, treated with the indicated concentration of monkey interferon, were infected with SV40 at a multiplicity of 5, 5, 15, and 75 PFU/cell, respectively. At 40 hr p.i., T-antigen-positive cells were counted as described in Materials and Methods. The number of positive cells in untreated culture was taken as 100.

AGMK and Cl4 cells. GC7 and CV-1 cells were less sensitive to interferon, but at a concentration of 480 units/ml, the number of T-antigen-positive cells was reduced to less than 1% of the control. G9186 cells, which were most sensitive to interferon for VSV plaque formation, were also most sensitive to inhibition by interferon for synthesis of SV40 T-antigen (Table 1). However, G9186 cells were least susceptible to SV40 infection among these cell lines so that a much higher input multiplicity was required for induction of more than 90% of Tantigen-positive cells in culture. Similar dose-dependent inhibition was observed for synthesis of viral DNA and virally induced cellular DNA when secondary cultures of AGMK cells were pretreated with increasing concentrations of interferon (Fig. 3), suggesting that interferon acts at a very early stage of infection. Interferon-induced inhibition of early SV40 functions was not due to the inability of the virions to penetrate into the nuclei of infected cells, where uncoating is carried out (Barbanti-Brodano et al., 1970). The

62

YAMAMOTO,

YAMAGUCHI TABLE

AND

1

THE FOHMA.IXONOF T ANI) V ANTIGENS m INFECTIOK SV40-DNA Cell

Infecting agent

NumberofT-anligenpositive cells’

ODA

Percent 01 control

IN INTEHFEHOS-TREATED CEI.LS” Numberot’V-antigenpositive cells

Percent of control

+

4 G9186

DNA (0.75 /.~a) DNA (0.075 /.~gl Virion (5 PFU/cell)

286 82 719

215 66 4

75.3 81.0 0.5

265 82 719

173 62 8

65.2 75.1 1.1

GC7

DNA (0.75 fig) DNA (0.075 pg) Virion (5 PFU/cell)

150 87 469

95 53 21

63.3 60.9 4.5

130 94 481

98 56 14

15.4 59.6 2.9

0 Subconfluent cultures of G9186 and GCi cells (ca. 1 x lo5 cellsicoverslip), treated with 320 units/ml of monkey interferon, were infected with the indicated concentration nf SV40-DNA, as described in Materials and Methods. At 36 hr p.i., T and V antigens were stained and the total number of the positive cells in ten different fields at 100 x magnification is given. e +, Interferon treated; ~~, untreated.

FIG. 3. Dose-response curve for the interferoninduced inhibition of early SV40 functions. Secondary cultures of AGMK cells, treated with the indicated concentrations of monkey interferon, were infected with SV40 at 5 PFUicell and labeled for 10 hr with 2.5 rCi/ml of [3H]thymidine (13 Ci/mmol) beginning 18 hr p.i.. Aliquots of labeled DNA extracted were hybridized with excess amounts of cellular (40 rg) and viral (2 pgl DNA as described in Materials and Methods. The radioactivity hybridized with labeled DNA from the infected cells untreated with interferon was taken as 100. In the case of cellular DNA, the radioactivity hybridized with labeled DNA from mock-infected cells was subtracted. T-antigen-positive cells were counted at 24 hr p.i., and the number of T-antigen-positive cells in the infected culture untreated with interferon was taken as 100. -O-, Cellular DNA; -A-, viral DNA; -m-, T-antigenpositive cells.

FIG. 4. Uptake of labeled SV40-DNA into the nuclei of infected cells pretreated with interferon. (a). Secondary cultures of AGMK cells (ca. 2 x 10” cells/F-cm dish), treated with 320 units/ml of monkey interferon, were infected with [3H]thymidine-labeled SV40 at 20 PFU/cell (input. 48,000 cpm). (b). Subconfluent cultures of G9186 cells (ca. 1.5 x lo6 cell&cm dish). treated with 320 units/ml of monkey interferon, were infected with 3H-labeled SV40-DNA (23,000 cpm/l.6 &dish) as described. At the times indicated, the TCA-insoluble radioactivity incorporated into the nuclei was measured as described in Materials and Methods. The values represent an average of duplicate determinations. -O--, Interferon-treated; a-, untreated.

radioactivity incorporated into the nuclei of 3H-labeled SV40-infected cells peaked at about 4 hr p.i. and then decreased thereafter. By pretreatment with interferon, the radioactivity incorporated was decreased 10-E% but was not significantly different from that incorporated into the nuclei of untreated cells (Fig. 4a).

