Inhibitory effects of vesicular stomatitis virus on cellular and influenza viral RNA metabolism and protein synthesis

VIROLOGY

172,274-284

Inhibitory

(1989)

Effects of Vesicular Stomatitis Virus on Cellular and Influenza Viral RNA Metabolism and Protein Synthesis DEBRA W. FRIELLE, PAUL B. KIM, AND JACK D. KEENE’

Department

of Microbiology

and Immunology,

Duke University Medical Center, Durham, North Carolina 277 10

Received March 9, 1989; accepted May 22, 1989 Infection with vesicular stomatitis virus (VSV) results in the rapid inhibition of cellular macromolecular synthesis, including transcription, translation, and maturation of the Ul and U2 snRNPs. Unlike infection with VSV, influenza virus infection did not result in the inhibition of either the processing of Ul and U2 snRNAs or the assembly of the RNPs. Although influenza virus relies on the cellular splicing apparatus to generate viral mRNAs, the maturation of snRNPs was inhibited during double infections with VSV. However, this inhibition of snRNP maturation had no effect on the splicing of a cellular pre mRNA in extracts prepared from VSV-infected HeLa cells. Thus, the effects of VSV on the processing and assembly of snRNPs appear to involve virus-specific functions and unique cellular targets. Coinfection with VSV and influenza virus resulted in the dramatic inhibition of influenza virus transcription; polyadenylated mRNAs corresponding to the influenza virus NP and NSl proteins could not be detected by Northern blot analysis. However, reduced levels of the influenza virus replicative templates were still synthesized during double infection. Coinfection with VSV also resulted in the inhibition of influenza viral mRNA translation, even when superinfection with VSV was delayed until 3 or 6 hr after influenza virus infection. VSV required at least 2 hr to affect the inhibition of translation; this corresponded to the time after VSV infection when inhibition of cellular protein synthesis was evident. These results demonstrate that, in contrast to adenovirus, the VSV-mediated inhibition of cellular macromolecular synthesis may be effective SgSinSt infkmza virus. 0 1989Academic Press, Inc.

their 3’termini during assembly into a complete snRNP (Eliceiri, 1980; Eliceiri and Sayavedra, 1976; Madore et a/., 1984; Fisher et al., 1985; Wieben et a/., 1985). Infection by VSV results not only in the accumulation of the precursor forms of these snRNAs but also in the failure of these pre snRNAs to assemble with the appropriate proteins to form a complete snRNP. Inhibition of 3’ processing and assembly of the pre Ul and pre U2 snRNAs proceeds more rapidly than the general inhibition of cellular transcription. In baby hamster kidney (BHK) cells, this inhibition can be detected 15-30 min after VSV infection (Fresco et a/., 1987). Synthesis of the VSV leader RNA has also been correlated with the inhibition of maturation of Ul and U2 snRNPs (Crone and Keene, in press). The relationship between the inhibition of processing of these snRNAs and the generalized disruption of cellular RNA metabolism by VSV is not clear. It is also not known whether the inhibition of cellular snRNA metabolism is a common result of infection with other negative-strand viruses, such as influenza virus. Some unique features of influenza virus replication involve its dependence on cellular RNA metabolism. First, synthesis of viral mRNAs requires continual cellular transcription as a source of capped primer (Plotch et a/., 1979; Krug et a/., 1979). Since VSV infection results in decreased transcription, VSV may also inhibit influenza virus transcription during a double infection.

INTRODUCTION Infection by vesicular stomatitis virus (VSV) results in the rapid and complete inhibition of both cellular RNA and protein synthesis. Viral transcription has been shown to be required for the inhibition of these cellular functions (Marvaldi et al., 1978; Week and Wagner, 1979; Wu and Lucas-Lenard, 1980; Dunigan and Lucas-Lenard, 1983). The VSV leader RNA, a 47-nucleotide transcript synthesized from the 3’ terminus of the genome, has been considered in this inhibition (Marvaldi et a/., 1978; Week et a/., 1979; McGowan et al., 1982; Dunigan and Lucas-Lenard, 1983; Dunigan et a/., 1986). Additional evidence suggests that translation of the viral nucleocapsid protein, N, is also involved in the inhibition of cellular macromolecular synthesis (Marvaldi et al., 1978; Wu and Lucas-Lenard, 1980; Dunigan and Lucas-Lenard, 1983; Poirot et al., 1985). Our laboratory has described an additional specific effect of VSV infection on cellular RNA metabolism: the inhibition of maturation of Ul and U2 snRNPs (Fresco et al., 1987). These RNAs are components of the small nuclear ribonucleoprotein particles (snRNPs) which mediate the splicing of cellular pre mRNAs. The U 1 and U2 snRNAs are synthesized as precursors which are processed by the removal of 3-10 nucleotides from ’ To whom requests for reprints should be addressed. 0042-6822/89

$3.00

Copyright 0 1989 by Academic Press. Inc. All rights of reproduction in any form reserved.

