Metabolism of host and viral mRNAs in frog virus 3-infected cells

VIROLOGY

186,435-443

(1992)

Metabolism

of Host and Viral mRNAs in Frog Virus 34nfected V. G. CHINCHAR’

Department

of Microbiology,

University

Received September

of Mississippi

AND

WEI YU

Medical

9, 199 1; accepted

Cells

Center, Jackson, Mississippi

392 16

October 2 1, 199 1

Treatment of purified frog virus 3 (FV3) with nonionic detergent and high salt released an endoribonucleolytic activity and confirmed earlier findings of a virion-associated endonuclease. This observation, coupled with evidence implicating host and viral message destabilization in herpesvirus and poxvirus biogenesis, raised the question of what role, if any, mRNA degradation plays in FV3 replication. To answer this question, Northern analyses of mock- and virus-infected cells were performed using probes for representative host and viral messages. These studies demonstrated that the steady state level of host messages progressively declined during the course of productive FV3 infection, whereas the steady state level of viral messages was not affected. To determine whether the decline in the steady state level of host mRNA was due to virus-induced degradation or to normal turnover coupled to virus-mediated transcriptional shut-off, actin mRNA levels were examined in mock- and virus-infected cells in the presence and absence of actinomycin D. Under these conditions, actin mRNA levels declined more quickly in actinomycin D-treated, virus-infected cells, than in mock-infected cells incubated in the presence of actinomycin D suggesting that the decline in the steady state level of actin mRNA was due to degradation. However, although it appears as if host message degradation is responsible for virus-mediated translational shut-off, the ability of heat-inactivated FV3 to block cellular translation without destabilizing cellular messages indicates that message degradation is not required for translational inhibition. As noted above, the degradation of early FV3 messages was not involved in controlling the transition from early to late gene expression. Furthermore, the presence of abundant, but nontranslated, early messages late in infection, coupled with the inefficient translation of late messages in vitro supported earlier suggestions that FV3 gene expression is controlled, at least in part, at the translational level. Taken together, these results suggest that FV3 regulates gene expression in a unique manner and may be a good model to examine the mechanics of translational control. o 1992 Academic

Press,

Inc.

INTRODUCTION Virus infection is often accompanied by the rapid and selective inhibition of host cell protein synthesis (Kozak, 1986; Schneider and Shenk, 1987; Sonenberg, 1987). Over the past several years, a variety of mechanisms responsible for host translational shut-off have been proposed. These include (a) the proteolytic inactivation of initiation factors required preferentially by host messages (Etchinson, et a/., 1984), (b) the competitive displacement of host transcripts from the cellular translational apparatus by either abundant or highly efficient viral messages (Lodish and Porter, 1980; Walden et al., 1981; Rosen et al., 1982) (c) changes in the ionic environment that favor viral over host translation (Carrasco and Smith, 1976), and (d) the selective degradation of host messages (Rice and Roberts, 1983; Mayman and Nishioka, 1985; Schek and Bachenheimer, 1985; Agy et a/., 1990). In our laboratory, we are currently exploring the basis for translational shut-off in frog virus 3 (FV3)-infected cells. Recently we have suggested that translational shut-off is a consequence of two events: (a) the phosphorylation (and functional inactivation) of the (Y subunit of eukaryotic initiation factor 2 (elF-2) and (b)

the ability of highly efficient viral messages to outcompete less efficient host transcripts for the remaining translational capacity of the cell (Chinchar and Dholakia, 1989; Chinchar and Yu, 1990a,b). Although the two mechanisms cited above appear to be the key determinants of FV3-mediated shut-off, other events, such as message destabilization, may also influence host and viral protein synthesis. Early work showed that purified virions contained an endoribonuclease (Kang and McAuslan, 1972). However, it is not known whether this protein is virus- or host-coded, nor is it clear what role, if any, it plays in FV3 replication. Furthermore, since heat-inactivated FV3 blocks host translation without degrading cellular messages (Raghow and Granoff, 1979) it appears that a direct role for the virion-associated ribonuclease in virus-mediated shut-off is unlikely. In view of the uncertainty surrounding the role of message destabilization in FV3 biogenesis, we wished to examine in more detail the metabolism of host and viral messages during the course of a productive FV3 infection.