ACTION

OF INTERFERON

Multiplicity-Dependent Leakiness of the Interferon-Induced Inhibition of Early SV40-mRNA Synthesis

multiplicity-dependent leakiness of interferon-induced inhibition of early SV40 functions was observed in nonpermissive cells as will be shown in a later section.

Interferon-induced inhibition of the multiplication of SV40 seems to act at the level of transcription (Oxman and Levine, 1971). To elucidate the mechanism by which transcription is blocked, the effect of multiplicity on synthesis of early SV40-mRNA was first examined in the infected GC7 cells pretreated with 320 units/ml of monkey interferon. As shown in Table 2, the degree of inhibition of early mRNA synthesis depended on the multiplicity of infection. Infection of the cells pretreated with interferon at 4 PFU/cell resulted in a complete inhibition, whereas infection at 100 PFU/cell resulted in synthesis of about 20% of mRNA as compared with the amount of mRNA synthesized in the untreated cells. A similar multiplicity-dependent effect of interferon was observed on the formation of T antigen. Infection of the untreated cells at 100 PFU/cell resulted in the formation of T antigen in all the cells. The proportion of T-antigen-positive cells in culture was slightly reduced to about 80% when infected at 4 PFU/cell (Table 2). These results indicate that the inhibition of the expression of all the known early SV40 functions by interferon is caused by the inhibition of the transcription of all the early regions of SV40-DNA and that this inhibition can be overcome by infecting the cells at a higher multiplicity. A similar

Failure of Complementation of SV40-DNA Replication in SV40-Transformed Monkey Cells To know the state of SV40-DNA in the infected cells pretreated with interferon, a complementation experiment was performed for the replication of SV40-DNA by early SV40 gene products present in the SV40-transformed AGMK cells, clone T22. In T22 cells, 40% of the total SV40-DNA is transcribed, and the early functions necessary to form T antigen and to support growth of human adenoviruses are expressed (Shiroki and Shimojo, 1971; Hashimoto et al., 1973). It has been known that the synthesis of T antigen in SV40-transformed cells is not affected by the presence of interferon (Oxman et al., 1967a). As shown in Fig. 5, synthesis of viral DNA in the infected T22 cells was almost completely inhibited by interferon. Insensitivity of Infectious Interferon

Multiplicity (PFUicell)

INHIBITION

Early SV40-mRNAb (cpm hybridized)

563 536 374

122 22 4

to

2

OF EARLY SV46 mRNA SYNTHESIS BY INTERFERON’

Percent of control

Number of T-antigenpositive cells

i 100 20 4

SV40-DNA

Interferon-treated G9186 cells were infected with infectious SV40-DNA after treatment with DEAE-dextran, and synthesis of viral DNA and mRNA was compared with that in the cells similarly treated but infected with SV40 virions.

TABLE MULTIPLICITY-DEPENDENT

63

ON SV40

Percent of control

+ 21.6 4.1 1.1

812 737 660

362 103 60

44.5 14.1 9.1

u Confluent monolayer cultures of GC7 cells (ca. 8 x l(Y), treated with 320 units/ml of monkey interferon, were infected with SV40 at a multiplicity of 100. 20 or 4 PFU/cell. The cells were incubated in Eagle’s medium containing 25 @g/ml of cytosine arabinoside. At 8 hr p.i., the cells were labeled with 30~Ci/ml of [3H]uridine (20 Ci/mmol) for 8 hr. The labeled RNA extracted was hybridized with 5 pg of SV40-DNA as described. The values represent an average of duplicate determinations. T antigen was stained at 20 hr p.i., and the total number of T-antigen-positive cells in ten different fields at 400 x magnification is given. D +, Interferon treated: -, untreated.

