Inhibition of Sindbis Virus Replication in HeLa Cells by

ANTIMICROBIAL AGENTS AND CHEMOrHERAPY, Jan. 1974, p. 55-62 Copyright 0 1974 American Society for Microbiology Vol. 5, No. 1 Printed in U.S.A. Inhibition of Sindbis Virus Replication in HeLa Cells by Poliovirus T. SREEVALSAN AND H. ROSEMOND-HORNBEAK' Department of Microbiology, Georgetown University Medical and Dental Schools, Washington, D.C. 20007 Received for publication 7. September 1973 Concomitant infection of HeLa cells with poliovirus and Sindbis (SB) virus allowed replication of poliovirus only. The poliovirus-induced interference with the replication of SB virus was dependent partially on the multiplicity of SB virus used for infection. The observed interference was not sensitive to guanidine. Translation, but not replication of poliovirus, appears to be needed for the restriction of SB virus. Superinfection of SB virus-infected cultures with poliovirus was followed by inhibition of protein, but not by viral ribonucleic acid (RNA) synthesis. Polyribosomes present in SB virus-infected cells were disaggregated subsequent to superinfection. The nascent SB viral RNAs synthesized in cells subsequent to superinfection were not associated with the ribosomal structures. These results indicate that the block in protein synthesis, possibly at the level of initiation, may be the basis of the observed interference. Infection of HeLa cells with poliovirus leads to a rapid restriction of cellular ribonucleic acid (RNA) and protein synthesis (1, 10, 23). The inhibition of cellular protein synthesis induced by the virus appears to be specific since translation of RNA from poliovirus or other enteroviruses can occur in the cells (1, 10, 14, 23). The restriction of translation of messenger RNAs other than poliovirus RNA appears to be the rule, because in cell cultures infected with herpes simplex virus (HSV), Newcastle disease virus (NDV), or vesicular stomatitis virus (VSV), virus-specific RNAs are not translated subsequent to superinfection with poliovirus (6, 12, 19). The only exception to the aforementioned dominance of poliovirus over other viral infections is the report that production of the paramyxovirus SV5 in monkey kidney cells was unaffected by superinfection with poliovirus (4). Apparently poliovirus did not restrict the translation of SV5-specific RNA. At present it is not known whether poliovirus can interfere with the multiplication of arboviruses. Arboviruses resemble picornaviruses in some respects. Virions of both groups contain single-stranded RNA which is infectious. The mode of translation of viral RNA of both groups of viruses appears to be similar, viz., the individual viral polypeptides appear to be formed in vivo by the cleavage of a large polypeptide (1, 20-22). Therefore we studied the virus-virus interaction 'Present address: Laboratory of Biology of Viruses, National Institutes of Health, Bethesda, Md. 20014. in cells dually infected with polio and Sindbis (SB) viruses. A strain of HeLa cells which can support the multiplication of both poliovirus and SB virus was used. The results show that poliovirus interfered with the multiplication of SB virus in doubly infected cells. Possible mechanisms for this interference are discussed. MATERIALS AND METHODS Cells. The strain of HeLa cells used and conditions for their growth have been described earlier (18). Primary chicken embryo (CE) cells were prepared and used for assaying the infectivity of SB virus according to methods described previously (18). Viruses. Guanidine-sensitive poliovirus type I was used throughout the study. Stocks were prepared by infecting confluent monolayers of HeLa cells at a multiplicity of infection (MOI) of 0.10 and incubating them for 24 h at 37 C. Then the infected cultures were harvested, frozen, and thawed three times. Cellular debris was removed by low-speed centrifugation. The supernatant fluid was assayed for infectious virus by the method of Holland and McLaren (9). The infectivity of the supernatant fluid varied from 5 to 10 x 108 plaque-forming units (PFU)/ml. The virus stocks were stored frozen at -40 C until used. Stock preparations of SB virus (HR strain) were prepared by infecting confluent monolayers of CE cells at a MOI of 0.10. The cultures were harvested at the end of a 24-h incubation at 37 C. Cellular debris was removed, and the supernatant fluid was assayed for infectivity according to methods described earlier (18) and usually contained 5 to 8 x 109 PFU/ml of SB virus. The virus stocks were kept frozen at -40 C until used. Dual infection. HeLa cell monolayers in 60-mm 55