’ To whom correspondence dressed.

Fathead Minnow (FHM) cells were grown at 30” in Eagle’s minimum essential medium containing Hank’s

and reprint requests

MATERIALS

AND METHODS

Cells and virus

should be ad-

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CopyrIght 0 1992 by Academic Press, Inc. All rights of reproduction in any form resewed.

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salts supplemented with 5% fetal calf serum (GIBCO). Frog virus 3 was grown and titered in FHM cells as described previously (Raghow and Granoff, 1979). FV3 was heat-inactivated at 56” for 30 min (Goorha and Granoff, 1974).

Preparation of a soluble virion extract Preparation of a soluble virion extract (SVE) was as described by Aubertin et a/. (1977, 1980) with slight modification. FV3 virions (6.7 X 10”) were centrifuged through 43% (w/v) sucrose prepared in 10 mM TrisHCI, pH 8.5 (T8.5) for 4 hr at 25,000 rpm in a Beckman SW27 rotor at 4”. The pellet was resuspended in T8.5, layered over 1O-40% (w/w) sucrose gradients prepared in T8.5, and centrifuged for 45 min at 11,000 rpm in a Beckman SW27 rotor at 4”. The diffuse virus band was removed and diluted with T8.5. Virions, recovered by centrifugation at 29,000 rpm for 60 min at 4” in a Beckman Type 30 rotor, were layered over 3565% (w/v) sucrose gradients and banded by isopycnic centrifugation (16 hr, 25,000 rpm, Beckman SW 27 rotor, 4”). The compact virus bands were removed, pooled, diluted with T8.5, and virions recovered bycentrifugation (60 min, 29,000 rpm, Beckman Type 30,4’). Purified virions were resuspended in 2 ml T8.5, made 0.5% in Nonidet P-40, and incubated at room temperature for 5 hr. The sample was subsequently dialyzed for 3 days against 25 mM Tris-HCI, pH 8.5 at 4”, then made 5 M with respect to LiCl and incubated on ice for 6 hr. Following LiCl treatment, virions were dialyzed for an additional 3 days against 25 mM Tris-HCI, pH 8.5, and the soluble virion extract (SVE) separated from viral cores by centrifugation (40,000 rpm, 60 min, Type 65 rotor, 4”). The SVE was concentrated about lo-fold using an Ultracent(Bio-Rad) ultrafiltration membrane.

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Isolation of total cellular RNA FHM cells (x4.0 x 1O7 cells/l 00-mm dish) were infected as described above and total RNA was isolated by the method of Paucha and Condit (1985). Briefly, cells were washed once with 10 ml ice-cold phosphate buffer saline (PBS) and lysed by adding 1 ml 4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sodium lauryl sarcosinate, and 0.1 M 2-mercaptoethanol to each loo-mm dish. Replicate lysates were pooled, CsCl(O.8 g) was added, and the samples were vortexed to shear DNA. Cell extracts were layered over 2 ml of 5.7 M CsCI, 100 mM EDTA, pH 7.5, and centrifuged in a Beckman SW50.1 rotor at 35,000 rpm for 15 hr at 20”. The RNA pellet was resuspended in 1 ml TE buffer (10 mM Tris-HCI pH 8.0; 1 mM EDTA), extracted with 1 vol of butanol:chloroform (1:4), and precipitated with 3 vol 1009/o ethanol. The resulting pellet was resuspended in TE at 5 pg/ml and stored at -20” until use.

In vitro cell-free translation Total cellular RNA (400 pg/mI), extracted from mockand FV3-infected FHM cells, was added to messagedependent wheat germ extracts (Promega) and translated in the presence of 130 mM KOAc, 2.5 mM magnesium acetate, and 300-400 &i/ml [35S]methionine as directed by the supplier (Chinchar and Yu, 1990a). Reactions were incubated for 60 min at 25” and stopped by adding 3 vol 125 mM Tris-HCI, pH 6.8, 20% glycerol, 2% SDS, and 2% 2-mercaptoethanol (Laemmli, 1970). Samples were boiled for 3 min and equal volumes of lysates analyzed by electrophoresis on 1Oq/o SDS-polyacrylamide gels. Radiolabeled proteins were visualized by autoradiography.