YAMAMOTO.

d

YAMAGI:CHI

I I

T,me

hr

FIG. 5. Synthesis

of viral DNA in interferontreated T22 cells infected with SV40. Subconfluent cultures of T22 cells were treated with either 160 units/ml of monkey interferon or the same amount of mock-interferon proteins (25 &ml). The cells were infected with SV40 at 10 PFU/cell. At the times indicated, the cells were labeled with 2 rCi/ml of [SH]thymidine (13 Ci/mmol) for 2 hr. and aliquots of labeled DNA extracted were hybridized with SV40DNA as described in Fig. 3. -O--, Interferon treated; -0--, mock-interferon treated.

Treatment of the cells with DEAE-dextran reduced the number of T-antigen-positive cells in the virion-infected culture to a somewhat variable degree (to ca. one-tenth of the untreated culture) depending on the respective cell lines. As shown in Fig. 6, synthesis of SV40-DNA was completely inhibited by interferon in the virioninfected cells for 40 hr p.i., whereas in the DNA-infected cells, about 60% of SV40DNA was synthesized in the interferontreated cells as compared with the amount of SV40-DNA synthesized in the untreated cells. Similar insensitivity of infectious SV40-DNA to interferon was observed for synthesis of viral mRNA (Fig. 7). The gradual increase in synthesis of viral DNA observed at 40-50 hr p.i. in the interferontreated cells infected with the virions indicates that the interferon-induced inhibition begins to disappear at this time. This increase, however, was not observed for viral mRNA in repeated experiments.

AND

ODA

FIG. 6. Synthesis of viral DNA in interferontreated G9186 cells infected with infectious SV40DNA. (a). Subconfluent cultures of G9186 cells (ca. 1.5 x lo6 tells/6-cm dish), treated with 320 units/ml of monkey interferon, were infected with infectious SV40-DNA (0.75 rg/dish) as described in Materials and Methods. (b), G9186 cells, similarly treated with interferon and DEAE-dextran, were infected with SV40 virions at 5 PFUicell. At the times indicated, the cells were labeled with 8 pCi/ml of [3H]thymidine (13 Ci/mmol) for 6 hr, and the labeled DNA extracted was hybridized with 2 fig of SV40-DNA as described in Materials and Methods. -O--. Interferon treated; -0--, untreated.

Fro. 7. Synthesis of viral mRNA in interferontreated G9186 cells infected with infectious SV40DNA. G9186 cells were similarly infected with either infectious SV40-DNA (a) or SV40 virions (b) as described in Fig. 6. At the times indicated, the cells were labeled with 20 wCi/ml of [3H]uridine (20 Ci/ mmol) for 6 hr, and the labeled RNA extracted was hybridized with 2 c(g of SV40-DNA as described in Materials and Methods. -O--. Interferon treated; -0--, untreated.

The effect of interferon on the formation of SV40 T- and V-antigens was also compared between the virionand DNAinfected cells. As shown in Table 1, the average number of T- and V-antigen-positive cells in G9186 cell culture infected

ACTION

OF INTERFERON

with the DNA was reduced only to 70-80% of that in control cells by pretreatment with interferon, whereas the number was reduced to 1% of that in control cells when the interferon-treated cells were infected with the virions at 5 PFU/cell. Essentially the same results were obtained with another cell line, GC7. Infection of the cells with SV40-DNA at a 10 times higher concentration resulted in an approximately threefold increase in the number of T-antigen-positive cells in G9186 cell culture and an approximately twofold increase in GC7 cell culture. The percent of the inhibition of T-antigen formation by interferon was, however, not altered by the concentration of DNA used for infection. The penetration of 3H-labeled SV40-DNA into the nuclei of infected G9186 cells peaked at 4-6 hr p.i., irrespective of interferon treatment, but was reduced to 70-75% of the control value by pretreatment of the cells with interferon (Fig. 4b). The decrease in the amounts of SV40-DNA, -mRNA and T-antigen synthesized in the DNA-infected cells by treatment with interferon may therefore be ascribed mostly to the relatively inefficient penetration of the infectious DNA into nuclei or to the relative instability of the incorporated DNA in nuclei. Multiplicity-Dependent terferon-Induced SV40 Functions Cells

