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Originally published In Press as doi:10.1074/jbc.M501156200 on February 10, 2005

J. Biol. Chem., Vol. 280, Issue 14, 13512-13519, April 8, 2005
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Inhibition of Host and Viral Translation during Vesicular Stomatitis Virus Infection

eIF2 IS RESPONSIBLE FOR THE INHIBITION OF VIRAL BUT NOT HOST TRANSLATION*

John H. Connor{ddagger} and Douglas S. Lyles

From the Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157

Received for publication, February 1, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In cells that allow replication of vesicular stomatitis virus (VSV), there are two phases of translation inhibition: an early block of host translation and a later inhibition of viral translation. We investigated the phosphorylation of the {alpha} subunit of the eIF2 complex during these two phases of viral infection. In VSV-infected cells, the accumulation of phosphorylated (inactivated) eIF2{alpha} did not begin until well after host protein synthesis was inhibited, suggesting that it only plays a role in blocking viral translation later after infection. Consistent with this, cells expressing an unphosphorylatable eIF2{alpha} showed prolonged viral protein synthesis without an effect on host protein synthesis inhibition. Induction of eIF2{alpha} phosphorylation at early times of viral infection by treatment with thapsigargin showed that virus and host translation are similarly inhibited, demonstrating that viral and host messages are similarly sensitive to eIF2{alpha} phosphorylation. A recombinant virus that expresses a mutant matrix protein and is defective in the inhibition of host and virus protein synthesis showed an altered phosphorylation of eIF2{alpha}, demonstrating an involvement of viral protein function in inducing this antiviral response. This analysis of eIF2{alpha} phosphorylation, coupled with earlier findings that the eIF4F complex is modified earlier during VSV infection, supports a temporal/kinetic model of translation control, where at times soon after infection, changes in the eIF4F complex result in the inhibition of host protein synthesis; at later times, inactivation of the eIF2 complex blocks VSV protein synthesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Many viruses can selectively inhibit host translation and still use the host translation machinery to synthesize their own proteins (for reviews, see Refs. 1–3). This provides two advantages for viral replication. It facilitates the rapid production of viral proteins, and it can also inhibit the production of host antiviral proteins (4). Both factors are important in the race of the virus against the cellular antiviral response. On the other hand, a critical part of the cellular antiviral response is the global inhibition of translation that blocks viral protein synthesis, effectively dampening virus production (5). The inhibition of both host and viral translation are apparent during infection with the prototype rhabdovirus vesicular stomatitis virus (VSV).1 During VSV infection, there is a rapid inhibition of host mRNA translation early after infection. This block of host translation is followed by a later inhibition of viral mRNA translation (6). The experiments presented here address the question of whether these two events reflect differing sensitivities of viral and host mRNAs to a single translation inhibition response or whether there are separate mechanisms controlling each inhibitory event.

A substantial body of evidence implicates the phosphorylation of the initiation factor eIF2{alpha} as a major player in inhibiting viral translation (5). eIF2{alpha} is a subunit of eIF2, the multiprotein complex that is responsible for recruiting the initiator tRNA to the 40 S subunit of the ribosome in a GTP-dependent manner. Phosphorylation of eIF2{alpha} inhibits the exchange of GDP for GTP and inhibits translation initiation by blocking the recruitment of the initiator methionine tRNA. The phosphorylation of eIF2{alpha} can be carried out by several different kinases (7), but the double-stranded RNA-activated kinase PKR is believed to be responsible for the phosphorylation of eIF2{alpha} during viral infection (5).

Early studies showed that eIF2 was inactivated in VSV-infected cells (8, 9), and in vitro experiments showed that adding eIF2 to lysates from VSV-infected cells could restore translational activity (10). More recent studies using cells derived from PKR knock-out mice suggested that the inactivation of eIF2 was caused by eIF2{alpha} phosphorylation and was primarily a function of PKR (11, 12), although a recent publication has demonstrated that there is an additional contribution from pancreatic endoplasmic reticulum kinase (13).