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because splicing of several influenza Virus mRNAs is accomplished by the cellular splicing apparatus (Lamb and Lai, 1980; Lamb eta/., 1981), the VSVinduced accumulation of pre Ul and pre U2 snRNAs may have an effect on the splicing of both cellular and influenza virus mRNAs. These features of influenza virus replication suggest that, like cellular macromolecular synthesis, influenza virus replication may be subject to inhibition by VSV. In support of this suggestion, it has been reported that the yield of influenza virus was reduced during mixed infections of VSV and influenza virus in Madin-Darby canine kidney cells (Roth and Compans, 198 1). Cellular protein synthesis is also dramatically reduced during infection with VSV. This inhibitory effect may be mediated by the VSV-induced activation of the double-stranded RNA-dependent protein kinase which phosphorylates the initiation factor elF2 (Centrella and Lucas-Lenard, 1982; Whitaker-Dowling and Youngner, 1983). Infection with several other viruses, including adenovirus, vaccinia, and influenza virus, results in the suppression of the activation of this kinase (WhitakerDowling and Youngner, 1983; Rice and Kerr, 1984: Paez and Esteban, 1984; Schneider et al., 1985; Siekierka et al., 1985; Katze et a/., 1986). We have determined what effects VSV and influenza virus have on cellular and viral translation during double infections. Thus, in this study we have examined VSV and influenza virus RNA and protein metabolism during double infections in order to determine whether either virus has a replicative advantage.

Second,

MATERIALS

AND METHODS

Cells and viruses Stocks of the WSN strain of influenza Avirus and VSV were grown in Madin-Darby canine kidney cells and BHK cells, respectively, from plaque-purified inocula. Cell lines were maintained as monolayer cultures in Joklik’s modified minimal essential medium supplemented with 5%fetal calf serum, glutamine, nonessential amino acids, and vitamins. Some experiments were performed in BHK and HeLa cells grown in suspension culture. Virus infections Influenza virus or VSV were diluted in medium to yield a multiplicity of infection of 3 PFU per cell. Cells grown in suspension culture were pelleted and resuspended in the inoculum at a density of 1 X 1O7 cells/ ml, virus was adsorbed for 0.5 hr at 37”, and cells were diluted to 1 X 10” cells/ml. Influenza virus-infected cells

275

were superinfected with VSV at various times by the addition of the VSV inoculum to the culture. Virus-specific

protein synthesis

Labeling of viral protein synthesis was carried out in methionine-free medium containing 50 &i/ml [35S]methionine. At the end of the labeling period, the cells were washed twice with PBS and lysed in Laemmli sample buffer (Laemmli, 1970). Viral proteins were analyzed by electrophoresis in SDS-containing 13% acrylamide-0.12% BIS gels (Lesnaw et al., 1979) with buffers described by Laemmli (1970). Bands were visualized with the aid of salicylate fluorography (Chamberlain, 1979). Northern

blot analysis of viral mRNA synthesis

Infected cells were resuspended in 5 packed cell volumes of 10 mM Tris, pH 7.4, 150 mM NaCI, 2 mn/r MgCI,. Cells were lysed by incubation in 0.5% NP-40 on ice for 10 min. After centrifugation to remove nuclei, SDS was added to a concentration of 0.2% and the cytoplasm was extracted with phenol and chloroform. Polyadenylated RNA was isolated by oligo(dT) cellulose chromatography. Viral RNA was separated on 5.5% acrylamide-7 M urea gels, electrophoretically transferred to Nytran membrane, and visualized by hybridization to nick-translated probes corresponding to influenza virus genes (Maniatis et a/., 1982). Plasmids containing the influenza virus NP and NS genes were kindly provided by J. S. Youngner. Analysis

of Ul and U2 snRNAs

Uninfected and infected BHK or HeLa cells were labeled with 30 &i/ml [3H]uridine for l-2 hr. Cells were lysed by sonication in 50 mM Tris, pH 7.4, and 150 mn/r NaCl and nuclei were removed by centrifugation. For each immunoprecipitation, extract representing 1.25 x 1O7 cells was incubated with human autoimmune antiserum for 15 min on ice and then with Pansorbin for 30 min on ice (Fresco et a/., 1987). Immune complexes were pelleted and washed five times with 50 mM Tris, pH 7.4, 150 mM NaCI, and 0.05% NP40. Following phenol-chlorform extraction, RNA was precipitated with ethanol and analyzed on 10% acryamide-7 M urea gels with 50 m M Tris-borate, pH 8.3 buffer (Kurilla and Keene, 1983). In vitro splicing Nuclear extracts for in vitro splicing were prepared according to Dignam et al. (1983). In vitro splicing reactions were carried out as described by Krainer et al. (1984). The substrate for splicing reactions was a trun-

