Translational Control in Murine Hepatitis Virus Infection - CiteSeerX

J.

gen. Virol. (1986), 67, 923-932. Printedin GreatBritain

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Key words: MHV/proteinsynthesis inhibition/actinmRNA degradation

Translational Control in Murine Hepatitis Virus Infection By ANNE HILTON,' LEE MIZZEN, 2 GEORGINA MACINTYRE, 2 STEVE CHELEY 3 AND ROBERT ANDERSON 2. 'Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1, Canada, 2Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta T2N 4N1, Canada and 3CoM Spring Harbor Laboratories, Box 100, Cold Spring Harbor, New York 11724, U.S.A.

(Accepted 7 February 1986) SUMMARY High multiplicity infection of mouse fibroblast L-2 cells with mouse hepatitis virus (MHV) resulted, within 6 h, in a decline in total protein synthesis to about 7 ~ of that observed in uninfected cells. The amount of intracellular total translatable RNA, however, increased approximately threefold, as a result of the accumulation of virusencoded mRNAs. MHV-infected cells could be superinfected with vesicular stomatitis virus, demonstrating that MHV infection did not irreversibly alter the cellular translational machinery to the exclusion of non-MHV mRNAs. Comparative polysome analysis from MHV-infected and uninfected L-2 cells showed that MHV infection resulted in an increase in single 80S ribosomes and in a shift from longer to shorter polysomes. These observations suggest first, that MHV infection inhibits total protein synthesis at a very early stage, as evidenced by the increase in 80S ribosomes, and, second, that the increased number of viral m R N A s produced after infection compete with cellular m R N A s for cellular ribosomes. In vitro translation of R N A extracted from MHV-infected and mock-infected cells suggested that levels of cellular m R N A s were decreased after infection. This suggestion was confirmed by demonstrating the loss of cellular actin m R N A , using a radiolabelled c D N A probe, as a consequence of MHV infection.

INTRODUCTION Inhibition of host cell protein synthesis is one of the major mechanisms by which virus infection results in host cell death. Translational inhibition by virus infection can occur in a number of ways, including degradation of cellular mRNAs (Nishioka & Silverstein, 1978; Rice & Roberts, 1983), disaggregation of cellular po]ysomes (Nishioka et aL, 1983), alteration of translational specificityin favour of viral mRNAs (Kaufmanet al., 1976; Traschselet al., 1980; Skup et al., 1981), or competitivedisplacement of cellular mRNAs by viral mRNAs for host translational machinery (Lodish & Porter, 1980, 1981; Golini et al., 1976; Jen et al., 1978). The coronavirusmouse hepatitis virus (MHV), for which the mechanism of host translational inhibition is unknown, producesinfectionsof varyingseverityin differenthost cell types (Lucas et al., 1978). In some cases, severity of infection is probably due to the degree of cytopathic effect, particularlycell fusion (Mizzen et al., 1983). A fullypermissive cell line such as the L-2 cell, however,also shows rapid inhibition of host protein synthesis within a few hours of MHV infection(Andersonet aL, 1979; Siddellet aL, 1980). Since, in other systems, host translational inhibition is a cell-killingfunction (Marvaldi et al., 1977),it is likelythat an analogous function is operativein MHV infectionwhich determines, at least in part, the severityof virus infection. We describe here studies aimed at elucidating the mechanism of MHV-induced inhibition of host cell translation in permissive L-2 cells. 0000-6898 © 1986 SGM

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A. HILTON AND OTHERS METHODS