56 SREEVALSAN AND ROSEMOND-HORNBEAK plastic petri dishes (Nunc) were prepared by seeding 1 x 106 cells/plate in Eagle minimum essential medium (MEM) containing 10% fetal calf serum. The cultures were incubated for 24 h at 37 C in a 5% CO,-humidified atmosphere before use. Cultures (2 x 101 cells/ plate) were infected singly or dually with the viruses at the multiplicities indicated in the experiment. Virus adsorption was allowed to take place for 1 h, and then the cultures were washed five times with phosphate-buffered saline (PBS). They were then incubated at 37 C with MEM containing 3% fetal calf serum. Duplicate cultures were harvested at the various times indicated and frozen at -40 C. Virus was released from the infected cells by three cycles of freezing and thawing. Because poliovirus is unable to form plaques in CE cells, SB virus contained in dually infected cultures was assayed selectively by using CE cells. Poliovirus present in dually infected cultures was assayed as follows. The efficiency of plaque formation of SB virus in HeLa cells was only 5% of that in CE cells. Therefore samples of culture fluids from dually infected cultures were neutralized for 1 h at 37 C with 0.1 % the volume of an antiserum against SB virus and then were used for assay of poliovirus in HeLa cells according to the method of Holland and McLaren (9). Ultraviolet irradiation of poliovirus. Three milliliters of the stock virus was placed in a petri dish (100 mm) and exposed at a distance of 10 cm to 17,W of ultraviolet irradiation source (Gates Raymaster) per cm2 for 10 min. This permitted the survival of only 0.1% of the original infectivity in the irradiated samples. Chemicals and isotopes. Actinomycin D was obtained as a gift from Merck, Sharp and Dohme (West Point, Pa.) and used at a concentration of 5 Ag/ml to inhibit synthesis of cellular RNA. Guanidine hydrochloride (2 x crystallized) was purchased from Eastman Kodak Co. and used at a concentration of 2 mm. Isotopically labeled amino acids and uridine were purchased from Nuclear-Chicago Corp. (Des Plaines, Ill.). The specific activities of tritiated leucine and uridine were >20,000 and >30,000 mci/mmol, respectively. ["4C]uridine (specific activity >50 mci/ mmol) was purchased from New England Nuclear Corp. (Boston, Mass.). RNA and protein synthesis. Total protein or RNA synthesized in infected cultures was determined as follows. Cell cultures were washed three times with warm PBS and incubated for 10 min with 1 ml of MEM containing either [3H ]leucine (25 ACi) or ['H]uridine (10 XCi). After a washing with chilled PBS, the cells were lysed by adding 0.4% sodium dodecylsulfate (SDS) in distilled water. Portions of the sample were incubated with cold 10% trichloroacetic acid for 1 h. Samples containing labeled uridine were immediately processed by filtration on membranes by methods described earlier (18). Samples containing labeled proteins were incubated in a boiling water bath for 10 min before they were filtered on membranes. Radioactivity present in the membrane was assayed in a Packard scintillation counter. The composition of the scintillation fluid as well as the details of counting samples containing mixtures of 'H and '4C isotopes have been described in detail earlier (18). Preparation of cytoplasmic extract and centrifugation on sucrose density gradients. Methods used for isolation of cytoplasmic extracts and the conditions used for centrifugation on a sucrose density gradient have been described previously (18). Isolation and analysis of viral RNA. Details concerning the techniques used for the isolation of viral RNAs and subsequent electrophoretic analysis have been described elsewhere (18). The composition of the polyacrylamide gel used for electrophoresis was similar to that described earlier (18). RESULTS Multiplication of SB and poliovirus in dually infected HeLa cells. Preliminary experiments indicated that superinfection of SB virus-infected HeLa cells with poliovirus reduced the yield of SB virus without any reduction in the yield of poliovirus. To determine whether the above interference was dependent on the MOI of the infecting viruses, the following experiment was done. HeLa cell cultures were dually infected with poliovirus and SB virus by using different MOI (Table 1). The virus yields from the doubly infected cultures at 24 h postinfection were determined. Cell cultures infected with polio- TABLE 1. Effect of multiplicity of infection on poliovirus-induced interference of sindbis virus replicationa Multiplicity of viruses used for infecting cells ANTIMICROB. AG. CHEMOTHER. Yield of virus in 24 h (PFU/cell) 200 1,245 200 10 1,200 <1.0 1 1,335 1 10 750 60 200 600 10 200 1,200 114 1 750 10 1 1,175 42 a HeLa cell cultures containing 1 x 106 cells were infected singly or dually with Sindbis (SB) and polioviruses. The virus inoculum, 0.5 ml/culture in phosphate-buffered saline (PBS), consisted of varying concentrations of SB virus or poliovirus or a mixture of both viruses. Adsorption of virus to cultures was allowed to occur at 37 C for 1 h, at the end of which they were washed with PBS and incubated with minimal essential medium for 24 h. The virus yields in cultures at the end of 24 h of incubation were determined. Multiplicity of infection (MOI), as used here and in the following experiments, is calculated on the basis of the amount of plaque-forming units (PFU)/cell added for infection. So the values represent only the apparent MOI.