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FRIELLE, KIM, AND KEENE TIME OF LABELING 1-3 HOURS

TOTAL

B-10 HOURS

Ul/UZ

snRNP

TOTAL

FIG. 1. Processing of Ul and U2 snRNAs In VSV and influenza virus-infected cells. BHK cells were infected as indicated and labeled with [3H]uridine from 1 to 3 or 8 to 10 hr after Infection. Whole cell extracts were prepared and RNAs analyzed before (TOTAL lanes) and after immunoprecipitation with Ul/U2 snRNP antiserum (Ul/U2 snRNP lanes). The RNAs were analyzed on a 10% acrylamide-7 M urea gel. Arrowheads mark the mature U 1 and U2 snRNAs and asterisks mark the orecursors.

cated p-globin pre mRNA containing the first exon, the first intron, and most of the second exon; 32P-labeled substrate was synthesized in vitro using SP6 RNA polymerase. Products of the in vitro splicing region were analyzed on 5.5% acrylamide-7 M urea gels. RESULTS Processing of Ul and U2 snRNAs during infections with influenza virus and VSV Infection with VSV results in the rapid and complete inhibition of processing of the pre Ul and pre U2 snRNAs to their mature size (Fresco et al., 1987). The unprocessed forms of U 1 and U2 snRNAs which accumulate are not associated with their normal complement of proteins. In particular, the proteins recognized by the Sm autoimmune sera do not bind to the pre Ul and pre U2 snRNAs which accumulate during VSV infection (Fresco eta/., 1987). Because several influenza virus mRNAs are generated by splicing, it was of interest to determine whether influenza virus also had an effect on U snRNA processing and snRNA assembly. BHK cells were infected with VSV or influenza virus and RNAs were labeled with [3H]uridine from either l-3 hr after infection or 8-10 hr after infection. The Ul and U2 snRNAs were analyzed both before and after immunoprecipitation with an Ul/U2 snRNP antiserum. As shown in Fig. 1, VSV infection resulted in the complete inhibition of processing of the Ul and U2 snRNAs by 3 hr after infection. In contrast, the levels of precursor and mature Ul and U2 snRNAs in influenza virus-in-

fected cells were indistinguishable from those in uninfected cells. At the later time examined, transcription of snRNAs in VSV-infected cells was reduced to a very low level, while the RNA that was synthesized accumulated as the precursor. However, in cells infected with influenza virus alone, transcription was slightly reduced and the Ul and U2 snRNAs were processed to the mature size. This result demonstrated that the inhibition of U snRNA processing and RNP assembly seen during VSV infection is not a common effect of infection with negative-strand RNA viruses. Rather, these data suggest that a VSV-specific factor is responsible for the inhibition of U snRNA metabolism. While VSV infection results in a rapid and dramatic inhibition of cellular RNA and protein synthesis, infection by influenza virus does not affect these cellular processes until later in infection. In addition, because of the striking difference between VSV and influenza virus with respect to the inhibition of processing of pre Ul and pre U2 snRNAs, we examined whether VSV was affecting the snRNP processing and assembly through its inhibition of cellular transcription or translation. The level of U snRNA processing was examined in cells treated with actinomycin D or cycloheximide to directly inhibit cellular RNA and protein synthesis, respectively. Uninfected cells were treated with 0.1 pg/ml actinomytin D for 1 hr and were labeled with [3H]uridine in the presence of the drug for 2 hr. This treatment decreased the TCA-precipitable incorporation of “H by greater than 80% (data not shown). Processing of Ul and U2 snRNAs in these cells was unaffected by the inhibition of transcription (Fig. 2). The level of U snRNA processing was also measured in cells treated with 250 pg/ml cycloheximide to reduce protein synthesis by greater than 95% (data not shown). Again, snRNA processing was complete in the absence of cellular protein synthesis (Fig. 2). In addition, the processed RNAs were precipitable with both Sm and Ul/U2 snRNP antisera, indicating that the processed snRNAs were being properly assembled into RNPs in the absence of cellular translation. These results suggest that the inhibition of processing observed during VSV infection is a specific viral effect directed at the metabolism of the snRNAs and not simply an indirect result of decreased cellular transcription or translation. Because infection with influenza virus, but not VSV, requires cellular pre mRNA splicing, we examined the state of U 1 and U2 snRNAs in cells which were doubly infected with influenza virus and VSV. At 4 hr post-influenza virus infection, cells were labeled with [3H]uridine for 1 hr, whole cell extracts were prepared, and the Ul and U2 snRNAs were precipitated with Ul/U2 snRNP antiserum (Fig. 3). The processing of Ul and

INHIBITORY ACTINOMYCIN

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EFFECTS OF VESICULAR

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not essential to splicing as measured in vitro. These data also suggest that VSV-mediated inhibition of U snRNP maturation would not affect the production of influenza virus spliced mRNAs.

CYcLotlExIMIM

Influenza virus RNA synthesis infections with VSV

-u1/u2 E

--i7iz I: snRNP

Sm

277

snRNP

and actinomycin D treatment FIG. 2. The effect of cyclohexrmrde on Ul and U2 snRNA processing in the Hela cells. HeLa cells were treated with either cycloheximide (250 pg/ml) or actinomycin D (0.1 pg/ml) for 1 hr and then labeled with [3H]uridine in the presence of the appropriate drug for 2 hr. Whole cell extracts were prepared and snRNAs were analyzed before (lanes marked “2”) and after immunoprecipitation with either Sm antiserum or Ul/U2 snRNP antiserum. Precipitated snRNAs were analyzed on a 10% acrylamrde-7 M urea gel.