The phosphorylation of eIF2{alpha} and inhibition of eIF2 function during VSV infection suggests two different models for the biphasic inhibition of host and then viral protein synthesis. The first is that eIF2 is the dominant complex that controls host and viral translation in VSV-infected cells, but that VSV messages are more resistant to eIF2 inhibition than are host messages. This would be a situation similar to that seen for cricket paralysis virus mRNAs and some cellular mRNAs (14, 15). The second model is that there are distinct mechanisms that regulate the early inhibition of host and the late inhibition of viral protein synthesis, and that eIF2 is responsible for only one of these events.

Support for the latter model has come from recent studies showing the alteration of the eIF4F initiation factor complex in VSV-infected cells (16). The eIF4F complex is a multiprotein complex that contains the cap-binding protein eIF4E, the scaffolding protein eIF4G, the helicase eIF4A, the protein kinase MNK1, and additional proteins (17). This complex is responsible for binding to the m7-GTP cap of mRNAs and bringing them to the 40 S ribosome complex. We showed that after VSV infection, there is a rapid alteration of the eIF4F complex, indicated by the dephosphorylation of the eIF4E subunit (16), which was correlated with the inhibition of host protein synthesis. The dephosphorylation of eIF4E was associated with the activation of the eIF4E binding protein eIF4E-BP1/PHAS1, a protein that is known to dissociate eIF4E from the eIF4F complex, although additional mechanisms also contribute to eIF4E dephosphorylation (16). These studies, however, did not determine whether there were concurrent alterations in eIF2{alpha} phosphorylation that might also contribute to the inhibition of host protein synthesis.

In this study, we analyzed the role of eIF2{alpha} phosphorylation in the inhibition of host versus viral translation during VSV infection. The results showed that eIF2{alpha} phosphorylation occurring after host protein synthesis is inhibited, suggesting that eIF2{alpha} phosphorylation does not play a role in inhibiting host protein synthesis. Consistent with this, a dominant active form of eIF2{alpha} that cannot be phosphorylated had no effect on host translation inhibition. EIF2{alpha} phosphorylation did seem to be sufficient for the inhibition of viral protein synthesis, in that pharmacological inducers of eIF2{alpha} phosphorylation blocked viral protein synthesis even early after infection, and a viral mutant that expresses viral proteins for a longer period of time than the wild-type virus did not induce the phosphorylation of eIF2{alpha} to the same extent as the wild-type virus, showing that the viral M protein can play a role in the induction of eIF2{alpha} phosphorylation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Chemicals, unless otherwise stated, were purchased from Fisher Scientific. Thapsigargin was purchased from Calbiochem. Antibodies against eIF2{alpha} and phospho-eIF2{alpha}, were purchased from Cell Signaling Technologies. Okadaic acid and microcystin were purchased from Alexis Pharmaceuticals. Wild-type mouse embryonic fibroblasts (wt MEFs) expressing wt eIF2{alpha} and MEFs expressing a form of eIF2{alpha} in which the phosphorylated serine 51 residue was mutated to alanine (S51A MEFs) were kindly provided by Donalyn Scheuner and Randall J. Kaufmann (18).

Virus Infections—BHK cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum and 2 mM glutamine. Cells were grown to 80–90% confluence and were infected with wt VSV (Indiana serotype, Orsay strain), recombinant wild-type (rwt), or recombinant M51R M protein mutant (rM51R-M) virus (19) in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at a multiplicity of 10 plaque-forming units/cell in a small volume (500 µl per well for a 6-well dish). At 1 h after infection, the culture volume was doubled by addition of Dulbecco's modified Eagle's medium + 10% fetal bovine serum.

Immmunoblotting—Infected or mock-infected cells were lysed in 6-well dishes using 400 µl of EBC buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 100 nM okadaic acid, and 100 nM microcystin (16). Lysates were spun at 10,000 x g for 8 min in a refrigerated centrifuge, and 360 µl of the supernatant was added to 40 µl of 10x sample buffer for SDS-PAGE. Equal volumes of lysate were electrophoresed on 12% SDS-PAGE gels. After electrophoresis, gels were electroblotted onto nitrocellulose and blocked in Tris-buffered saline, pH 7.5, + 5% dry milk. Antibodies were diluted as recommended by the manufacturers. Band intensities were quantitated by first scanning the film and then analyzing the images with Quantity One software (Bio-Rad).