Cells, virus and culture conditions. Monolayer cultures of mouse L-2 cells (Rothfels et al., 1959) were cultured at 37 °C in MEM supplemented with 5 ~ foetal calf serum (FCS). Suspension cultures of L-2 cells were grown at 37 °C with rotary shaking (140 r.p.m. ; New Brunswick Psycrotherm) in MEM containing 0.1 ~ methyl cellulose (15 centipoises) and 5 ~ FCS. Viruses used were the A59 strain (Manaker et al., 1961) of MHV and the Indiana serotype of vesicular stomatitis virus (VSV). Radiolabelling. Confluent cultures of L-2 cells grown in 35 mm plates (1 × 108 cells/plate) were inoculated at a m.o.i, of 10 with MHV, absorbed for 30 min at room temperature and then incubated at 37 °C in 2 ml MEM supplemented with 5 ~ FCS. For cultures to be superinfected with VSV the inoculation was repeated in analogous fashion with VSV at an m.o.i, of 10. Radiolabetling was performed by replacing the medium at appropriate times with 0.5 ml of methionine-free medium containing [35S]methionine (50 gCi/ml, 985 Ci/mmol; Amersham). After renewed incubation at 37 °C for 30 min, cultures were harvested and examined either for TCA precipitable radioactivity or for radiolabelled polypeptides by ftuorographic SDS PAGE (Cheley & Anderson, 1981). RNA extraction and in vitro translation using reticulocyte lysate. Total RNA was extracted (Cheley et al., 1981) from monolayer cultures of MHV-infected and mock-infected L-2 cells. RNA was quantified spectrophotometrically by monitoring absorbance at 260 nm. In vitro translation was performed in incubation mixtures of 30 gl using a reticulocyte lysate kit (New England Nuclear), containing ~3sS]methionine (1 mCi/ml) and various amounts of extracted RNA. Incubation conditions were those recommended by the manufacturer, with KC1 and magnesium acetate concentrations of 87 mM and 1.0 mM respectively, found to be optimal for translation of extracted RNA. Following 60 min incubation at 37 °C, the reactions were terminated by the addition of dissociation buffer (Cheley & Anderson, 1981) and the mixtures were subjected to gel electrophoresis followed by fiuorography. Polysome analysis. Monolayer cultures of L-2 cells (1 x 106 cells) in 35 mm dishes were incubated for 24 h at 37 °C with 2 ml MEM supplemented with 5 ~ FCS and [5-3H]uridine (3 ~tCi/ml). Thereafter, the labelling medium was removed, the cultures washed with non-radioactive medium, and subsequently 'chased' for 48 h at 37 °C in 2 ml MEM supplemented with 5 ~ FCS. Cultures were then inoculated with MHV (m.o.i. 10) or mock-infected and harvested at 6 h post-infection. One mock-infected culture was treated hypertonically by exposure to medium containing 335 mM-NaCI (Saborio et al., 1974) for 20 rain prior to harvesting. Post-nuclear supernatants were prepared and subjected to sucrose gradient centrifugation, according to the method of Jaye et al. (1982), with the exception that a Beckman SW56 rotor (2 h at 45000 r.p.m.) was used instead of an SW27. Following centrifugation, the gradients were fractionated by drop-collection and radioactivity determined in each fraction by liquid scintillation spectrometry (using Atomlight; New England Nuclear). Hybridization assay Jbr cellular actin mRNA. A cloned cDNA insert encoding the chicken fl-actin sequence (Cleveland et al., 1980) was radiolabelled by nick translation using [3,,P]dCTP (3000 Ci/mmol; Amersham) by the method of Rigby et al (1977). RNA samples (approx. 0.1 gg), prepared from mock-infected and MHV-infected (m.o.i. 10) L-2 ceils, harvested at various times after infection, were subjected to dot-blot hybridization (Cheley & Anderson, 1984). Parallel RNA samples (25 gg) were electrophoresed in a formaldehyde agarose gel and subjected to Northern blot analysis essentially according to the procedure of Rice & Roberts (1983). Completeness of transfer was verified by methylene blue staining of the nitrocellulose blots. As a negative control, 9 gtg of a HindIII digest of phage lambda DNA was included in the gel electrophoresis and transfer procedure.