U2 snRNAs was completely inhibited when cells were infected with VSV and influenza virus simultaneously or when influenza virus-infected cells were superinfected with VSV after 3 hr. Thus, it is possible that VSV may interfere with influenza virus replication by disruption of cellular splicing. Although snRNPs have a relatively long half-life, we investigated the possibility that inhibition of the processing of the U 1 and U2 snRNAs by VSV interfered with the splicing of pre mRNAs. Plotch and Krug (1986) have demonstrated that nuclear extracts prepared from uninfected HeLa cells were not able to produce the spliced influenza virus NS2 mRNA. Therefore, the ability of nuclear extracts prepared from VSV-infected cells to splice a cellular pre mRNA was assessed. Nuclear extracts were prepared from uninfected HeLa cells and from cells which had been infected with VSV for 3 hr. Control experiments showed that by 3 hr after infection with VSV, processing of pre Ul and pre U2 snRNAs and the assembly of snRNPs in HeLa cells was completely inhibited (data not shown). Splicing of the first 2 exons of the P-globin pre mRNA transcript was compared in extracts prepared from uninfected and from VSV-infected cells. The results, presented in Fig. 4, show that nuclear extracts prepared from VSV-infected cells were able to splice the @-globin pre mRNA transcript as well as the extracts prepared from uninfected cells. Therefore, the inhibition of processing and assembly of the U 1 and U2 snRNPs by VSV did not have a short-term effect on in vitro pre mRNA splicing. Because snRNPs have a long half-life, continued synthesis of snRNPs is apparently

during double

In addition to the effect on snRNA metabolism, infection with VSV results in the inhibition of cellular mRNA transcription. In contrast, synthesis of influenza virus mRNA is dependent on newly synthesized cellular mRNAs as a source of 5’ cap and primer (Plotch et al., 1979; Krug eta/., 1979). Influenza virus replication may serve as a simple model system in which to study the VSV-mediated inhibition of cellular transcription. Therefore, we investigated the effect of VSV infection on influenza virus mRNA synthesis. BHK cells were singly infected, doubly infected, or infected with influenza virus and superinfected with VSV after 1 or 3 hr. Both polyadenylated poly(A)’ and nonpolyadenylated poly(A)- RNAs were isolated from infected cells 5 hr after influenza virus infection and were analyzed by Northern blot hybridization. Figure 5 shows blots which were probed with cDNAs representing the influenza virus NP and NS genes. When cells were infected with both influenza virus and VSV simultaneously (FL.U X VSVJ, synthesis of influenza virus RNA was not detected. This inhibition affected both transcription of influenza

FIG. 3. Processing of Ul and U2 snRNAs In singly and doubly infected BHK cells. BHK cells were either singly infected with influenza virus or VSV, coinfected with both viruses (FLU X VSVJ, or infected with influenza vtrus and supennfected with VSV after 3 hr (FLU X VSVJ. Cells were labeled with [3H]uridrne from 4 to 5 hr after influenza virus Infection. Whole cell extracts were prepared 5 hr after influenza virus infection. The Ul and U2 snRNAs and their precursors were immunopreciprtated wrth Ul/U2 snRNP antiserum and separated on a 10% acrylamide-7 M urea gel. RNAs before: immunoprecipttation are shown In lanes marked TOTAIL

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FRIELLE. KIM. AND KEENE HELA

IN VITRO

SPLICING

HELA EXTRACT PREPARED AT 3H P.I.

ATPICP

MOCK I_+ -

VSV + -

622 527

-Pre-mRNA

-Ligated

513

Exons

360

Synthesis of viral protein during double infection VSV and influenza virus

242

147

These results suggest that influenza virus mRNA synthesis was not directly inhibited by VSV. The greater reduction in influenza virus primer-dependent (mRNA) transcription during a double infection with VSV suggests that the inhibition may have resulted from VSV-mediated inhibition of cellular mRNA synthesis. Infection with VSV results in the inhibition of cellular transcription; hence, there is a reduction in the availability of cellular capped mRNAs to donate their 5’ termini to the influenza virus transcriptase. The decreased transcription of influenza virus non-primerdependent RNAs (vRNAs and cRNAs) may be a secondary effect, resulting from the decreased synthesis of viral mRNA.

-Lariat

143

FIG. 4. In vitro splicing by extracts of VSV-infected HeLa cells. Nuclear extracts were prepared from uninfected or VSV-infected HeLa cells 3 hr after infection. The nuclear extracts were incubated with a 32P-labeled transcript containing the first two exons and the first intron of the human P-globin pre mRNA. Creatine phosphate and ATP (ATP/CP), which are required for splicing, were omitted from the reactions in the lanes indicated. Products of the in vitro splicing reaction were analyzed on 5.5% acrylamide-7 M urea gels. The positions of molecular weight markers are indicated on the left.