Thapsigargin Treatment—BHK cells were treated with thapsigargin for 1 h before 35S labeling or lysis. For 35S labeling, thapsigargin was included in the initial methionine depletion step. Thapsigargin was made as a 1 mM stock in Me2SO.

Metabolic Labeling and Rate of Protein Synthesis Determination—BHK cells were mock-infected or infected with VSV and then labeled with [35S]methionine for 10 min at varying times after infection, as described previously (16). After labeling, cells were washed three times with phosphate-buffered saline and lysed in 400 µl of radioimmunoprecipitation assay buffer for 10 min at 4 °C. Lysates were spun at 10,000 x g for 8 min. 360 µl of supernatant was added to 40 µl of 10x SDS-PAGE sample buffer, and 10 µl was electrophoresed on a 12% SDS-PAGE gel. Gels were stained with Coomasie blue to determine their protein content, then were analyzed by phosphorescence imaging (Amersham Biosciences). Phosphorescence intensities were analyzed using ImageQuant software. The rate of viral protein synthesis was determined from experiments by quantitation of the radioactivity in the viral L, G, N/P, and M bands in each lane. The rate of host protein synthesis was determined by quantitation of the radioactivity between the L and G bands, the N/P and M bands, and below the M band. For all experiments, three separate experiments were analyzed in this manner to determine an average.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
EIF2{alpha} Phosphorylation Occurs after Host Protein Synthesis Is Inhibited in VSV-infected Cells—Fig. 1A illustrates the changes in both viral and host protein synthesis during VSV infection of BHK cells. Cells were pulse labeled with [35S]methionine and analyzed by SDS-PAGE and phosphorimaging (an image of the Coomassie-stained gel is shown in Fig. 1B to demonstrate protein loading). The synthesis of host proteins, apparent as a ladder of bands in the lane from mock-infected cells, decreased over the first few hours of VSV infection, and was largely inhibited by 6 h after infection. Synthesis of the VSV proteins L, G, N, P, and M was apparent by 4 h after infection, approximately the time that the inhibition of host protein synthesis began. VSV proteins were synthesized at high levels between 6 and 8 h after infection, but by 12 h after infection, the synthesis of VSV protein was noticeably diminished. Fig. 1C shows a quantitation of the host and viral protein synthesis in VSV-infected BHK cells. Host protein synthesis, determined by the sum of the radioactivity between the viral L and G bands, the P and M bands, and the region below the M band, showed a decrease by 4 h after infection and was only 30% of mock-infected control by 6 h after infection. In contrast, viral protein synthesis began to increase dramatically at 4 h after infection, approximately the same time that host protein synthesis began to decrease. We have shown previously that this increase parallels the increase in viral mRNA in infected cells (16). Viral protein synthesis reached a maximum at 8 h after infection, and then decreased to only 20% of its maximum at 12 h after infection.



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  		FIG. 1.
Impact of VSV infection on protein synthesis and eIF2{alpha} phosphorylation. A, BHK cells were mock-infected (M) or infected with VSV for the indicated times, then labeled with [35S]methionine for 10 min. Cell lysates were electrophoresed on a 12% SDS-PAGE gel, a phosphorescence image of which is shown. Viral proteins are indicated to the right of the image. B, Coomassie-blue stain of autoradiograph gel. C, quantification of host (open diamonds) and viral (closed squares) protein synthesis. The rates of protein synthesis were determined from images similar to A as detailed under "Experimental Procedures." D, extracts from mock- and VSV-infected cells were analyzed by Western blotting with antibodies against eIF2{alpha}, phosphorylated at serine 51 (eIF2{alpha}-P), and total eIF2{alpha}. E, the levels of eIF2{alpha} phosphorylation were determined by densitometry of phospho-eIF2{alpha} specific Western blots and were quantitated as a ratio to total eIF2{alpha}. Data shown are means ± S.D. for three experiments.