VOL. 5, 1974 virus or SB virus alone served as controls. The results (Table 1) suggest that the interference induced by poliovirus on SB virus replication was partially dependent on the MOI of SB virus used for infection. Thus when cells were infected at a MOI ratio of 20 poliovirus-1.0 SB virus, there was little replication of SB virus. However, when the MOI of SB virus used was increased (1 poliovirus-20 SB virus), there was partial replication of SB virus. There was only a twofold decrease over controls in the replication of polio by Sindbis virus under the above conditions. Thus, poliovirus interferes with the replication of SB virus in HeLa cells. The above interference can be partially overcome by increasing the multiplicity of SB virus. SB virus was unable to interfere with replication of polio to the same extent as poliovirus could on SB virus replication. Requirement for poliovirus-induced interference with SB virus replication. It is known that the interference induced by poliovirus on the replication of viruses like VSV, NDV, or HSV is not dependent on the active replication of poliovirus in the dually infected cells (6, 12, 19). Therefore, the following experiment was done to determine whether replication of poliovirus was necessary for the inhibition of SB virus. Cells were infected with SB virus (MOI of 10) and polioviruses (MOI of 200) under the conditions described in Table 1, except for the incorporation of 2 mm guanidine to the dually as well as singly infected cultures. Guanidine was used in the MEM to inhibit the replication of poliovirus. Also, cells were double infected with SB virus (MOI of 10) and ultraviolet-irradiated poliovirus (MOI of 200 PFU/ ml before irradiation). The 24-h yield of virus in the culture was determined. The results indicate that the interference induced by poliovirus can occur in the absence of its active replication (Table 2). However, it appears that expression of the poliovirus genome may be necessary for interference, because irradiated poliovirus was unable to interfere with the replication of SB virus. The degree of inhibition of SB virus replication was dependent on the time at which infected cultures were superinfected with poliovirus. Figure 1 compares the percent virus yields obtained at 24 h in cultures infected with SB virus alone (MOI of 10) and with those superinfected with poliovirus (MOI of 200) at various intervals after the initial infection. There was no replication of poliovirus (data not shown), because guanidine was used throughout the experiment. Superinfection during the 1st 6 h after SB virus infection reduced the final yield INHIBITION OF SINDBIS VIRUS REPLICATION 57 TABLE 2. Requirements for poliovirus-induced interferencea Conditions of infection Virus yield (PFU/cell) Polio SB Poliovirus alone with MEM 1,150 Poliovirus with 2 mm guanidine < 1.0 Poliovirus + SB virus with 2 mm guanidine... < 1.0 < 1.0 SB virus alone with MEM 650 SB virus with 2 mm guanidine 635 Irradiated polio virus.with MEM < 1.0 Irradiated polio virus + SB virus with MEM.<.<1.0 500 SB virus alone with MEM 625 'Cultures of HeLa cells (1 x 106/culture) were incubated with 0.5 ml of phosphate-buffered saline containing 1 x 107 plaque-forming units (PFU) of Sindbis (SB) virus or 2 x 108 PFU of polioviruses. The inocula for cultures to be doubly infected contained both viruses at the above concentration. Wherever applicable, guanidine (2 mm) was added during adsorption of the virus to cells. The multiplicity of infection of irradiated poliovirus is based on the PFU/ml present before irradiation. The cultures were handled by methods similar to those described in Table 1 except for incubation at 37 C for 24 h. The minimal essential medium (MEM) with or without 2 mm guanidine was used wherever necessary. so go. 20 2 4 6 s 10 12 Tm hr.of chall.gew. PoS wiru6 FIG. 1. Growth of Sindbis (SB) virus in dually infected HeLa cells. Cultures were infected with SB virus at a multiplicity of infection (MOI) of 10 and incubated with minimal essential medium containing 2 mm guanidine. At indicated times, the duplicate cultures were superinfected with poliovirus at a MOI of 200. The 24-h yields of SB virus in SB-infected and the doubly infected cultures were determined. The virus yields in the doubly infected cultures are expressed as the percentage of virus produced in cultures infected with SB virus alone. The yield of virus in SB virus-infected cultures was 750 plaque-forming units per cell.