65

ON SV40

by treatment of the cells with interferon. No significant stimulation of cellular DNA synthesis was observed in the mockinfected cells. The effect of multiplicity and interferon concentration on the SV40induced synthesis of cellular DNA is shown in Fig. 9. When the cells were infected at 15 PFU/cell (Fig. 9a), pretreatment of the cells with 500 units/ml of interferon resulted in 65% inhibition in synthesis of cellular DNA. There was, however, very little further inhibition with higher doses of interferon. The extent of interferoninduced inhibition of cellular DNA synthesis was decreased linearly by increasing multiplicities of infection (Fig. 9b). Similar results were obtained in terms of the efficiency of T-antigen formation in SV40infected C3H2K-C4 cells. As shown in Table 3A, the inhibition of T-antigen formation was also dependent on multiplicity of infection. The number of T-antigen.,

b

Leakiness of Inof Early Inhibition in Nonpermissive

The dependency of the interferoninduced inhibition of early SV40 functions on the multiplicity of infection was also examined in nonpermissive cells. The rate of SV40-induced synthesis of cellular DNA in the mock interferon-treated C3H2K-C4 cells and in untreated cells reached a maximum at 30-40 and 20-30 hr p.i. when the cells were infected at 15 or 100 PFU/ cell, respectively (Fig. 8). In interferontreated cells, synthesis of cellular DNA was reduced to 50% of the control when the cells were infected at 15 PFU/cell (Fig. 8a) but almost no inhibition was observed in the cells infected at 100 PFU/cell (Fig. 8b). At both multiplicities, the time required for reaching the maximum rate of cellular DNA synthesis was delayed by about 10 hr

FIG. 8. Synthesis of cellular DNA in interferontreated C3H2K-C4 cells infected with SV40. Confluent monolayer cultures of C3H2K-C4 cells, maintained in serum-free Eagle’s medium for 4 days, were treated with either 1000 units/ml of mouse interferon or the same amount of mock-interferon proteins (20 &ml). The cells were infected with SV40 at a multiplicity of either 15 PFU/cell (a) or 100 PFIJ/cell (h) and incubated at 37” in serum-free Eagle’s medium. At the times indicated, the cells were labeled with 1.25 FCi/ml of [3H]thymidine (14.5 Ci/mmol) for 2 hr as described in Materials and Methods. The cells (ca. 5 Y 10’1 were lysed in 1 ml of 0.15 M NaCl, 0.05 M EDTA (pH 8.0). 0.5% SDS and incubated at 37” for 10 min. Aliquots of 0.1 ml were assayed for radioactivity after TCA precipitation -O--, Interferon treated, infected; -A-. mock-interferon treated, infected; -m--, infected; -0--. interferon treated, mock infected: -A-, mock-interferon treated, mock infected; -Cl--. mock infected.

66

YAMAMOTO. TABLE

YAMAGUCHI

AND

11

ODA

1

EFFECT OF INTEHPEIWN COXCESTKATION ANI) MULXPLI~ITY OF INFWTION ON WE FOKM~TION ob SV40

T-ANTIGEN

IN CSH‘JK-C4 (loo0 units/ml)

A. Interferon

Multiplicity (PFUicell)

C~r.r.s”

NumberofT-antigenpositive cells’

Percent of control

.

t

10 30 100

46.6 72.4 129.8

12.0 27.8 87.8

B. Multiplicity Interfemn concentration (units/ml) 0 500 1000 2000

25.7 38.3 67.6

(15 PFlJ/cell)

Number ofT-antigenpositive cells 56.4 45.4 24.7 12.7

Percent of control 100 80.4 43.4 25.4

a Confluent monolayer cultures of C3H2K.C4 cells on coverslips, maintained in serum-free Eagle’s medium for 4 days. were treated with the indicated concentrations of interferon. The cells were then infected with SV40 at the indicated multiplicities. T antigen was stained at 40 hr p.i. The values represent an average number of T-antigen-positive cells counted in five different fields at 200 x magnification. ’ t , Interferon treated; -, untreated.