Analysis of mRNA distribution on polysomes In vivo protein synthesis FHM cells (=2 X 1O6 tells/35-mm dish) were infected with FV3 at 50 PFU/cell and incubated at 30”. After allowing 1 hr for adsorption, the inoculum was removed and incubation continued in Basal Media Eagle (Sigma) containing 2% fetal calf serum. At the indicated times after infection, the cells were radiolabeled in methionine-free Eagle’s minimum essential medium containing Earle’s salts (Sigma) and 20 &i/ml [“?S]methionine (Amersham). After 1 hr, the cells were lysed in buffer containing sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and radiolabeled proteins separated by electrophoresis on 10% SDS-polyacrylamide gels (Laemmli, 1970). Radiolabeled proteins were visualized by autoradiography using Kodak Xomat RP film.

Polysome profiles were obtained by modification of the method of Pachter et a/. (1987). One hundred-millimeter dishes of FHM cells at subconfluent density were infected with FV3 at 50 PFU/cell. At 2, 6, and 10 hr postinfection, three loo-mm dishes were washed with 10 ml ice-cold PBS, and the cells from each dish lysed with 200 ~1 10 mM Tris-HCI, pH 8.4, 10 mM NaCI, 3 mM MgCI,, 0.5% v/v Nonidet-P40, 100 pg/ml cycloheximide, and 660 U/ml RNasin (Promega). Lysates were transferred to 1.5-ml microcentrifuge tubes, vot-texed for 25 set, centrifuged at low speed to remove nuclei, and stored at -80”. To determine the distribution of mRNA on polysomes, lysates (containing -200 pg RNA) were layered over 15-4096 (w/v) sucrose gradients prepared in 10 mM Tris-HCI, pH 8.4, 10 mM NaCI, and 1.5 mM MgCI, and centrifuged

METABOLISM

OF HOST AND VIRAL mRNAs IN FROG VIRUS 3-INFECTED

at 33,000 rpm for 2 hr at 4” in a Beckman SW41 rotor. One-milliliter fractions were collected into sterile tubes containing 10 ~1 500 mll/l EDTA and precipitated overnight at -20” by adding 3 ml lOOOh,ethanol. Precipitates were collected by centrifugation, resuspended in 10 mM NaCI, 1 mlLl EDTA, 10 mM Tris-HCI, pH 8.0, extracted once with 1 vol of phenol chloroform and once with 1 vol of chloroform, and reprecipitated with ethanol. Subsequently the RNAfrom each fraction was resuspended in 10 ~1 TE buffer. Northern

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To determine the steady state levels of host and viral mRNA and the polysomal distribution of messages, 10 pg of total cellular RNA from mock- and virus-infected cells, or RNAfrom the entire polysomefraction, respectively, were electrophoresed on 1o/oagarose gels containing 2.2 M formaldehyde (Maniatis et a/., 1982). To ascertain that all lanes contained equal amounts of RNA, samples were prepared as described by Maniatis er al. (1982) except that ethidium bromide was added to a final concentration of 125 pg/ml and the samples denatured at 55” for 15 min. After electrophoresis, the 18s and 28s ribosomal RNA bands were visualized by uv light and the relative levels of RNA compared. Ethidium bromide did not adversely affect the transfer of RNA to nitrocellulose and provided a simple visual method to determine whether all lanes contained equivalent amounts of RNA. After transfer, the membranes were prehybridized for 4 hr, then hybridized for 14-20 hr with [32P]cDNA probes for /3-actin, and the FV3 18-kDa early protein (18K) and the FV3 48-kDa late protein (48K). After washing, specific annealing was detected by autoradiography. [32P]cDNA probes for actin, 18K and 48K mRNA were prepared by random priming (Feinberg and Vogelstein, 1983) using a commercial kit (Multiprime, Amersham). The chicken &actin probe was obtained from D. W. Cleveland (Cleveland et al., 1980) and the FV3 early and late probes from D. B. Willis (unpublished data). The 18K early probe (pSP6-18XB2), contains a 2 kbp Xbal-Bglll fragment (derived from the 3.5 kbp FV3 Xbal-K fragment) cloned downstream from the SP6 polymerase promoter of pSP18 (BRL). The late gene probe (PBSICR534) contains a 1.6 kbp fragment bearing just the 48-kDa capsid protein coding region (excised from the FV3 Sall-F fragment), cloned downstream from the T3 polymerase promoter of “bluescript M 13” (Stratagene). RESULTS Virion-associated

endonucleolytic

activity

To confirm that FV3 virions contain an endoribonuclease activity and to determine whether this activity