58 SREEVALSAN AND ROSEMOND-HORNBEAK of SB virus considerably. However, superinfection at the 8th or the 12th h resulted in only partial inhibition of SB virus synthesis. These results indicated that events occurring in HeLa cells during the 1st 6 h after infection with SB virus are affected by superinfection with poliovirus. Synthesis of virus-specific RNAs and proteins is initiated in HeLa cells 2 h after addition of SB virus to cultures and continues to be synthesized for 6 to 8 h. (T. Sreevalsan, unpublished data). Therefore, the synthesis of viral RNAs and proteins was examined in cell cultures infected with SB virus and superinfected 4 h later with poliovirus. The conditions of infection were similar to those used in the previous experiment, except that all cultures were preincubated with actinomycin D before the start of the infection. Uninfected cultures or cultures infected singly with polio or SB virus served as controls. The amount of [3H Juridine incorporated by dually infected cultures was similar to that obtained in control SB virus-infected cultures (Fig. 2A). Superinfection of infected cultures with poliovirus at the 4th h after the initial infection did not affect the synthesis of SB virus-specific RNA, because it continued to be synthesized for at least 3.5 h in the dually infected cells. Unlike the results obtained on RNA synthesis, there was a dramatic decrease in the rate of incorporation of [3H]leucine by dually infected cells (Fig. 2B). There was a ten-fold reduction in the rate of incorporation of radioactive leucine by dually infected cells over the control cultures by 3.5 h after superinfection. Thus, protein synthesis was drastically reduced in the cultures subsequent to superinfection with poliovirus, although viral RNA synthesis was unaffected. These experiments were repeated several times with results similar to those presented here. The observed reduction in the synthesis of proteins in dually infected cells may be due to several reasons. Some of the possibilities are that: (i) poliovirus selectively inhibits the synthesis of the SB virus-specific messenger RNAs; (ii) the synthesis of certain specific SB virus polypeptides is inhibited in dually infected cells; or (iii) there is a general inhibition in the synthesis of SB viral polypeptides. The following experiment was done to test the first possibility. The plan of experiment was similar to that used in the previous experiment. Guanidine and actinomycin D were used as before. Cultures infected with SB virus alone or those dually infected were incubated with [3H ]uridine 94 WIr JI 1Cs ' 20 la5 ANTIMICROB. AG. CHEMOTHER. -I I F>ZA VA I- -l yzi A 30 -ioli0120 15 210 OTim.0-"*,bh PW W * l FIG. 2. Ribonucleic acid and protein synthesis in dually infected HeLa cells. HeLa cell cultures were incubated with actinomycin D for 1 h at the end of which time they were infected with Sindbis (SB) virus at a multiplicity of infection (MOI) of 10. Some of the cultures received phosphate-buffered saline (PBS) instead of SB virus and served as uninfected cultures. At the end of 1 h at 37 C all cultures were washed and incubated at 37 C with minimal essential medium (MEM) plus 2 mm guanidine. Four hours later some of the uninfected and SB-infected cultures were incubated for 1 h at 37 C with poliovirus (MOI of 200) in the presence of 2 mm guanidine. The cultures were then washed with PBS and incubated at 37 C with MEM containing 2 mm guanidine. Duplicate cultures were incubated with 20 /Ci of [8H]uridine or 10 uci of [3HJleucine per culture for 10 min at the indicated intervals. The acid-insoluble radioactivity per culture was determined. A, [3Hluridine incorporated. B, [3H]leucine incorporated. O, Cultures infected with SB virus only. The values for each time interval were obtained after subtraction of the radioactivity present in uninfected cultures in the presence of actinomycin. 0, Dually infected cultures. Appropriate values of radioactivity incorporated in poliovirus-infected cultures were substracted from similar values obtained from dually infected cultures. for 30 min. Cultures infected with poliovirus were labeled with ["C luridine for 30 min. The [3H ]uridine-labeled RNAs were co-electrophoresed with [4C ]uridine-labeled RNA from poliovirus-infected cells. The results are presented in Fig. 3. The nature of the various forms of SB viral RNAs appearing on polyacrylamide gels have been described recently (18). There was little difference in the types or amounts of viral RNAs appearing in singly or dually infected cultures. Recently it has been reported that the 26S and 16 to 18S form of viral RNAs function as messenger in arbovirus-infected