RESULTS

Decline in total cellular protein synthesis following M H V infection Previous observations by ourselves (Anderson et al., 1979) and others (Siddell et al., 1980) have shown that MHV infection often results in an apparently selective inhibition of host protein synthesis. During the course of the present investigation, we noted in addition that levels of total protein synthesis declined markedly following MHV infection. This feature is illustrated in Fig. 1, in which TCA-precipitable counts from [35S]methionine-labelled L-2 cells (expressed as a percentage relative to uninfected controls) are recorded against increasing time of infection with MHV. Several features are worthy of note. First, total protein synthesis declined considerably, starting at 3 h post-infection, eventually reaching only 7 ~ of its original level by 6 h. Second, the decline in total protein synthesis was due essentially to diminished translation of host cell polypeptides, seen most clearly by the abrupt decline in synthesis between 2 and 4 h after infection. Third, the synthesis of virus-specific polypeptides, which was first evident at 3 h post-infection, proceeded rapidly until (by 5 and 6 h) it accounted for virtually all protein synthesis in the infected cell.

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2 3 4 5 6 1 2 3 Time post-infection (h) RNA (gg) Fig. 1 Fig. 2 Fig. 1. Decline in protein synthesis in L-2 cells following MHV infection. MHV-inoculated cultures were radiolabelled with [3SS]methionine for 30 min at various times post-infection. Total incorporated radioactivity (F3), assayed as TCA-precipitable c.p.m., is expressed as a percentage of the uninfected sample (0 b). The relative amounts of viral (O) and cellular (©) polypeptides were determined densitometrically from SDS-PAGE fluorographs of the individual samples. Fig. 2. Increase in amount of translatable RNA in L-2 cells following MHV infection. Total RNA was extracted from MHV-inoculated cultures at 0 (O), 4 (O) and 6 (A) h post-infection. Aliquots of 1,2 or 3 gg total RNA were subjected to in vitro translation with [3sS]methionine in a reticulocyte lysate-derived system. The products of translation were detected by fluorographic SDS PAGE and quantified by densitometric scanning.

Levels of translatable RNA are increased after M H V infection A common feature of virus infections is that there is an increase in intracellular m R N A levels as a result of the production of viral R N A transcripts. The resultant translational competition between cellular and viral m R N A s for a limiting number of host ribosomes and associated factors may result in increased synthesis of viral polypeptides at the expense o f host-encoded o n e s (Lodish & Porter, 1980). In order to examine total m R N A levels in an infection with M H V , total R N A was extracted from uninfected and MHV-infected cells and translated in an m R N A dependent reticulocyte lysate system, using [35S]methionine as radiolabelled amino acid. Following incubation, the translation mixtures were subjected to autoradiographic S D S P A G E , and the relative amounts of incorporated [3SS]methionine were quantified by densitometric scanning. As shown in Fig. 2, there was a m a r k e d increase in total translatable R N A levels with increasing time of M H V infection. By 4 h post-infection, the amount of translatable R N A was found to be about 2.5-fold higher than that found in uninfected cells; by 6 h, the amount had increased to three-fold.

Superinfectibility of" MHV-infected cells by V S V Certain viruses, such as poliovirus, alter the translational specificity of the host cell so that only viral m R N A s are translated into proteins ( K a u f m a n et al., 1976). In order to investigate whether the translational machinery of MHV-infected cells retains the capacity to translate m R N A s other than those specified by M H V , protein synthesis was examined in MHV-infected cultures which had been superinfected with VSV. VSV was chosen as the superinfecting virus since it has been shown that it gives rise to an infection in which its encoded m R N A s are able to

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A. H I L T O N A N D O T H E R S

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Fig. 3. Superinfectibility of MHV-infected cells by VSV. Cultures of MHV-inoculated L-2 cells were superinfected with VSV at 5 (lane 3), 4 (lane 4), 3 (lane 5) or 2 (lane 6) h post-infection. Parallel cultures were mock-infected (lane 1) or infected singly with MHV (lane 2) or VSV (lane 7). All cultures were radiolabelled with [35S]methionine for 30 min at 6 h after MHV inoculation. Equal volumes of sample were applied to lanes 2 to 7; lane 1 received a one-tenth amount. Cell extracts were analysed by fluorographic SDS-PAGE.