 
To determine the change in the phosphorylation of eIF2{alpha} during the inhibition of host and viral protein synthesis, we assessed eIF2{alpha} phosphorylation over a time course similar to that shown in Fig. 1A. Lysates were analyzed by Western blotting using antibodies specific for the phosphorylated and total forms of eIF2{alpha}. As a control, cells were treated for 1 h with 1 µM thapsigargin, a pharmacological agent that induces eIF2{alpha} phosphorylation by the pancreatic endoplasmic reticulum protein kinase through destabilization of Ca2+ stores in the endoplasmic reticulum (20). The results are shown in Fig. 1D. A low level of phosphorylated eIF2{alpha} was detectable in mock-treated cells and, as expected, thapsigargin caused the rapid phosphorylation of eIF2{alpha} (Fig. 1D, top left). VSV infection did cause an increase in eIF2{alpha} phosphorylation, as reported previously (11, 12). However, this occurred long after the inhibition of host protein synthesis seen in Fig. 1A. At early times after infection, eIF2{alpha} phosphorylation was at the level of mock-infected cells. Phosphorylation of eIF2{alpha} did not begin to noticeably increase until well after host protein synthesis was inhibited, becoming clearly elevated at 10 and 12 h after infection.

The extent of eIF2{alpha} phosphorylation was quantitated as a ratio of signal from the phosphospecific antibody to the signal from the total eIF2{alpha} antibody and normalized to the amount of eIF2{alpha} phosphorylation that is caused by thapsigargin treatment (Fig. 1E). In this analysis, the phosphorylation of eIF2{alpha} in response to VSV infection did not increase significantly above mock-infected controls until 8 h after infection, but the level of phosphorylation by 12 h after infection was approximately equal to that that of thapsigargin-treated cells. This lack of significant phosphorylation until later after infection suggests that the inactivation of eIF2 through eIF2{alpha} phosphorylation is not involved in VSV's inhibition of host protein synthesis. However, the increase in eIF2{alpha} phosphorylation did correlate with the decrease in VSV protein synthesis, consistent with the idea that it is involved in blocking viral protein synthesis.

Host and Viral Protein Synthesis Are Similarly Sensitive to eIF2{alpha} Phosphorylation—It has been proposed that translation of VSV messages during infection is more resistant than host messages to the phosphorylation of eIF2{alpha} (9). A resistance of viral protein synthesis to eIF2{alpha} inactivation could account for the results seen in Fig. 1A. For example, host protein synthesis would be inhibited early after infection by a level of eIF2{alpha} phosphorylation too low to be detected by the phosphospecific antibody, whereas later after infection, higher levels of eIF2{alpha} phosphorylation would block viral protein synthesis. To determine whether the translation of VSV messages was more resistant than cellular messages to the induction of eIF2{alpha} phosphorylation, VSV- and mock-infected cells were treated with 200 nM thapsigargin, and the levels of protein synthesis were determined. Consistent with previous reports, the effects of thapsigargin were transient; protein synthesis was inhibited for ~2 h but thereafter began to recover (21). This allowed us to determine both the short-term effects of eIF2{alpha} phosphorylation and the recovery of protein synthesis. Rates of protein synthesis were determined at 6 h after infection for both VSV- and mock-infected cells treated for various times with thapsigargin; a diagram of the experimental procedure is shown in Fig. 2A. Thapsigargin was added either coincident with infection (6 h Tgn), 2 h after infection (4 h Tgn), 4 h after infection (2 h Tgn), or 5 h after infection (1 h Tgn), and cells were then either pulse-labeled to determine the rate of protein synthesis or lysed and analyzed for the state of eIF2{alpha} phosphorylation by Western blotting.