Infection with either VSV or influenza virus results in the inhibition of cellular protein synthesis. This effect is presumed to be at the level of translation rather than a result of enhanced degradation of cellular mRNAs during infection. Work from several laboratories indicates that infection by both VSV and influenza virus can modulate the level of phosphorylation of the initiation factor elF2 (Centrella and Lucas-Lenard, 1982; WhitakerDowling and Youngner, 1983; Katze et al., 1986). Infection by VSV apparently results in the activation of the double-stranded RNA-dependent protein kinase, while influenza virus infection causes a suppression of kinase activation. We examined protein synthesis in doubly infected cells to determine whether either virus interferes with protein synthesis by the other. BHK cells

POLY A + RNA

virus poly(A)+ mRNA and synthesis of the nonpolyadenylated full-length plus strand cRNAs and full-length minus strand vRNAs. When influenza virus-infected cells were superinfected with VSV after 1 hr (FLU x VSV,), synthesis of a small amount of influenza virus poly(A)- RNA was observed, but no poly(A)+ RNA was detected. Synthesis of influenza virus mRNAs could not be detected, even when the influenza virus infection was allowed to proceed for 3 hr before VSV superinfection (FLU x VSVJ. Under these conditions, poly(A)- RNAs corresponding to the NP and NS genes were observed; however, their synthesis was reduced relative to a single infection (Fig. 5). This result is not surprising because the synthesis of these RNAs occurs by an apparently different mechanism than synthesis of the viral mRNAs, in that synthesis of the cRNAs and vRNAs is not dependent on the presence of capped primer (Hay et al., 1982; Beaton and Krug, 1984).

by

POLY A -RNA

w NSl

FIG. 5. Northern blot analysis of influenza virus RNA synthesis during double infections with VSV. Cells were singly infected with influenza virus (FLU) and VSV, infected with both viruses simultaneously (FLU X VSV,), or infected with influenza virus and superinfected with VSV after 1 (FLU x VSV,) or 3 (FLU VSVJ hr. Cytoplasmic RNA was isolated 5 hr after infection with influenza virus. Both polyadenylated and nonpolyadenylated RNAs were analyzed in a 5.5% acrylamide7 M urea gel. After transfer to Nytran membrane, RNAs were hybridized to nick-translated probes for the influenza virus NP and NS RNAs.

INHIBITORY

EFFECTS OF VESICULAR

G Ns N

M NSl

STOMATITIS

with VSV after 1 or 3 hr. Duplicate cultures were labeled with [35S]methionine either from 4 to 5 hr or from 7 to 8 hr after influenza virus infection (Fig. 7). When influenza virus was given a 1 hr head start before VSV superinfection, synthesis of the influenza virus proteins remained inhibited. The synthesis of NP, Ml, and NSl was severely reduced by 5 hr post-influenza virus infection and was completely inhibited by 8 hr after influenza virus infection. Under these conditions of infection, all VSV proteins were synthesized at the same level as during a single infection. A reduction in influenza virus-specific protein synthesis was also observed when superinfection with VSV occurred 3 hr after influenza infection. For example, the synthesis of NP between 4 and 5 hr post-influenza virus infection (or 1 to 2 hr after VSV superinfection) was slightly reduced relative to a single infection (Fig. 7). In contrast, when cells were labeled between 7 and 8 hr

TIME OF LABELING

FIG. 6. Vrral protein syntheses during double

infections with VSV and influenza vrrus. BHK cells were either singly or doubly infected with VSV and influenza vrrus (FLU) each at a multiplicity of 3 PFU/ cell. Infected cells were labeled with [%]methronine from 3 to 5 hr after infection, lysed wrth SDS, and analyzed on a 13% SDS-polyacrylamide gel. The positions of the major Influenza virus proteins NP, M 1, and NSl are indicated on the left; the VSV proteins are marked on the right.

were singly or doubly infected with influenza virus and VSV at a multiplicity of infection of 3 PFU per cell. Infected cells were labeled with [35S]methionine from 3 to 5 hr after infection. The proteins synthesized in cells singly infected with either VSV or influenza virus are shown in Fig. 6. Cells infected with influenza virus continued to synthesize cellular proteins, as well as the major influenza viral proteins NP, M 1, and NSl In contrast, synthesis of cellular protein was dramatically inhibited during VSV infection while VSV proteins were synthesized in large amounts. The pattern of protein synthesis in double infected cells was nearly indistinguishable from that seen during infection with VSV alone, while the synthesis of influenza proteins was dramatically reduced (Fig. 6). Thus, the synthesis of VSV proteins was dominant over that of both influenza virus and the cell. The inhibitory effect of VSV on the level of influenza virus protein synthesis was also examined in BHK cells which were initially infected with influenza virus and subsequently superinfected with VSV. Cells were simultaneously infected with both viruses or were singly infected with influenza virus and then superinfected

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4-5 HR 013

013

G NS NP

M NSl

FIG. 7. lnhrbrtron of Influenza virus protern synthests followrng coinfection or supennfectron with VSV. Duplrcate cultures of BHK cells were singly Infected with influenza vrrus (FLU) or VSV, infected with both viruses (FLU X VSV,), or Infected with Influenza vrrus and superinfected with VSV after either 1 (FLU x VSV,) or 3 (FLU x VSV,) hr. A multiplrcity of 3 PFU/cell was used for each vrrus. Infected cells were labeled with [%]methionine from either 4 to 5 or 7 to 8 hr after rnfluenza virus infectron. Labeled proteins were analyzed by electrophorests In 13% SDSacrylamide gels.