compete against cellular (i.e. non-VSV) m R N A s for the host translational machinery (Lodish & Porter, 1981). Thus, any synthesis of VSV-specified polypeptides by cells which had been preinoculated with M H V would suggest that M H V did not irreversibly alter the translational specificity of the host cell. MHV-infected L-2 cells were superinfected with VSV at 2, 3, 4 and 5 h post-infection. The cells were labelled with [35S]methionine for 30 min immediately prior to harvesting at 6.5 h, and radiolabelled polypeptides were analysed by fluorographic S D S - P A G E (Fig. 3). The three major structural polypeptides of M H V (E2, N and PEI/E~) (Cheley & Anderson, 1981 ; Cheley et al., 1981) were evident in cells infected with M H V (Fig. 3, lane 2). Synthesis of cellular polypeptides was strongly inhibited by M H V infection (compare lanes 1 and 2). A somewhat less strong inhibition of cellular protein synthesis was observed after VSV infection (lane 7). Superinfection by VSV of MHV-infected cells, at progressively earlier times

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40 50 10 20 30 40 50 Fraction number Fig. 4. Comparative polysome analysis of MHV-infected cells (a) and hypertonically treated uninfected cells (b). L-2 cell cultures were radiolabelled for 24 h with [3H]uridine and then 'chased' for 48 h; replicate cultures were then MHV-infected (a, O), mock infected (a and b, O) or mock-infected and treated with hypertonic medium for 20 min prior to harvesting (b, O). Cell extracts were fractionated by sucrose gradient centrifugation, and radioactivity was determined in collected fractions. Fraction 0 represents the bottom of the gradient. P designates polysomes. 0

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(lanes 3 to 6) led to progressively higher levels of synthesized VSV polypeptides. Most importantly, the results clearly show that M H V infection, unlike that of poliovirus, did not permanently alter the translational specificity of the host cell to the exclusion of non-MHV mRNAs.

Comparative polysome analysis of uninfected and MHV-infected cells The increase in total translatable R N A observed in L cells after M H V infection (Fig. 2) suggests that viral and cellular m R N A s would compete for the host ribosomes. If the number of host ribosomes is limiting, one would expect to see a reduction in polysome size in infected, as compared to uninfected, cells. As shown in Fig. 4(a), there was indeed a shift from heavier to lighter polysomes in L-2 cells as a result of M H V infection. In addition, however, there was a decrease in the total amount of polysomes and an increase in the amount of single 80S ribosomes. For comparison, Fig. 4(b) shows a polysome profile from L-2 cells inhibited at the stage of translational initiation by treatment with hypertonic medium (Saborio et al., 1974). In this latter case, there were no detectable polysomes; virtually all ribosomes were in the single 80S state. Thus, it would appear that although increased m R N A competition may account for the decreased polysome size found in MHV-infected cells, the substantial increase in 80S ribosomes is suggestive of inhibition at the initiation stage of translation. Such inhibition is likely to be responsible for the large decline in total protein synthesis observed in L-2 cells following M H V infection (Fig. 1).

Evaluation of competition in vitro between MHV-induced and cell mRNAs An estimate of the degree of virus-induced host translation inhibition which could be attributable to competition among cellular and viral m R N A s was obtained by the following experiment. Total R N A extracted from uninfected and MHV-infected L-2 cells was subjected to

A. HILTON AND OTHERS

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Fig. 5. In vitro translation analysis of RNA from uninfected and MHV-infected cells. Total RNA was extracted from MHV-infected cultures at 0 (a) 4 (b) and 6 (c) h post-infection, and samples of 1, 2 and 3 ~tg RNA were translated in vitro using a reticulocyte lysate-derived system. ( - ) designates no RNA added. in vitro translation using a reticulocyte lysate system. Viral polypeptides identified by in vitro translation of MHV-infected L cell R N A included the N and PE1 polypeptides as well as an approx. 150000 mol. wt. polypeptide. This latter polypeptide had a lower apparent molecular weight than E2 and is probably an incompletely glycosylated precursor (pre-Ez). A similar E2 precursor has been identified in cell-free translation studies by others (Leibowitz et al., 1982; Rottier et al., 1981; Siddell, 1983). In vitro translation of isolated R N A from MHV-infected cells, harvested at 0, 4 and 6 h postinfection, showed a progressive decline in the synthesis of host cell-specified polypeptides (Fig. 5). Densitometric scanning of the autoradiogram allowed quantification of the synthesis of cellular polypeptides. R N A extracted from MHV-infected cells at 4 h post-infection directed the synthesis of numerous proteins, of which 6 0 ~ of the total were host cell-encoded. Of the total protein translated from R N A extracted at 6 h post-infection (Fig. 5), only 1 0 ~ was host cellencoded. Surprisingly, this observed reduction in translation of host cell m R N A was noted under conditions of non-saturating amounts of translatable R N A (i.e. 1 or 2 gg R N A ; see Fig. 2), suggesting that cellular m R N A s may be selectively lost as a consequence of M H V infection. Hybridization assay for cellular actin m R N A in cells infected with M H V