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  		FIG. 2.
Effect of thapsigargin on host and viral protein synthesis in VSV-infected cells. A, schematic of experimental procedure showing times of thapsigargin addition after viral infection (left) and total time of thapsigargin treatment (right). B, Western blot analysis of phosphorylated and total eIF2{alpha} after treatment of VSV-infected cells with 1 µM thapsigargin for the indicated times. Experiment is representative of three independent blots. C, phosphorescence image of SDS-PAGE analysis of mock- or VSV-infected cells treated with thapsigargin for the times indicated in B and then labeled with [35S]methionine for 10 min. D, mock-infected cells treated with thapsigargin for the times indicated and then labeled with [35S]methionine for 10 min. E, quantitation of viral (filled bars) and host (open bars) protein synthesis in thapsigargin-treated cells. For viral protein synthesis, results from three independent experiments were quantified and are expressed as a percentage of the untreated 6 hpi control. For host protein synthesis, levels are expressed as a percentage of the mock-treated control.

 
Western blot analysis of VSV-infected, thapsigargin-treated cells (Fig. 2B) showed that eIF2{alpha} was extensively phosphorylated after the 1-h Tgn treatment, but little eIF2{alpha} phosphorylation was seen in cells infected with VSV alone for 6 h. Phosphorylation of eIF2{alpha} decreased over longer periods of TGN treatment, consistent with the transient effects of the drug treatment. In virus-infected cells that were pulse labeled with [35S]methionine (Fig. 2C), a 1-h treatment with thapsigargin resulted in a dramatic inhibition of both VSV protein synthesis and host protein synthesis (lane 3). Treatment of cells with thapsigargin for longer times resulted in a lessening of this effect (lanes 4–6) because of the cells' recovery from thapsigargin treatment. A similar effect was seen in mock-infected cells (Fig. 2D). A 1- or 2-h treatment with thapsigargin resulted in a significant inhibition of protein synthesis, but at 4 and 6 h of treatment, recovery of protein synthesis was evident, consistent with the reversal of eIF2{alpha} phosphorylation. The inhibition and recovery of VSV protein synthesis was quantitated and compared with the recovery of host protein synthesis in mock-infected cells that were treated with thapsigargin (Fig. 2E). The rates of recovery for both viral and host protein synthesis were very similar for 1, 2, and 4 h, indicating that the inhibition and recovery of viral and host protein synthesis was correlated with the phosphorylation and subsequent dephosphorylation of eIF2{alpha}. In some cases, the treatment of cells with thapsigargin coincident with VSV infection (6 h) resulted in a level of VSV protein synthesis (Fig. 2E) and protein accumulation (data not shown) that was variable and somewhat greater than values for uninfected cells for reasons that are not well understood. The conclusion to be drawn from Fig. 2 is that the rates of viral and host protein synthesis are similarly sensitive to the effects of eIF2{alpha} phosphorylation. Thus, the different timing of viral and host translation inhibition seen in Fig. 1 cannot be explained by different sensitivities to the level of eIF2{alpha} phosphorylation.

Host Protein Synthesis Inhibition by VSV Is Not Altered in Cells Overexpressing a Nonphosphorylatable eIF2{alpha}—To further test the conclusion that the phosphorylation of eIF2{alpha} was not a critical factor in the inhibition of host protein synthesis during VSV infection, we used MEF cells expressing eIF2{alpha} that contained a serine-to-alanine mutation at residue 51 (S51A knock-in cells; kindly provided by Donalyn Scheuner and Ronald Kaufman), which prevents the phosphorylation and inactivation of eIF2{alpha}. This S51A mutation has previously been shown to block the phosphorylation of endogenous eIF2{alpha} and the associated decrease in host protein synthesis in response to several different cell stress events (22, 23). Control and knock-in MEFS were infected with VSV for 4, 6, 8, and 10 h or treated with 1 µM thapsigargin for 1 h. In cells expressing wild-type eIF2{alpha} (Fig. 3A), thapsigargin treatment resulted in the inhibition of protein synthesis as expected. Infection with VSV also resulted in the inhibition of host protein synthesis by 6 h after infection, similar to the effects seen in BHK cells. In these cells, there was a more rapid inhibition of viral protein synthesis than in BHK cells, so that maximal levels of viral protein synthesis occurred at 6 hpi. Assessing the level of eIF2{alpha} phosphorylation in these cells by Western blotting (Fig. 3D), we found that there was a rapid phosphorylation of eIF2{alpha} that became apparent at 6 hpi and increased up to 10 hpi consistent with a role in blocking viral protein synthesis (band intensities of phospho eIF2{alpha} signal normalized to total eIF2{alpha} are printed at the bottom of Fig. 3D).