-

.

.

JO0 1000 2000 Inttxrferon units ml

I I

.

*

.

100 10 30 Sl”lt,~,llr,t~ I’b.1’ ct.11

FIG. 9. Effects of interferon concentration and multiplicity of infection on the induction of cellular DNA synthesis by SV40 in C3H2K-C4 cells (a), Dose response to interferon. Confluent monolayer cultures of C3H2K-C4 cells, treated with the indicated concentrations of mouse interferon, were infected with SV40 at a multiplicity of 15 PFU/cell as described in Fig. 8. (b), Effect of multiplicity. The cells, treated with 1000 units/ml of mouse interferon, were infected with SV40 at the indicated multiplicities. At 40 hr p.i.. the cells were labeled with [3H]thymidine, and aliquots of 0.05 ml of the cell lysates were assayed for radioactivity as described in Fig. 8.

ent in SV40-transformed monkey cells; (d) insensitivity of naked SV40-DNA to function in interferon-treated cells. Interferon-induced inhibition of SV40mRNA synthesis may be caused by either 1) incomplete uncoating, 2) degradation of positive cells in cultures infected at 15 DNA after uncoating or 3) degradation of The latter two PFU/cell decreased linearly with increas- mRNA after transcription. are suggested by the recent ing doses of interferon (Table 3B). The possibilities poor sensitivity of C3H2K-C4 cells to interexperiments of Marcus et al. (1975) who demonstrated that interferon induces the feron and to SV40 infection as compared with monkey cells made it difficult to synthesis of an alkaline RNase, which estimate early mRNA synthesis quantitadegrades viral mRNA. The results of the tively. The formation of T antigen by experiments of the uptake of SV40-DNA infectious SV40-DNA was too inefficient to into nuclei (Fig. 4) indicated that SV40DNA peaks at 4-6 hr p.i. and then declines estimate in nonpermissive cells. both in interferon-treated and untreated DISCUSSION control cells. This parallel increase and The following phenomena observed in decrease in the amount of the TCA-insoluble form of SV40-DNA in the nuclei sugthe present study indicate that interferon of a specific inhibitis multiplication of SV40 at a very gests that the induction DNase by interferon is not the case in the early stage of infection: (a) Complete inhiSV40 system. The induction of a specific bition of SV40-mRNA synthesis by interferon; (b) this inhibition can be overcome RNase is also unlikely since with this by infecting the cells at a higher multiplicmodel it is difficult to explain the differenity; (c) inability of SV40-DNA in the tial response of naked SV40-DNA and infecting virions to be complemented for its intact virions to the action of interferon for replication by the early gene products pres- expression of early viral functions. It may

ACTION OF INTERFERON ON SV40 be possible that, in interferon-treated cells, uncoating of the infecting virions takes place to an extent at which DNase can attack DNA but RNA polymerase is unable to bind to a proper site of DNA for early transcription. However, attempts to detect the uncoating intermediate of SV40 virions in secondary cultures of AGMK cells according to Barbanti-Brodano et al. (1970) were unsuccessful. [3H]thymidine-labeled SV40 at an input multiplicity of about 100 PFU/cell remained in a single band in CsCl density gradient located at a density of 1.33 g/cm3 throughout the early phase of infection. Difficulty in detection of the uncoating intermediate is probably due to either an inefficiency of uncoating of SV40 virions or a rapid association of proteins in the infected cells with the uncoated DNA. It has been recently reported by Howe et al (1975) that a proportion of infecting SV40-DNA replicated after uncoating is only 2% of the total and the remaining unreplicated parental DNA is present as uncoated DNA complexed with proteins present in the infected cells. The following points should be argued in relation to the present results. 1) The feature of inability of SV40 virions to function in interferon-treated cells resembles that of a SV40 temperature-sensitive mutant, SVlOl which cannot be complemented by any other group of mutants at nonpermissive temperature. The naked SVlOl-DNA is not temperature sensitive (Robb and Martin, 1972). Insensitivity of SV40 T-antigen formation by adeno 7-SV40 hybrid viruses to the action of interferon in AGMK cells (Oxman et al., 1967b) has been ascribed to the polycistronic transcription of SV40-mRNA coding for T antigen with adenovirus mRNA whose translation is relativly insensitive to interferon action. However, it is possible that the uncoating step of the hybrid virus is insensitive to interferon action so that the viral genome is able to be converted to an active form for transcription or that the uncoated hybrid virus DNA is rapidly converted to a form which is resistant to the action of DNase. 2) The second step of uncoating of vac-