Virus-induced degradation of cellular m R N A s has been reported in certain virus infections (Rice & Roberts, 1983; Nishioka & Silverstein, 1978; Plotch et al., 1981) but not in others (Gallwitz et al., 1977; Fernandez-Munoz & Darnell, 1976; Lodish & Porter, 1980). Since the results from in vitro translation (Fig. 5) indicated an apparent decrease in the levels of cellular m R N A s , the possibility was investigated that M H V infection might cause the degradation of

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Translational control in M H V infection (a)

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Fig. 6. Dot-blot (a) and Northern blot (b) hybridization analysis of actin mRNA in L-2 cells, either mock-infected (C) or MHV-infected, harvested at 2, 4 or 6 h post-infection. The mock-infected sample was also harvested at 6 h. As a negative control, 9 gg of a HindIII digest of lambda DNA (L) was also subjected to the Northern blot analysis. Hybridization was performed with 32p-labelled fl-actin cDNA. The blots were washed according to standard procedures and exposed for autoradiography.

cellular m R N A such as that encoding actin. Using a radiolabelled actin c D N A probe, actin m R N A was assayed in mock-infected and MHV-infected cells by dot-blot hybridization and Northern blotting (Fig. 6). As shown in Fig. 6 (a), the level of actin m R N A remained relatively constant for the first 2 h of infection, but started to decline by 4 h, and fell off rapidly by 6 h (to less than 1 0 ~ of its level in mock-infected cells). The degree of reduction in the level of actin m R N A could not be explained on the basis of turnover, since parallel experiments on L-2 cells infected with VSV, which is known to inhibit cellular R N A synthesis (Wagner & Huang, 1966), only showed a slight decrease in actin m R N A over a 6 h period. Analysis of actin m R N A by Northern blotting (Fig. 6b) confirmed the dot-blot results and suggested that the degradative removal of actin m R N A proceeded by an efficient pathway, since no smaller actin-related R N A fragments were detected. DISCUSSION Certain viruses utilize relatively specific means to ensure inhibition of translation of hostencoded m R N A s . Such means include the operation of viral functions to apply translational discrimination against cellular m R N A s [as in poliovirus infection (Kaufman et al., 1976) or in vaccinia virus infection (Coppola & Bablanian, 1983)] or to effect selective degradation of cellular m R N A s (as in herpesvirus infection: Nishioka & Silverstein, 1978). In the cases of certain RNA-containing viruses such as VSV, mengovirus and encephalomyocarditis virus, it is generally agreed that alteration of cellular protein synthesis involves one or more processes such