VOL. 5, 1974 INHIBITION OF SINDBIS VIRUS REPLICATION 59 C" 42S 26S 2 12 RF 10 42S 41- z21 ORI"I RF 26S 16-ISS ~~~~~~~~16-BSS 10 4 5 7 FRACTIONS FIG. 3. Viral ribonucleic acid (RNA) synthesized in dually infected HeLa cells. Cultures were infected with Sindbis (SB) virus and superinfected with poliovirus under conditions identical to those described in Fig. 2. Both singly infected and doubly infected cells were labeled with [3H]uridine (20 MCi/culture) at 2 h after superinfection. Cultures infected with poliovirus and incubated with guanidine received [14C]uridine (10 gci/culture) for 1 h at 2 h after superinfection. The [3H] or ["4C]uridine-labeled viral RNAs were isolated, and samples were analyzed by electrophoresis on 2.2%1o polyacrylamide gels by using 6 ma/gel for 4 h. The bottom panel (SB alone) represents a mixture of [3H]uridine-labeled viral RNA from SB virus-infected cells and ["4C]uridine-iabeled RNA from poliovirus-infected cells. The upper panel (SB plus polio) represents a mixture of [3Hjuridinelabeled RNA from dually infected cells and [14C]uridine-labeled RNA from poliovirus-infected cells. 0, [8H]uridine; 0, ["4C uridine. The captions RF, 42S and 26S in the figures represent the positions where the replicative form, 42S, and 26S RNA from SB virus infected cells appear in polyacrylamide gels under identical conditions (18). cells (13, 16, 18). Specifically, the synthesis of SB virus-specific messenger RNAs, viz., the 26S and the 16 to 18S RNA (fraction 45 and 64 in bottom panel, Fig. 3), were not significantly affected subsequent to superinfection with poliovirus. It may be noted here that cultures infected with poliovirus and incubated with guanidine synthesized a species of RNA which possessed mobility slightly higher than that of the SB virus replicative form. Although the total labeled RNA synthesized in the presence of guanidine in poliovirus-infected cultures represented only 0.2% of that in control cultures, the majority of the guanidine-resistant RNA was ribonuclease resistant (data not shown). Recently it has been reported that ribonucleaseresistant poliovirus replicative form is the only species of viral RNA accumulating to any appreciable extent in HeLa cell cultures infected with poliovirus in the presence of guanidine (2, 17). The present results indicate that the electrophoretic behavior of viral RNA species appearing in poliovirus-infected cultures in the presence of guanidine was similar to those reported by Noble and Levintow (17). So it is possible that the poliovirus-specific RNA appearing in guanidine-treated cultures is the virus-specific replicative form. The types of SB virus-specific polypeptides synthesized in singly or dually infected cells in the presence of guanidine were analyzed by electrophoresis on polyacrylamide gels. The results (data not shown) indicated that although the amount of proteins synthesized in dually infected cells was drastically reduced, there was little difference in the types of SB virus polypeptides synthesized in control or interfered cultures. Thus, it appears that the poliovirus-induced interference with SB virus synthesis is due to a general inhibition of protein synthesis. The inhibition of protein synthesis in dually infected cultures could result in interference with the attachment of SB v virus-specific messenger RNA to ribosomes or disaggregation of virus-specific polyribosomes. The following experiment was performed to determine such possibilities. Polyribosomes and ribosomes were labeled with [3H ]uridine before infection with SB virus. The sedimentation patterns of labeled polyribosomes and ribosomes in cells infected with SB virus alone or superinfected with poliovirus were determined. Additionally, some of the singly and dually infected cultures were pulse ["C]uridine to label the nascent labeled with viral RNA synthesized. Actinomycin and guanidine were used in the same fashion as in previous experiments. The sedimentation patterns of the radioactivity contained in prelabeled polyribosomes in singly or doubly infected cells are shown in Fig. 4. Comparison of the sedimentation patterns indicates that disaggregation of polyribosomes is evident in the dually