67

cinia virus, the release of viral DNA from the core, is inhibited by interferon in chick cells (Magee et al., 1968). Vaccinia virus DNA in the core can serve as template for a partial transcription of the early region (Kates and McAuslan, 1967), and the translation product of this mRNA is supposed to act on the uncoating of the core permitting a transcription of the remaining early region. However, the presence of this type of proto-early region in SV40-DNA is unlikely, since the transcription of SV40DNA was almost completely inhibited by interferon when interferon-treated monkey cells were infected with SV40 at a relatively low multiplicity (Table 2). 3) A dependency of the leakiness of interferon-induced inhibition of early SV40 functions on the input multiplicity is evident, since this phenomenon was observed over the input multiplicity of 4 PFU/cell at which almost all the cells exhibited T antigen and 60-70% of the maximal level of mRNA was synthesized (Table 2). A quantitative estimation of the inhibition of T-antigen formation was difficult to perform at the lower range of multiplicities, since a proportion of the T-antigen-positive cells was decreased to less than 5% by interferon treatment even in the relatively interferon-insensitive cell line, GC7 (Fig. 2). There may exist a limit to the capacity of interferon action that induces the alteration in the cellular system so as to inhibit the infecting virions to function. With increasing input multiplicities, a proportion of the infecting virions which escapes from this inhibition may therefore be increased. The capacity of interferon expressed in cells may be a particular property of the species of cell and of an individual cell line. In mouse cells, clone C3H2KC4, a much higher concentration of interferon and a lower input multiplicity were required for effective inhibition of early SV40 functions as compared with monkey cells. When the cells were infected with SV40 at 100 PFU/cell, pretreatment of the cells with 1000 units/ml of interferon failed to inhibit virally induced synthesis of cellular DNA (Figs. 8 and 9). Todaro and Green (1967) also failed to show the inhibition of SV40-induced cellular DNA synthe-

68

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sis by interferon in 3T3 cells under similar conditions. The multiplicity-dependent reversibility of interferon-induced inhibition of virus multiplication has been reported in VSVinfected chick cells (Ho, 1962). Our results coincide with this observation but are contradictory to those of Hallum and Youngner (1966) who showed that a given concentration of interferon reduces the yield of progeny virus in VSV-infected chick cells regardless of the size of the challenge dose. This discrepancy, however, could be explained by the difference in the range of input multiplicities employed in the experiments. Hallum and Youngner used input multiplicities ranging from 12 to 1.2 x lOme PFU/cell, while we used from 100 to 4 PFU/cell. 4) The synthesis of SV40-DNA and -mRNA and the formation of T and V antigens by infectious SV40-DNA were much less inhibited by interferon. The 50-20% decrease in the amounts of these macromolecules synthesized could be explained by an ineffective penetration of infectious DNA into the nuclei or by an instability of the incorporated DNA in the nuclei of interferon-treated cells (Fig. 4b). Under the same conditions, whole virions did not induce these functions. Inability of interferon to block the function of infectious SV40-DNA in monkey cells cannot be ascribed to the high multiplicity of infection, since the number of T-antigen-positive cells in the DNA-infected G9186 cells (0.075 pg/coverslip) was about 10% of that in the virion-infected cells at 5 PFU/cell (Table 3). Graessmann et al. (1974) recently reported that T-antigen formation in monkey cells injected with either SV40-DNA or complementary RNA was inhibited by human interferon and suggested that interferon inhibits primarily the translation of SV40-mRNA. The results are divergent from those of Oxman and Levine (1971) and of ours. A reason for this difference is presently obscure; however, under the conditions they used, the synthesis of early SV40-mRNA by the infecting virions was reduced to 20% of the control.