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FIG. 1. Effect of SVE on Protein Synthesis in vitro. (A) SVE was prepared by sequential detergent/high salt drssociation of purified FV3. After centrifugation to remove viral cores, the protein composition of the SVE was determined by SDS-polyacrylamide gel electrophoresis. Lane S, SVE; lane V, purified FV3; lane M. mol wt markers (size in kilodaltons). (B) BMV RNA was translated in wheat germ extracts supplemented with SVE (S, lane 4) or heat-denatured SVE (A, lane 3) and the products were analyzed by SDS-polyacrylamide gel electrophoresis. As a control to ensure that dialysis removed LiCl and Nonidet P-40 (NP-40) and did not introduce adventious nucleases and/or inhibitors, buffer alone (T, 25 mM Tris-HCI, pH 8.0. lane 1) or buffer with 0.59/o NP-40 (TN, lane 2) was treated in parallel with virus. Arrowheads indicate the positions of the three authentic BMV translation products, whose mol wt (in kilodaltons) is shown to the right of(C). (C) BMV RNA was translated in message-dependent wheat germ extracts supplemented with SVE (SVE, lanes 7 and 8) or heat-denatured SVE (ASVE, lanes 5 and 6) and the products were analyzed by SDS-polyacrylamide gel electrophoresis. Where indicated, reactions were incubated in the presence (+, lanes 5 and 7) or absence (-, lanes 6 and 8) of RNAsin.

was responsible for the translational inhibition observed by Aubertin and her co-workers (Cordier et a/., 1981) in a soluble virion extract (SVE), purified virus was dissociated by sequential detergent/high salt treatment and the SVE assayed for its protein composition and its ability to block translation in vitro. Figure 1(A) shows that the SVE contained predominantly the 48-kDa major capsid protein along with smaller amounts of other viral polypeptides. When SVE was added to a wheat germ cell-free translation system, translation of brome mosaic virus (BMV) RNA was markedly inhibited (Fig. 1 B, lanes l-4). Consistent with earlier work (Cordier et al., 1981), we found that heat denaturation eliminated SUE’s ability to block BMV RNA translation. To determine whether the SVE contained (or induced) a ribonuclease-like activity, the above experiment was repeated except that the in vitro reactions were supplemented with RNasin, a commercially available ribonuclease inhibitor. As shown in Fig. 1C, lanes 5-8, the addition of RNasin blocked the ability of SVE to inhibit BMV translation. This result sug-

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FIG. 2. FV3 Protein Synthesis in viva and in v&-o. Protein synthesis was monitored in mock-infected (Mock) or in FV3-infected FHM cells as described under Methods. Proteins were radiolabeled in viva for 1 hr beginning at 2, 6, and 10 hr after infection and analyzed by SDSpolyacrylamide gel electrophoresis (a). In addition, total RNA, prepared from mock-infected or FV3-infected FHM cells at the indicated times, was translated in message-dependent wheat germ extracts and the products were analyzed by SDS-polyacrylamide gel electrophoresis (b). The positions of mol wt markers (in kilodaltons) are shown to the left of the panel. The FV3 18.kDa (18K) early protein and the 48.kDa (48K) late protein are identified along with several other early (double arrowheads) and late (single arrowheads) proteins.

gests that the inhibitory ability of SVE is due primarily to the presence of a ribonuclease-like activity and is consistent with earlier observations that purified virions contain an endoribonuclease.

mRNA stability: Protein synthesis assays To determine the role of ribonuclease in FV3 biogenesis, we analyzed indirectly the complexity of functional cellular and viral mRNA in mock- and FV3-infected cells by monitoring protein synthesis in vivo and in vitro. FHM cells were mock-infected or infected with FV3, and at 2, 6, and 10 hr after infection, replicate cultures were either labeled with [35S]methionine or processed for RNA extraction. Fig. 2 (lane a) shows the result of in vivo labeling. It is clear from Fig. 2 that mock-infected cells synthesized a wide range of polypeptides, and that virus infection resulted not only in the progressive inhibition of cellular protein synthesis but also in the synthesis of more than 20 virus-coded proteins. When total RNA, prepared from replicate cultures, was translated in wheat germ extracts (Fig. 2, lane b) or rabbit reticulocyte lysates (data not shown), abundant amounts of viral proteins were synthesized. Note, however, that in viva and in vitro polypeptide profiles were not identical. For example, late polypeptides, which were abundantly synthesized in vivo at 6