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  		FIG. 3.
Effect of a dominant active mutation in eIF2{alpha} on host and viral protein synthesis inhibition in VSV-infected cells. WT MEFs (A) or S51A eIF2{alpha} knock-in MEFs (B) were mock-infected (M), treated with 1 µM thapsigargin, or infected with VSV for the indicated times and labeled with [35S]methionine for 10 min. Lysates were electrophoresed on a 12% SDS-PAGE gel, a phosphorescence image of which is shown. Viral proteins are indicated to the right of the image. C and D, extracts from mock- and VSV-infected MEFs at the times indicated in A and B were analyzed by Western blotting with antibodies against eIF2{alpha}, phosphorylated at serine 51 (top), total eIF2{alpha} (middle), and actin (bottom). Quantitation of host (E) and viral (F) protein synthesis in wt (filled bars) and S51A eIF2{alpha}-expressing cells (open bars). Host protein synthesis was determined from images like A and B as explained under "Experimental Procedures." Data represent the results of three separate experiments ± S.D.

 
In cells expressing the eIF2{alpha} S51A mutant, protein synthesis was not markedly inhibited after thapsigargin treatment, consistent with previous results showing that the S51A mutant does not allow the phosphorylation of eIF2{alpha} and inactivation of protein synthesis. However, when these cells were infected with VSV, the time course of host protein synthesis inhibition was very similar to that seen in MEFs expressing wt eIF2{alpha}, so that by 6 h after infection, host protein synthesis was largely inhibited (Fig. 3B, lanes 3–6), although viral protein synthesis continued for much longer. As expected, Western blotting for eIF2{alpha} phosphorylation showed no signal in these cells (Fig. 3D).

Fig. 3E shows a quantitative comparison of the rate of host protein synthesis in control cells and cells expressing the S51A mutant eIF2{alpha}. For control cells, thapsigargin treatment reduced the rate of protein translation to ~20% of control, but the protein synthesis rate remained above 80% of control in cells overexpressing the nonphosphorylatable eIF2{alpha}. VSV infection resulted in the inhibition of host protein synthesis in control cells, dropping to 30% of mock-infected cells by 8 h after infection. This pattern was virtually identical in cells expressing the S51A mutant eIF2{alpha}, further supporting the idea that eIF2{alpha} does not play a significant role in the inhibition of host protein synthesis. However, viral protein synthesis (Fig. 3F) was both increased and persisted for longer times in the S51A MEFs. This was particularly noticeable at 10hpi, where viral protein synthesis in the knock-in MEFs was 4-fold that seen in MEFs expressing wt eIF2{alpha}, consistent with previous data showing that eIF2{alpha} phosphorylation plays a role in blocking viral protein synthesis (24).

EIF2{alpha} Phosphorylation Is Disregulated in Cells Infected with the VSV M51R M Protein Mutant—The data shown above suggest that independent factors control viral and host protein synthesis during VSV infection. To further test this hypothesis, we used a virus containing a point mutant in the VSV matrix protein that is delayed in the inhibition of both host and viral translation. Fig. 4A shows an autoradiograph of lysates from cells that were mock-infected or infected with isogenic recombinant viruses derived from cDNA clones containing either wt or M51R M proteins (rwt or rM51R-M virus, respectively) for the indicated times, after which they were pulse-labeled with [35S]methionine. Two important results are illustrated by this image: first, the M51R virus is defective in its ability to inhibit host protein synthesis. Quantitation of three separate experiments (Fig. 4B) showed that although the rwt virus inhibited host protein synthesis by more than 80% by 12 hpi, the rM51R-M virus inhibited only approximately 50% of host protein synthesis at the same time. The second important result is the fact that viral protein synthesis was not inhibited to the same extent in cells infected with the rM51R-M virus as it was in cells infected with the rwt virus (Fig. 4A, compare lane 4 with lane 8).