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ODA

5) Two distinct sites of interferon action have been suggested. One is an alteration of the protein synthesizing system such that ribosomes in interferon-treated cells are selectively inhibited in their ability to translate viral mRNA and the other is inhibition of transcription of viral genome (references are cited in the Introduction). The present results confirmed that the latter site is also the target of interferon action. Insensitivity of infectious SV40DNA to interferon for the formation of both T and V antigens suggests that translation of both early and late SV40-mRNA is not inhibited by interferon. In contrast to our results, the replication of infectious poliovirus RNA is inhibited by interferon (Ho, 1961; Grossberg and Holland, 1962; Hallum and Youngner, 1966). Interferon may induce the alteration in the cellular system at multiple sites, and the site at which viral multiplication is blocked may differ among different species of cells and challenge viruses. It is important to answer the questions whether the multiplication of interferon-sensitive RNA viruses is inhibited at the level of translation in monkey cells and whether SV40-mRNA can be translated by in vitro protein synthesizing system prepared from interferon-treated monkey cells. ACKNOWLEDGMEXTS This work was supported by research grants from the Ministry of Education of ,Japan and by a research grar from the Mitsubishi Foundation to H. Shimojo. REFEREXCES BARBANTI-BROUANO, G., SWF.TI.Y, P., and KOPROWKI. H. (1970). Early events in the infection of permissive cells with simian virus 40: Adsorption, penetration, and uncoating, J. Viral. 6, ‘iA-86. BIALY, H. S., and COI,BY, C. (1972). Inhibition of early vaccinia virus ribonucleic acid synthesis in interferon-treated chicken embryo fibroblasts. J. Viral. 9, 286-289. BORUN, T. W;.. SHARFF, M. D., and ROBBINS, E. ( 1967). Preparation of mammalian polyribosomes with the detergent Nonidet P-40. Biochim. Biophys. Acta 149, 302-304. DENHARDT, D. T. (1966) A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646.

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OF INTERFERON

FALCOFF, E., FALCOFF, R., LEBLEU, B., and REVEL, M. (1973). Correlation between the antiviral effect of interferon treatment and the inhibition of in vitro mRNA translation in noninfected L cells. J. Viral. 12, 421-430. FRIEDMAN, R. M., METZ, D. H., ESTEBAN, R. M., TOVEI.L, I>. R., BALL, I,. A., and KERR, I. M. (1972). Mechanism of interferon action: Inhibition of viral messenger ribonucleic acid translation in L-cell extracts. J. Viral. 10, 1184-1198. GILLESPIE, D., and SPIEGELMAN, S. (1965). A quantitative assay for DNA-RNA hybrids with DNA immoblized on a membrane. J. Mol. Biol. 12,829-842. GRAESSMANN, A., GRAESSMANN, M., HOFFMANN, H., NIEBEL, J., BRANDNER, G., and MUELLER, N. (1974). Inhibition by interferon of SV40 tumor antigen formation in cells injected with SV40 cRNA transcribed in uitro. FEBS Lett. 39, 2499251. GROSSBERG,S. E., and HOLLAND, J. J. (1962). Interferon and viral ribonucleic acid. Effect on virus-susceptible and insusceptible cells. J. Immunol. 88, 708-714. HALLUM, J. V., and YOUNGNER, J. S. (1966). Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacteriol. 92, 104771050. HASHIMOTO, K., NAKAJIMA, K., ODA, K., and SHIMOJO, H. (1973). Complementation of translational defect for growth of human adenovirus type-2 in simian cells by a simian virus 40 induced factor. J. Mol. Biol. 81, 2077223. Ho, M. (1961). Inhibition of the infectivity of polivirus ribonucleic acid by an interferon. Proc. Sot. Exp. Biol. Med. 107, 639-644. Ho, M. (1962). Kinetic considerations of the inhibitory action of an interferon produced in chick cultures infected with Sindbis virus. Virology 17, 262-275. HOWE, C. C., TAN, K. B., and SOKOL, F. (1975). Fate of parental simian virus 40 DNA in permissive monkey kidney cells. J. Gen. Viral. 27, 11-24. IIDA, H., and ODA, K. (1975). Stimulation of nonhistone chromosomal protein synthesis in SV40infected simian cells. J. Viral. 15, 471-478. JOKLIK, W. K., and MERIGAN, T. C. (1966). Concerning the mechanism of action of interferon. hoc.