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and 10 hr after infection, were synthesized less efficiently in vitro (compare lanes a and b in the 6- and 10-hr samples). This observation is consistent with the earlier suggestion of Raghow and Granoff (1983) that viral-coded or virus-induced factors are necessary for the efficient synthesis of late viral mRNAs in vitro. In contrast, we observed that at least two early proteins (the 18-kDa protein and second protein of about 31 kDa), whose synthesis was markedly reduced in viva at late times after infection, were among the major polypeptides synthesized in cell-free extracts programmed with RNA prepared from cells at 6 and 10 hr after infection. The latter observation suggests two tentative conclusions: (a) unlike events in other viral systems (Kwong and Frenkel, 1987; Rice and Roberts, 1983; Meis and Condit, 1991) early viral messages were not degraded in FV3-infected cells, and (b) the inability to translate some early messages (i.e., those encoding the 18- and 31 -kDa proteins) late in infection may be due to the presence of a transacting translational repressor which is removed from early viral messages upon phenol-chloroform extraction.

mRNA Stability: Northern analyses To extend these observations and determine the fate of host and viral messages during infection, total RNA from mock- and virus-infected cells was subjected to Northern analysis. Ten-microgram aliquots of total cellular RNA from mock- and virus-infected cells were electrophoretically separated, transferred to nitrocellulose, and hybridized to probes for chicken P-actin, and the cognate mRNAs for the FV3 18-kDa early protein (18K), and the 48-kDa late protein (48K). To ensure that the absence of hybridization was due to the degradation of specific mRNA species and not to loss of all RNA from the filter, the 18s and 28s ribosomal RNA bands were routinely visualized with ethidium bromide. In all cases, equal amounts of RNA were present in each lane (data not shown). As shown in Fig. 3A, FV3 infection resulted in a progressive and rapid drop in the steady state level of actin mRNA. Similar experiments using probes for ribosomal protein L7 and glucase-6-phosphate-dehydrogenase also showed a marked decrease in host messages in virus-infected cells (data not shown). In contrast, early 18K mRNA first appeared 2 hr after infection and accumulated throughout the virus life-cycle (Fig. 3B). As expected, the cognate mRNA for the 48-kDa late protein appeared at 6 hr after infection and increased in abundance thereafter. Taken together these results indicate that the steady state level of host mRNAs declined throughout infection, while viral mRNA levels remained high during the same time period. Furthermore, com-

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lanes 1 and 3 in A-C). However, the inhibition of protein synthesis was more marked in infected cells receiving AMD than in mock-infected, AMD-treated cells. Because viral genes are not expressed in the presence of AMD (Fig. 4) it is clear that the decline in cellular protein synthesis that is observed in AMDtreated, FVB-infected cells must be due to a virion component and not to newly synthesized viral products. When the steady-state level of actin mRNA was exam-

48K

FIG. 3. Northern blot analysis of host and viral mRNA. Total RNA was extracted from mock- and FV3infected FHM cells at 2, 6, and 10 hr after infection and separated by electrophoresis on 1% agarose gels containing 2.2 M formaldehyde. RNA was transferred to nitrocellulose and hybridized to r’P]cDNA probes for actin (A), the FV3 18K early protein (B), and the FV3 48K late protein (C). After washing, specific annealing was demonstrated by autoradiography. M, mock-infected cell RNA; 2, 6, and 10, total RNA prepared from FV3-infected cells at 2, 6, and 10 hr after infection. The positions of 28s rRNA (large arrowhead) and 18s rRNA (small arrowhead) are shown.