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  		FIG. 4.
Effect of M51R mutation in the viral M protein on eIF2{alpha} phosphorylation during VSV infection. A, cells were mock-infected (M) or infected with rwt or rM51R-M virus for the indicated times, then labeled with [35S]methionine for 10 min. Cell lysates were electrophoresed on a 12% SDS-PAGE gel, a phosphorescence image of which is shown. B, quantitation of host protein synthesis inhibition by rwt and M51R virus. C, lysates from mock-infected, rwt, or rM51R-M virus-infected cells (infected for the indicated times) were analyzed for eIF2 phosphorylation by Western blotting using antibodies against phosphorylated and total eIF2. The figure represents a single blot reprobed with the two different antibodies. D, quantitation of eIF2 phosphorylation, as per Fig. 1D. Data shown are means ± S.D. for three experiments.

 
Fig. 4C shows eIF2{alpha} phosphorylation in response to infection with rwt or rM51R-M virus. Quantitation of three separate experiments is shown in Fig. 4D. Cells were mock-infected or infected with rwt or rM51R-M virus, and at 4, 8, or 12 h after infection, cell lysates were prepared for SDS-PAGE and Western blotting. When these lysates were probed for levels of eIF2{alpha} phosphorylation (Fig. 4C, left), cells infected with rwt virus showed a pattern of phosphorylation at 8 and 12 h after infection that was similar to that of cells infected with the wild-type Orsay strain used in Figs. 1, 2, 3. In cells infected with the rM51R-M virus, eIF2{alpha} phosphorylation was observed at a level higher than that seen in the rwt virus-infected cells at 4 h after infection (Fig. 4C, lane 2 versus lane 5). This level of eIF2{alpha} phosphorylation in the rM51R-M virus-infected cells remained constant throughout infection but was only 60% of the level that was induced by the rwt virus at 12 hpi (Fig. 4D). Thus, the M protein mutant virus induced more eIF2{alpha} phosphorylation than the wt virus early after infection, but the inhibition of host protein synthesis by the mutant virus was less than that of the wt virus. This result is inconsistent with a role for eIF2 complex inactivation in the early inhibition of host protein synthesis. In contrast to early times after infection, the M protein mutant virus induced less eIF2{alpha} phosphorylation at late times after infection. This may account for the higher levels of protein synthesis seen in the rM51R-infected cells and is consistent with a role for eIF2 in blocking viral protein synthesis.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous reports have suggested a role for both eIF2 and eIF4 in the inhibition of translation in VSV-infected cells, but the relative roles of these complexes in the inhibition of host versus virus translation had not been established (9, 10, 16, 24–26). Of particular importance was the possibility that host and viral mRNAs might be differently susceptible to inhibition by eIF2{alpha} phosphorylation. The results presented here, combined with our earlier findings regarding how VSV infection impacts eIF4F function (16), support a model for understanding the differential inhibition of host versus viral protein synthesis as being caused by the alteration of two different translation initiation factors, eIF2 and eIF4F, at different times. Our model for the changes in these translation initiation factors after VSV infection is illustrated schematically in Fig. 5. In normal cells, the eIF2 and eIF4F complexes are fully functional, and host protein synthesis proceeds normally (far left), but after VSV infection, there are two discernible changes to the translation apparatus. The first is an early event after infection, illustrated in the middle column of Fig. 5, a change in the eIF4F complex, indicated by the dephosphorylation of eIF4E. This change in the eIF4F complex results in an exclusion of host messages while viral mRNAs are translated. The second effect on translation occurs later after infection and is depicted in the right column of Fig. 5, where eIF2{alpha} phosphorylation increases, inhibiting proper eIF2 function and blocking both host and viral translation. This model emphasizes the role of eIF2 and eIF4F; however, it is possible that other translation control mechanisms contribute to the inhibition of host and viral translation. With this model as a basis, similar approaches could be used to test the roles of other mechanisms.