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as inhibition of translational initiation (Colby et al., 1974; Nuss et al., 1975; Golini et al., 1976; Jaye et al., 1982), some degree of competition between viral and cellular mRNAs (Abreu & Lucas-Lenard, 1976; Lodish & Porter, 1980; Lawrence & Thach, 1974) and other less understood processes (Stanners et al., 1977; Schnitzlein et al., 1983; Jen & Thach, 1982). It is evident from the results presented in this study that while mRNA competition may play a role early (3 to 4 h post-infection) in MHV infection of L-2 cells, other factors, particularly the degradation of cellular mRNAs, are responsible for the overwhelming subversion of host cell protein synthesis which is observed at 5 to 6 h post-infection. In the face of cellular mRNA degradation, the observed threefold increase in total translatable RNA found at 6 h is all the more impressive, and we note that by this time about 90~o of the intracellular mRNA is viruscoded (Fig. 5). With respect to the degradation of cellular mRNAs, MHV shares characteristics associated with infections by the large DNA viruses herpes simplex virus (Nishioka & Silverstein, 1978) and vaccinia virus (Rice & Roberts, 1983) which have also been implicated in breakdown of cellular mRNA species. The degradation of cellular mRNAs has also been noted in infection with the RNA-containing influenza virus (Plotch et al., 1981), although this appears to be a specialized mechanism related to the requirement of influenza virus for scavenged 5' cap structures for use as primers in RNA transcription (Bouloy et al., 1980). We are currently investigating the mechanism behind the apparent selective degradation of cellular mRNAs which is a most interesting aspect of MHV infection. We also present evidence that MHV infection inhibits overall protein synthesis at a very early stage, as indicated by an increase in single 80S ribosomes in the cell following MHV infection. A similar increase in free 80S ribosomes was observed after hypertonically blocking the initiation stage of translation in uninfected L-2 cells (Fig. 4; see also Saborio et al., 1974). The increase in 80S ribosomes observed in MHV-infected cells may alternatively be due to inhibition of the normal dissociation of'run-off' 80S ribosomes into their 40S and 60S subunits. In either case the effect appears to be one in which the normal progression of free ribosomes into polysomes is inhibited. This inhibition is likely to be responsible for the gross decline in total protein synthesis observed following MHV infection (Fig. 1). In this regard, MHV infection shares similarities with infection by VSV (Lodish & Porter, 1980; Jaye et al., 1982) in which both an increase in free 80S ribosomes, and a shift from heavier to lighter polysomes is observed. Most cellular and viral mRNAs contain a 5' terminal cap (Shatkin, 1976). In the case of poliovirus which gives rise to uncapped mRNA, translational specificity is ensured by the inactivation of a host cap-recognition factor (Kaufman et al., 1976; Trachsel et al., 1980). Changes in the characteristics of host initiation factors have also been implicated in translation inhibition induced by viruses which give rise to capped (Centrella & Lucas-Lenard, 1982; van Steeg et al., 1981), uncapped (Jen & Thach, 1982) or both capped and uncapped (Ray et al., 1983 ; Skup et al., 1981) mRNAs. MHV mRNAs contain normal cap structures (Lai et al., 1982) and so it would appear unlikely that MHV-mediated inhibition of host protein synthesis involves, for example, inactivation of cap-binding factor. Furthermore, the results of the present study suggest that much of the apparent translational preference for viral mRNAs observed in MHV infection is determined by competition within the intracellular mRNA pool, particularly in view of the selective loss of cellular mRNAs. REFERENCES AI~REU, S. L. & LUCAS-LEYARD,J. (1~76). Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral m R N A ' s in extracts from uninfected and infected Ehrlich ascites tumor cells. Journal of Virology 18, 182 194. ANDERSON, R., CHELEY, S. & HAWORTH-HATHERELL,E. (1979). Comparison of polypeptides of two strains of murine hepatitis virus~ Virology 97, 492-494. BOULOY,M., PLOTCH,S. J. & KRUG, R. M. (1980). Both the 7-methyl and the 2'-O-methyl groups in the cap of m R N A strongly influence its ability to act as primer for influenza virus R N A transcription. Proceedings of the National Academy of Sciences, U.S.A. 77, 3952-3956. CENTRELLA, M. & LUCAS-LENARD,J. (1982). Regulation of protein synthesis in vesicular stomatitis virus-infected mouse L-929 cells by decreased protein synthesis initiation factor 2 activity Journal of Virology 41, 781-796.

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w. c. & TrlACH, a. E. (1983). Role of m R N A competition in regulating translation: further characterization of m R N A discriminatory initiation factors. Proceedings of the National Academy of Sciences, U.S.A. 80, 663667. RICE, A. P. & ROBERTS,B. E. (1983). Vaccinia virus induces cellular mR N A degradation. Journal of Virology 47, 529 539. RIGBY, P. W. J., DIECKMANN, M., RHODES, C. & BERG, P. (1977). Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with D N A polymerase I. Journal of Molecular Biology 113, 237-251.

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(Received 10 October 1985)