parison of in viva protein synthesis (Fig. 2) and mRNA levels (Fig. 3) revealed that the decline in host translational capacity was temporally correlated with the drop in the steady state level of host messages. However, whether this correlation was fortuitous or causal is not clear. Although the above observations suggest that FV3 infection resulted in the progressive degradation of host mRNAs, it is also possible that the resulting decline in actin mRNA levels was due to the combined effect of FV3-mediated transcriptional shut-off coupled with normal mRNA turnover. To address this question, we examined the steady state levels of actin mRNA in the presence and absence of actinomycin D (AMD). Our intent was to determine whether the decline in actin mRNA levels in AMD-treated cells was equal to that seen in FV3-infected cells. If it was, then the drop in steady state levels seen after infection may simply reflect the normal turnover rate of actin mRNA in the absence of replacement synthesis. However, if the steady state level of actin mRNA declined more rapidly in virus-infected cells than in AMD-treated cells, then at least part of the drop in actin mRNA levels must be due to enhanced mRNA degradation. Mock- and virus-infected FHM cells were incubated in the presence and absence of AMD and, at the indicated times, protein synthesis and actin mRNA stability were monitored by polyacrylamide gel electrophoresis and Northern blot analysis, respectively. As shown in the upper three panels of Fig. 4, inclusion of 10 pg/ml AMD in the growth (and labeling) medium had a general inhibitory effect on protein synthesis (compare

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FIG. 4. Steady state level of actin message in the presence and absence of actinomycin D. FHM cells were mock-infected or infected with FV3 and grown in the presence or absence of 10 pg/ml actinomycin D (AMD). At 2. 6, and 10 hr after infection replicate samples were either labeled with [%]methionine or total cellular RNA extracted. Upper three panels: SDS-polyacrylamide gel analysis. Radiolabeled proteins were analyzed by SDS-polyacrylamide gel electrophoresis and the results are depicted in (A, B, and C). Lane 1, mock-infected cells; lane 2, FVB-infected cells; lane 3, mockinfected cells grown in the presence of AMD; lane 4, FV3infected cells grown in the presence of AMD. Mol wtfnarkers (in kilodaltons) are shown to the left of the figure. Lower four panels: Northern analysis of total cellular RNA. RNA, prepared at 2, 6, and 10 hr after infection, mock-infection, or addition of AMD, was separated by electrophoresis, transferred to nitrocellulose, and hybridized to a [32P]cDNA probe for actin. The autoradiogram shows the location of actin mRNA, along with the position of 28s (small arrowhead) and 18s (large arrowhead) rRNA. Following autoradiography, actin mRNA levels were quantitated by densitometry using a Bio-Rad Model 620 densitometer. Mock, mock-infected cells; FV3, virus-infected cells; AMD, mock-infected cells incubated in the presence of AMD; Both, FVB-infected cells incubated in the presence of AMD.

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ined by Northern analysis (Fig. 4, lower four panels), we found that at 6 hr after infection there was a 25% decrease in actin message levels in mock-infected ceils treated with AMD. In contrast, in virus-infected cells incubated in the presence or absence of AMD, the level of actin mRNA declined about 65%. This result suggests that active degradation of cellular messages occurred during FV3 replication. The above results showing a decline in the steady state levels of host mRNAs are at variance with previous work indicating that host message degradation plays no role in FV3-induced translational shut-off (Raghow and Granoff, 1979). However, the latter conclusion was drawn after monitoring translation in vitro of total cellular mRNAs isolated following infection with heat-inactivated virus. Since heat-inactivated virus may operate via a different shut-off mechanism than live virus and because in vitro translation of total cellular RNA may not be sensitive enough to detect subtle changes in message levels, we monitored protein synthesis and actin mRNA levels at various times after infection with live and heat-inactivated virus and in infections conducted in the presence of cytosine arabinoside, a drug which blocks viral DNA replication and limits viral gene expression primarily to the synthesis of early proteins (Elliott and Kelly, 1980). By comparing protein synthesis under these conditions (Fig. 5, upper panel) to the steady state level of actin mRNA (Fig. 5, lower panel) we can correlate shut-off with the stability of host messages. As is clear from the figure, the potential to block host protein synthesis does not correlate with ability to degrade host messages. Infection with heat-inactivated FV3 blocked host translation, but did not result in the degradation of host messages (compare Fig. 5, lanes 2 and 4). In contrast, a productive infection, even one that did not result in the synthesis of late proteins or viral DNA, blocked host translation and degraded host messages. This result confirms earlier suggestions (Raghow and Granoff, 1979; Chinchar and Dholakia, 1989) that message degradation is not a requirement for translational shut-off mediated by heat-inactivated FV3. Subcellular