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FRIELLE. KIM, AND KEENE

post-infection very little influenza virus NP protein was seen. This result suggests that a VSV-specific product may affect the ability of influenza virus mRNA to be translated; the NP mRNA which was translated between 4 and 5 hr post-influenza virus infection was not translated during the later labeling period. The Northern blot analysis of influenza virus transcription presented in Fig. 5 demonstrated that superinfection by VSV did cause an inhibition of influenza virus-specific transcription. However, the effect on translation seen in Fig. 7 appears to be distinct from this inhibition of transcription. Clearly, a level of NP mRNA which was sufficient to direct detectable protein synthesis was synthesized at some point before 4 hr post-influenza virus infection, but was no longer translated between 7 and 8 hr after infection. Thus, VSV may exert separate inhibitory effects on both the transcription and the translation of influenza virus mRNA. The effect of VSV superinfection on influenza virus mRNA translation was also examined in replicate cultures which were infected with influenza virus and subsequently infected with VSV at 1-hr intervals, up to 6 hr post-influenza virus infection. All cultures were labeled from 7 to 8 hr post-influenza virus infection (Fig. 8). When influenza virus was allowed a 3-hr head start before superinfection with VSV, only low levels of influenza virus mRNA translation were observed. When VSV was added at later times, a gradual increase in synthesis of influenza virus proteins occurred. At the point when infection by VSV was delayed until 5 to 6 hr post-influenza virus infection, the level of influenza virus-specific protein synthesis was comparable to that in a single infection. Several interpretations of this data are reasonable. First, the possibility of superinfection exclusion must be considered. By 5 hr post-influenza virus infection, it is possible that VSV is excluded from superinfecting the cells. This is unlikely because VSV-specific proteins are present in cells superinfected at these late times. In Fig. 8, VSV-specific L, NS, N, and M proteins were synthesized at high levels in cells superinfected at 5 hr post-influenza virus infection. Second, the data may suggest that by 5 to 6 hr after infection, influenza virus reached a point in the replication cycle where it was no longer sensitive to VSV-mediated inhibition of translation. Alternatively, VSV may simply require more than 2 hr to completely exert its inhibitory effect on protein synthesis. To distinguish between these possibilities doubly infected cells were labeled at various times after VSV superinfection, as described below. Cells were singly infected as in the previous experiments and superinfection with VSV was delayed until 6 hr after influenza virus infection. Infected cells were labeled with [35S]methionine for 30 min ending 2, 5,

TIME OF VSV INFECTION

0123456

G NS NP

M NSl

FIG. 8. Inhibition of influenza virus protein synthesis following superinfection by VSV. Replicate cultures of BHK cells were infected with influenza virus at a multiplicity of 3 PFUkell. At hourly intervals, cells were superinfected with VSV. All cultures were labeled with [35S]methionine from 7 to 8 hr after influenza virus infection and labeled proteins were analyzed on a 13% SDS-acrylamide gel.

and 8 hr after infection by VSV; these times corresponded to 8, 1 1, and, 14 hr after infection with influenza virus. This protocol allowed VSV a longer interval to exert the inhibitory effect. As shown in Fig. 9, when cells were superinfected 6 hr after influenza virus infection and labeled from 7.5 to 8 hr, synthesis of influenza virus proteins was evident. When doubly infected cells were labeled at later times, a gradual decrease in influenza virus protein synthesis was observed. By 8 hr after infection by VSV, the level of synthesis of influenza virus proteins was very low and the VSV proteins were predominant. This experiment demonstrated that superinfection of influenza virus infected cells with VSV resulted in the disruption of ongoing influenza virus translation. Influenza virus-specific mRNAs clearly had been synthesized and were being translated at the earliest labeling period. These mRNAs were not translated during the later labeling times. Even in the absence of continued influenza virus transcription, translation of the preexisting mRNAs would be expected to continue. The decreased synthesis of influenza virus proteins

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vsv 258 time of labeling (hours):

VXF 250 81114

FLU

EFFECTS OF VESICULAR

M

81114(14)

post-VSV Dost-influenza

Ml NSI

FIG. 9. Inhibitron of Influenza virus protein synthesis at Intervals followrng VSV superinfection. BHK cells were singly infected or were infected with influenza virus and supennfected with VSV 6 hr later. Replicate cultures were labeled with [?S]methionine for 30 min ending at the times indicated. Proterns were analyzed on a 13% SDSacrylamide gel.