60 SREEVALSAN AND ROSEMOND-HORNBEAK K fs / 0.AS 45l 74S 745 30 ' 10 J I BOT 10 20 30 TOP BOT 10 20 30 TOP FRACTIONS FRACTIONS FIG. 4. Sedimentation pattern of polyribosomes after velocity sedimentation of cytoplasmic extracts. HeLa cells (4 x 106/culture) were incubated with minimal essential medium (MEM) containing [1H]uridine (5 ACi/culture) for 24 h. The cultures were then washed, incubated with MEM for 24 h, and incubated with actinomycin D (5 pg/ml) for 1 hr. Then the cultures were infected with Sindbis virus (MOI of 10) and incubated with MEM containing 2 mm guanidine. Four hours later some of the cultures were superinfected with poliovirus for 1 h at 37 C (MOI of 200) and incubated with MEM containing 2 MmMguanidine. The rest of the singly infected cultures serving as controls were also handled in fashions similar to those used for superinfection by using phosphate-buffered saline instead of poliovirus. Cytoplasmic extracts were prepared from the control and superinfected cultures at the end of 90 and 180 min after superinfection. The cytoplasmic extracts were centrifuged on 15 to 30%o sucrose density gradients prepared in a buffer containing 0.01 M tris(hydroxymethyl)aminomethane, 0.01 M NaCl, and 0.0015 M MgCl,, ph 7.4, at 24,000 rpm for 2 h at 4 C by using a 27.3 Spinco rotor. A and B, Cytoplasmic extracts at 90 and 180 min, respectively, from control cultures. C and D, Cytoplasmic extracts at 90 and 180 min, respectively, from superinfected cultures. infected cells within 1.5 h after superinfection with poliovirus, and by 3 h very few polyribosomes can be detected in the superinfected cultures. The rapidly sedimenting structures seen in Fig. 4A and B represent polyribosomes, because incubation of cytoplasmic extracts with RNase (1 pg/ml) before centrifugation resulted in a 80 to 90% loss of such structures (data not shown). Thus, superinfection of SB virusinfected cultures with poliovirus brings about a disaggregation of polyribosomes. Figure 5 shows the fate of ["4C Juridinelabeled viral RNAs synthesized in singly or doubly infected cultures. The time of centrifugation of the sucrose density gradients was long enough to separate the ribosomes and ribosomal subunits. The distribution of [1C ]uridine, representing viral RNAs in the SB virusinfected cells, shows the presence of viral RNA sedimenting with the ribosomes and ribosomal subunits. However, in the superinfected cultures very little of the [4C ]-labeled RNA appears to sediment with ribosomes or ribosomal units, a finding similar to that observed in culture infected with poliovirus in the presence of guanidine. The absence of labeled viral RNA associated with ribosomes and ribosomal subunits in cells infected with poliovirus was expected, because guanidine prevents a majority of viral biosynthetic activities (1, 3, 11). We have shown recently that in SB virus-infected cells the radioactive viral RNA cosedimenting with ribosomes and ribosomal units are true messenger RNA-ribosome complexes (18). Therefore, the apparent association of viral a i 2 ANTIMICROB. AG. CHEMOTHER. C OT TP WT TOP W TM FrmT" FUAU FrA FIG. 5. Sedimentation pattern of ribosomal structures from cytoplasmic extracts. The details of the experiment were identical to those described under Fig. 4, except that the HeLa cell cultures were labeled with ["4C]uridine for 30 min at 90 min after superinfection. An additional control consisting of HeLa cells infected with poliovirus alone in the presence of guanidine was also used. Cytoplasmic extracts were prepared and analyzed by centrifugation on sucrose density gradients in a manner similar to that described in Fig. 4, except that the gradients were centrifuged at 20,000 rpm for 14 h at 4 C. A, Sindbis (SB) virus-infected cells. B, SB-infected cultures superinfected with poliovirus. C, Poliovirus-infected cultures. 0, 'H; 0, 14C.