distribution

of host and viral mRNA

In the final series of experiments, we turned from examining host translational shut-off and host message instability to the metabolism of early viral messages. As discussed above, mRNA for the 18-kDa early protein was abundant throughout infection, yet was only translated efficiently in vitro at early times. To gain insight into this selective translational block, we examined the polysomal distribution of 18K mRNA at various times after infection. Cytoplasmic extracts, prepared from mock-infected cells and from virus-in-

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level of actin mRNA in FVB-infected cells. FIG. 5. Steady-state FHM cells were mock-infected (lane l), infected with heat-inactivated FV3 (lanes 2 and 4) or productively infected with FV3 in the presence (lane 5) or absence (lanes 3 and 6) of 30 pg/ml cytosine arabinoside. At 4 hr (lanes l-3) and 7 hr (lanes 4-6) after infection, replicate cultures were labeled for 1 hr with [35S]methionine or total cellular RNA was extracted. Upper panel: SDS-polyacrylamide gel analysis of radiolabeled proteins. The positions of three FV3 proteins are shown. Lower panel: Northern analysis. Total cellular RNA was separated by electrophoresis, transferred to nitrocellulose, and hybridized to a [3’P]cDNA actin probe. The autoradiogram shows only the actin mRNA band.

fected cells at early and late times after infection, were centrifuged through 15-40°~ sucrose gradients and lml fractions collected. RNA was isolated from each fraction, transferred to nitrocellulose, and hybridized to probes for chicken @actin, and the FV3 18K (early) and 48K (late) proteins (Fig. 6). In mock-infected cells, actin mRNA was found both on polysomes (fractions 6-l 0) and in free mRNA ribonucleoprotein (mRNP) complexes (fractions l-3). However, by 2 hr after infection, actin mRNA was localized mainly within the mRNP fraction, and by 10 hr actin mRNA could not be detected in any fraction. These results are consistent with the interpretation that host protein synthesis was blocked at initiation, and that, following translational shut-off, host messages were progressively destabilized. As before, ethidium bromide staining showed that 18s and 28s rRNAs were present throughout the gradient indicating that the absence of actin mRNA was not due to the nonspecific loss of RNA. In contrast to the distribution of actin mRNA, the cognate mRNA for the FV3 18K protein was present on polysomes at 2 hr after infection, and, although displaced to smaller polysomes and to free mRNPs as infection proceeded, was not degraded. Furthermore, because the bulk of 18K mRNA was found in the mRNP

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FRACTION NUMBER FIG. 6. Polysomal distribution of host and viral mRNA. Cytoplasmic extracts from mock-infected cells or from FV3infected cells prepared at 2, 6, and 10 hr after infection were centrifuged through 15-40% sucrose gradients as described under Methods. Approximately 10, 1-ml fractions were collected and the RNA was extracted, separated by electrophoresis, and transferred to nitrocellulose. Immobilized RNAs were hybridized to [32P]cDNA probes for actin (A), FV3 18K (B), or FV3 48K (C). The positions of the 28s (large arrowhead) and 18s (small arrowhead) rRNA markers are indicated. and fraction numbers are shown at the bottom of each autoradiogram-fraction 1 is at the top of each gradient.

fraction at 10 hr after infection, it is likely that the defect in 18K mRNA translation is at initiation and not due to a block in elongation. Finally, the distribution of messages encoding the 48-kDa late protein was examined. Message for this abundant late protein was not detected in mock-infected cells or in cells at early times after infection, but was present on polysomes at 6 hr after infection (Fig. 6C). The presence of 48K mRNA throughout the gradient at 10 hr after infection may reflect the fact that even viral messages are susceptible to translational shut-off initiated by infection. These results confirmed that actin mRNA levels declined during infection and indicated that the translational defect preventing synthesis of the 18-kDa early protein occurred at initiation and did not involve 18K mRNA degradation.