during the later labeling periods suggests that there is a VSV-mediated inhibition of translation which is independent of the inhibition of transcription. This experiment also suggests that influenza virus mRNA translation is sensitive to inhibition by VSV, even when superinfection by VSV is delayed until a late step in the influenza replication cycle. The results of the previous experiments can be explained by the observation that VSV requires more than 2 hr to fully exert an inhibitory effect on influenza virus protein synthesis. The requirement for at least 2 hr to affect the inhibition of influenza virus-specific translation is in agreement with the time required for VSV to cause the inhibition of cellulartranslation. DISCUSSION Influenza virus is unique among RNA viruses in having a nuclear phase during its replication cycle. This nuclear involvement reflects the virus’ dependence on several cellular metabolic processes. For example, transcription of influenza virus mRNA requires ongoing transcription of cellular genes. Newly synthesized cellular mRNAs are the source of the 5’capped primerthat

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is required by the viral RNA polymerase (Plotch et a/., 1979; Krug et al., 1979). In addition, at least two influenza virus mRNAs are generated by a splicing reaction which is accomplished by the cellular snRNPs (Lamb and Lai, 1980; Lamb et a/., 1981). In contrast, infection with another negative stranded RNAvirus, VSV, results in the inhibition of cellular transcription (Week and Wagner, 1979; Wu and Lucas-Lenard, 1980; Poirot et al., 1985). Furthermore, infection by VSV results in a more rapid inhibition of 3’ terminal processing and assembly of pre Ul and pre U2 snRNAs. Thus, VSV directly interferes with at least 2 cellular processes which are required by influenza virus. These observations are consistent with previous experiments which demonstrated that the replication of VSV is dominant during a double infection with VSV and influenza virus, resulting in a decreased yield of influenza virus (Roth and Compans, 1981). We have investigated the effect of coinfection and superinfection with VSV on transcription by influenza virus. Viral transcription was examined in cells which were infected with both viruses. Northern blot analysis of influenza virus NP and NSl mRNAs demonstrated that both coinfection and superinfection by VSV resulted in the inhibition of influenza virus mRNA transcription (Fig. 5). One possible explanation is that the VSV-mediated inhibition of cellular transcription resulted in the absence of a source of newly synthesized nuclear RNAs to donate capped primers to the influenza virus RNA polymerase. Transcription of influenza virus poly(A)) RNAs were also reduced in doubly infected cells, although to a lesser extent. This result IS not surprising because vRNAs and cRNAs differ from viral mRNAs in that they are not capped and their synthesis is not primer dependent (Hay et al., 1982; Beaton and Krug, 1984). The reduced level of synthesis of these RNAs is likely to be the result of the low level of mRNA synthesis in doubly infected cells. Adenovirus infection also results in the inhibition of cellular transcription and transport of cellular mRNA from the nucleus (Babich et al., 1983; Beltz and Flint, 1979). Unlike VSV, during double infections with adenovirus, influenza virus maintains transcription and transport of viral mRNAs. When cells are doubly infected with adenovirus and influenza vrrus, no inhibition of influenza virus-specific transcription was seen (Katze et a/., 1984). Rather, influenza vrrus mRNAs were synthesized which contained primers derived from both cellular and adenoviral mRNAs. Influenza virus mRNAs were transported to the cytoplasm and translated normally. Thus, influenza virus mRNAs are distinguished from cellular mRNAs and escape the adenovirus-induced inhibition of transcription and transport.

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Infection with VSV results in the rapid inhibition of 3’ terminal processing of the pre Ul and pre U2 snRNAs and the assembly of the snRNAs into nuclear RNPs (Fresco eta/., 1987; Fig. 1). Because this inhibition occurs within 30 min of infection, it is unlikely to be the result of VSV-induced inhibition of cellular transcription ortranslation. The inhibition of cellular macromolecular synthesis occurs more slowly, requiring several hours to be complete. As reported here, the 3’ terminal processing of the snRNAs was unaffected by drug-induced inhibition of cellular RNA and protein synthesis (Fig. 2). In addition, the snRNAs were precipitable with Sm antiserum, indicating that the snRNAs are apparently assembled into RNPs. The processing of cellular pre Ul and pre U2 snRNAs was also examined during double infections with VSV and influenza virus. In contrast to VSV infection, influenza virus infection had no effect on 3’ terminal processing of these RNAs (Fig. 1). However, precursor U snRNAs continued to accumulate in cells which were doubly infected with VSV and influenza virus regardless of the time of VSV superinfection (Fig. 3). The VSV-mediated inhibition of snRNP maturation may have no effect on splicing of influenza virus mRNAs because nuclear extracts prepared from VSV-infected cells, which contained precursor forms of the U snRNAs, were fully competent for the splicing of fi-globin pre mRNA transcripts (Fig. 4). SnRNPs synthesized prior to VSV infection are stable (unpublished observations) and apparently retain their splicing activity in cell-free extracts. Infection with VSV also results in the inhibition of cellular translation (Marvaldi et a/., 1978; Dunigan and Lucas-Lenard, 1983). This may be the result of virus-induced activation of the double-stranded RNA-dependent protein kinase which phosphorylates the initiation factor elF2. The initiator tRNA, GTP, and elF2 form the ternary complex which interacts with the 40 S ribosomal subunit forming the preinitiation complex. Phosphorylation of the (Y subunit of elF2 results in decreased initiation of protein synthesis because the guanine nucleotide exchange factor, elF2B, does not mediate exchange of GDP and GTPfrom the phosphorylated form of elF2. Under these conditions of limiting elF2, VSV mRNAs may be preferentially translated over the cellular mRNAs because they are efficient competitors for the few functional initiation complexes which form. In contrast, adenovirus, vaccinia virus, and influenza virus have all been shown to suppress the dsRNA-dependent activation of the kinase (Whitaker-Dowling and Youngner, 1983; Katze et al., 1986; Schneider et a/., 1985; Siekierka et al., 1985). The vaccinia virus product, the specific kinase inhibitory factor, appears to be