VOL. 5, 1974 INHIBITION OF SINDBIS VIRUS REPLICATION 61 RNAs with ribosomal subunits observed in Fig. 5A is not an artifact. It seems that in dually infected cultures the nascent viral RNAs do not appear to be associated with the ribosomal structures. DISCUSSION This study shows that poliovirus completely restricts the replication of SB virus in dually infected cultures. The poliovirus-induced interference can be partially overcome by increasing the MOI of SB virus. SB virus did not exert any interference with the replication of poliovirus. The instance of interference studied here did not differ from those studied by others concerning the dominance of poliovirus in cells infected with other animal RNA viruses (6, 12, 19). The present results indicate that the expression of the poliovirus genome may be necessary for the onset of interference in cell cultures. Irradiated poliovirus was unable to restrict the replication of SB virus in cell cultures. However, replication of the poliovirus genome is not required for the interference, because restriction of SB virus is observed even after poliovirus RNA synthesis is blocked by guanidine. Apparently the translation, but not the replication, of the parental poliovirus RNA can occur in the presence of guanidine (1-3, 11, 17). Thus, the example of interference studied here resembles the "intrinsic interference" occurring between NDV and SB virus (15). The mechanism by which poliovirus restricts replication of SB virus was examined in some detail. Poliovirus did not inhibit the synthesis of SB viral RNAs under the conditions used here. In this respect our results are similar to those observed by Doyle and Holland (6) on the effect of poliovirus on synthesis of RNAs in VSVinfected cells. Similar observations have been reported by Saxton and Stevens (16) on the continued synthesis of viral messenger RNAs in HSV-infected HeLa cells subsequent to superinfection with poliovirus. It is interesting to note that poliovirus inhibits host cell RNA synthesis soon after infection (1, 10, 23). Apparently the mechanism by which poliovirus restricts the host cell RNA synthesis does not extend to the synthesis of RNAs of viral origin. The probable mechanism involved in the restriction of SB virus by poliovirus in dually infected cells in the inhibition of protein synthesis directed by SB virus. Evidence is presented here indicating that polyribosomes present in SB-infected cells disaggregate soon after superinfection with poliovirus. Additionally, we have shown that the nascent SB viral RNAs synthesized in dually infected cells do not appear to be associated with the ribosomal structures. Thus, it seems possible that initiation of SB virus protein synthesis is blocked at the level of the formation of initiation complexes between SB viral RNAs and ribosomes after poliovirus superinfection. In this context, our conclusions are similar to those of Giorno and Kates (8) on the inability of vaccinia virus messenger RNAs to associate with ribosomes in cells doubly infected with vaccinia and adenovirus. At present the exact mechanism by which poliovirus can inhibit this step remains unclear. Recent observations suggest that double-stranded viral RNA of poliovirus inhibits the initiation of protein synthesis by rabbit reticulocytes in vitro (7). Also, it has been reported that enterovirus double-stranded RNAs are toxic to mammalian cells and can inhibit protein synthesis in vivo (5). The present results, as well as those reported previously, (2, 17) suggest that doublestranded RNA (replicative form) is formed in poliovirus-infected cells in the presence of guanidine. Thus, it is conceivable that the observed inhibition of SB viral proteins in dually infected cells may be due to the general inhibition of protein synthesis effected by poliovirus doublestranded RNA. If the double-stranded RNA is the intermediary molecule affecting restriction of translation by other viral RNAs, then it has to be assumed that such a property is limited to the RNAs of poliovirus or other enteroviruses and not to those of SB virus. The above conclusion stems from the observation that SB virus was unable to interfere with replication of poliovirus, although infected cells contain SB virus-specific double-stranded RNA. However, no direct evidence exists to suggest that only poliovirus or other enterovirus double-stranded RNAs has the unique ability to interfere with protein synthesis in vivo. Indeed it has been suggested that various double-stranded RNAs with different specificities can alter the synthesis of protein. Thus, it is not clear whether the dominance established by poliovirus over SB virus in dually infected cells is due to the poliovirus double-stranded RNA. ACKNOWLEDGMENT This investigation was supported by Public Health Service research grant A109355-02 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Baltimore D. 1969. The replication of picornaviruses, p. 101-176. In H. B. Levy (ed.), The biochemistry of viruses. Marcel Dekker, New York and London. 2. Caliguiri, L., and I. Tamm. 1968. Action of guanidine on the replication of polio virus RNA. Virology 35:408-417. 3. Caliguiri, L., and I. Tamm. 1968. Distribution and

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