DISCUSSION Following infection with vaccinia virus, human immunodeficiency virus type 1, Autographa californica nu-

clear polyhedrosis virus, and herpes simplex virus types 1 and 2, host messages are degraded (Rice and Roberts, 1983; Agy er al., 1990; Ooi and Miller, 1988; Kwong and Frenkel, 1987). In the HSV-1 system, degradation of cellular mRNAs is required not only for translational shut-off, but also for the timely expression of late viral genes (Strom and Frenkel, 1987; Oroskar and Read, 1989). Although translational shut-off is mediated by the HSV vhs gene (Fenwick and Owen, 1988) it is not known whether the vhs gene product is a virus-coded ribonuclease, or another protein that activates or modulates the activity of a preexisting host nuclease (Kwong and Frenkel, 1987). Vaccinia virus infection also leads to the progressive degradation of host transcripts (Rice and Roberts, 1983). However, in contrast to HSV-1, host mRNA degradation is not required for the inhibition of cellular translation (Cooper and Moss, 1979). Like the herpesvirus system, early viral mRNAs appear to be degraded late in poxvirus infection, thus mediating (or facilitating) the transition

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from early to late gene expression (Meis and Condit, 1991; Cooper and Moss, 1979). Our results, described above, indicate that FV3 infection was accompanied by a progressive decline in the steady-state level of cellular messages due most likely to the degradation of cellular transcripts. In contrast to the HSV-1 system, host mRNA instability was most likely not the direct cause of FV3-mediated translational shut-off since heat-inactivated FV3 blocked cellular protein synthesis without degrading host messages. Moreover, the decline in RNA levels following FV3 infection was confined primarily to cellular mRNAs. Thus, unlike HSV-1 and vaccinia virus, the transition from early to late FV3 gene expression was not dependent on the degradation of early FV3 messages. In fact, abundant amounts of early transcripts were present late in infection. At present, the molecular basis underlying the selective degradation of cellular transcripts is not known. Aside from a short 5’ untranslated region, early viral messages appear similar to host transcripts in overall structure (Willis et al., 1984; Beckman et a/., 1988; Schmitt eta/,, 1990). Perhaps specific proteins interact with defined regions within host (or viral) messages and mark them for (or protect them from) degradation. It is also possible that selective degradation is simply a manifestation of differences in subcellular compat-tmentalization. Translational shut-off and host mRNA degradation appear to be mediated by two distinct functions. Translational shut-off is catalyzed by a heat-stabile virion component, since host protein synthesis is blocked following infection with heat-inactivated virus or infection in the presence of actinomycin D. In contrast, another virion component is responsible for host message destabilization, but this component is likely heat-labile since heat-inactivated FV3 does not destabilize cellular transcripts. RNA metabolism in FV3-infected cells may be fundamentally different from that seen following infection with either HSV-1 or vaccinia virus. However, in the absence of additional data, we can only speculate on the significance of host mRNA destabilization to FV3 biogenesis. One possibility is that the degradation of host messages is required for the efficient translation of late viral messages. For example, if late viral messages are not translated as efficiently as their early counterparts, one way to enhance their expression would be to reduce the levels of competing cellular transcripts. In addition, our data support earlier findings that FV3 gene expression may be controlled, at least in part, at the translational level. These data are consistent with an earlier suggestion that virus-coded (or virus-induced) factors negatively regulate early

AND YU

mRNA translation late in infection, and that other viruscoded (or virus-induced) factors positively regulate late transcript translation (Raghow and Granoff, 1983). At present, the identity of such transacting regulatory factors in the FV3 system is problematic, but evidence in other systems points to the possibility of their existence (Najita and Sarnow, 1990; Costanzo and Fox, 1988; Winter et a/., 1987; Theil, 1990; Meerovitch et a/., 1989; del Angel et a/., 1989). Taken together our results suggest that efficient viral gene expression may require the interplay of several diverse mechanisms: the inactivation of elF-2 (Chinchar and Dholakia, 1989), the presence of highly efficient early virus messages (Chinchar and Yu, 1990a,b), the elimination of host messages by destabilizing preexisting messages and blocking the synthesis of new transcripts, and the modulation of early and late protein synthesis at the translational level. Elucidation of the precise mechanisms controlling FV3 gene expression will be the focus of future work.

ACKNOWLEDGMENTS The authors thank Kumud Srivastava for technical This work was supported by NSF Grant DMB-8502868.

assistance.

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