a protein which may act by binding double-strand RNA (Whitaker-Dowling and Youngner, 1983, 1984). The VA1 RNA of adenovirus has been shown to be a viral function which is required to suppress the activation of the kinase during infection with adenovirus (Schneider et al., 1985; Siekierka et al., 1985). Adenovirus infection results initially in the inhibition of both cellular and viral translation because of the activation of the kinase. Synthesis of the VA1 RNA allows suppression of the kinase and synthesis of the adenoviral proteins at the expense of cellular translation. During a double infection with adenovirus and influenza virus, translation of both adenovirus and influenza virus proteins was observed (Katze et al., 1984). This suggests that the influenza virus mRNAs are distinguished from cellular mRNAs. However, when cells were infected with influenza virus and the adenovirus mutant d133 1, which is deleted of VA1 RNA, synthesis of only influenza virus proteins was observed (Katze eta/,, 1984). In this case, infection probably resulted in the initial inhibition of translation with influenza virus subsequently establishing its own mechanism for maintaining viral translation. The influenza virus product involved in suppressing the kinase has not been identified. We have examined the state of protein synthesis in cells which were doubly infected with VSV and influenza virus to determine whether VSV inhibits influenza virus mRNA translation in a manner analogous to cellular translation or whether influenza virus is able to maintain translation as during a double infection with adenovirus. When BHK cells were doubly infected with VSV and influenza virus, there was a complete inhibition of influenza virus translation by 5 hr after infection (Fig. 6). When influenza virus-infected cells were superinfected with VSV after 1 hr, there was dramatic reduction of influenza virus-specific translation (Fig. 7). And when superinfection was delayed until 3 hr after influenza virus infection, synthesis of influenza virus proteins was only slightly inhibited. When duplicate cultures were labeled and harvested at 8 hr post infection, there was increased inhibition of translation of influenza virus mRNAs; however, some residual translation was observed. Inhibition of influenza translation was still evident when cells were superinfected as late as 6 hr after influenza infection (Fig. 9). Inhibition of influenza virus translation was not observed when cells were labeled 2 hr after VSV superinfection (or 8 hr after influenza virus infection), but was detected when the cells were labeled 5 and 8 hr after VSV infection (or 11 and 14 hr post-influenza virus infection). This experiment demonstrated that influenza virus mRNAs which were being translated 8 hr after influenza virus infection were no longer translated in cells which had been superinfected with VSV for an additional 3 hr. Thus, superin-

INHIBITORY

EFFECTS OF VESICULAR

fection of influenza virus-infected cells with VSV resulted in the disruption of ongoing influenza virus protein synthesis. This effect on influenza virus translation appears to be distinct from the inhibitory effect of VSV on influenza virus transcription; influenza mRNAs which had been transcribed priorto VSV superinfection were no longer translated following VSV superinfection. These results also suggested that there is not a point in the replication of influenza virus when protein synthesis was protected from the inhibitory effects of VSV. In addition, these results demonstrate that inhibition of influenza virus translation, like that of cellular translation, requires the VSV infection to proceed for at least 2 hr. One possible mechanism to account for these observations is that VSV infection results in the activation of the kinase, phosphorylation of elF2, and the subsequent inhibition of the initiation of protein synthesis. Influenza virus infection results in an activity which suppresses the activation of the kinase, allowing protein synthesis to be maintained at high levels. In both cases viral mRNAs may be preferentially translated over cellular mRNAs, because of their inherently high efficiency of initiation. During a double infection, one would expect that there would be a competition between the activation and suppression of activation of the kinase. Because the VSV mRNAs are preferentially translated during a double infection, it may be that they are able to successfully compete with the influenza virus and cellular mRNAs for the initiation complexes that are available. ACKNOWLEDGMENTS This work was supported by research grants from the National Instatutes of Health. J.D.K. IS a Pew Scholar In the Blomedlcal Sciences.

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