Nat. Acad. Sci. USA 56, 558-565. KATES, J. R., and MCAUSLAN, B. R. (1967). Messenger RNA synthesis by a “coated” viral genome. Proc. Nat. Acad. Sci. USA 57, 314-320. KELLY, T.. and ROSE, J. (1971). Simian virus 40 integration site in an adenovirus 7-SV40 hybrid DNA molecule. Proc. Nat. Acad. Sci. USA 68, 1037-1041. KERR, I. M. (1971). Protein synthesis in cell-free systems: An effect of interferon. J. Viral. 7, 448-459. LEVY, H. B.. and CARTER, W. A. (1968). Molecular

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basis of the action of interferon. J. Mol. Biol. 31, 561-577. MAGEE, W. E., LEVINE, S., MILLER, 0. V., and HAMILTON, R. D. (1968). Inhibition of interferon of the uncoating of vaccinia virus. Virology 35, 505-511. MANDERS, E. K., TILLES, J. G., and HUANG, A. S. (1972). Interferon-mediated inhibition of viriondirected transcription. Virology 49, 573-581. MARCUS, P. I., and SALB, J. M. (1966). Molecular basis of interferon action: Inhibition of viral RNA translation. Virology 30, 5022516. MARCUS, P. I., ENGELHARDT, D. L., HUNT, J. M., and SEKELLICK, M. J. (1971). Interferon action: Inhibition of vesicular stomatitis virus RNA synthesis induced by virion-bound polymerase. Science 174, 593-598. MARCUS, P. I., TERRY, T. M., and LEVINE, S. (1975). Interferon action. II. Membrane-bound alkaline ribonuclease activity in chick embryo cells manifesting interferon-mediated interference. Proc. Nat. Acad. Sci. USA 72, 182-186. MCCUTCHAN, J. H., and PAGANO, J. S. (1968). Enchancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminoethyl-dextran. J. Nat. Cancer Inst. 41, 351-357. MERIGAN, T. C., WINGET, C. A., and DIXON, C. B. (1965). Purification and characterization of vertebrate interferons. J. Mol. Biol. 13, 679-691. OXMAN, M. N., BARON, S., BLACK, P. H., TAKEMOTO, K. K., HABEL, K., and ROWE, W. P. (1967a). The effect of interferon on SV40 T antigen production in SV40-transformed cells. Virology 32, 122-127. OXMAN, M. N., ROWE, W. P., and BLACK, P. H. (1967b). Studies of adenovirus-SV40 hybrid viruses. VI. Differential effects of interferon on SV40 and adenovirus T antigen formation in cells infected with SV40 virus, adenovirus, and adenovirussSV40 hybrid viruses. Proc. Nat. Acad. Sci.

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Sci. USA 68, 299-302. PAUCKER, K., BERMAN, B. J., GOLGHER, R. R., and STANCEK, D. (1970). Purification, characterization, and attempts at isotopic labeling of mouse interferon. J. Viral. 5, 145152. PENMAN, S. (1966). RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17, 117-130. ROBB, J. A., and MARTIN, R. G. (1972). Genetic analysis of simian virus 40. III. Characterization of a temperature-sensitive mutant blocked at an early stage of productive infection in monkey cells. J.

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N.. and KUCHINO, T. (19751. Temperature-sensitive mutants of simian virus 10 selected by transforming ability. J. Viral. 15, 129;-1:101. YOSHIKIJRA, H.. and HIROKAWA,.Y. (1968). Induction of cell replication. Exp. Cell Res. 52, 4:W441. YAMAGUCHI,