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  		FIG. 5.
Model of translational control during VSV infection. Cartoon reflects three stages of translation control. The first, depicted on the left, is in uninfected cells, where the eIF2 complex is fully functional and the eIF4F complex is assembled and eIF4E is phosphorylated allowing for recognition and translation of host mRNAs. The second, illustrated in the middle, shows the conditions early in VSV infection, where host mRNA is excluded from translation, eIF4E is dephosphorylated but eIF2 is still active, and viral mRNA is efficiently translated. Right, last phase of translational control, where eIF2{alpha} is phosphorylated and eIF2 is inactive, leading to the inhibition of viral translation.

 
The data in Figs. 1, 2, 3, 4 support the model in Fig. 5. During the course of viral infection, eIF2{alpha} was phosphorylated in VSV-infected cells, but this occurred well after the inhibition of host protein synthesis and was coincident with the inhibition of viral protein synthesis (Fig. 1). Blocking eIF2{alpha} phosphorylation with a dominant active eIF2{alpha} did not affect the inhibition of host protein synthesis during VSV infection (Fig. 3). When eIF2{alpha} phosphorylation was induced by thapsigargin treatment during viral infection, it blocked the translation of both viral and host proteins (Fig. 2). The synthesis of both viral and host proteins was recovered on the same timescale, showing that neither viral nor host translation is more susceptible to eIF2{alpha} phosphorylation. In contrast to these results with eIF2{alpha} phosphorylation, the alteration of the eIF4F complex indicated by eIF4E dephosphorylation correlates very well with the inhibition of host translation.

The sensitivity of VSV translation to eIF2{alpha} phosphorylation is fully consistent with previous reports showing that the activation of PKR (12, 27) and the subsequent phosphorylation of eIF2{alpha} (24, 26) is an important host defense mechanism against VSV infection. These studies showed that removing PKR or keeping eIF2{alpha} phosphorylation from blocking eIF2 function allows enhanced VSV replication. One question raised by these findings is how VSV still manages to replicate well in animal hosts such as mice, where the PKR pathway is still intact. Our results showing a delay in eIF2{alpha} phosphorylation until after VSV has been producing both RNA and protein for a significant period of time (8 hpi in BHK cells) suggests that the kinetics of eIF2{alpha} phosphorylation may be important for viral replication. The reason that some cell types are resistant to VSV infection and others are sensitive may result from the fact that cells that rapidly phosphorylate eIF2{alpha} are more resistant to VSV infection, whereas those that have a significant delay in eIF2{alpha} phosphorylation block viral protein synthesis at a point that is too late to inhibit effective replication. This idea is supported by our results, where BHK cells that replicate virus at titers up to 109 plaque-forming units have a longer delay in eIF2{alpha} phosphorylation than do MEFs that only replicate VSV to titers of ~106 plaque-forming units.

The previous reports showing that VSV infection inactivates the eIF2 complex have all suggested that VSV infection rapidly induces the phosphorylation of eIF2{alpha} (28). Our results showing a delay in eIF2{alpha} phosphorylation until after VSV has been producing both RNA and protein for well over 6 h suggests a more complex interpretation. VSV infection has multiple triggers (dsRNA production, ER stress through G protein production, RNA synthesis inhibition, etc.) that could lead to the eIF2{alpha} phosphorylation seen in the wt virus infection. Because eIF2{alpha} phosphorylation is not seen until later after infection, it is possible that one or more VSV gene products act to delay this inhibitory event. Our results suggest that the M protein is a good candidate protein for this effect, in that cells infected with an M protein mutant virus (rM51R-M) showed more eIF2{alpha} phosphorylation early after infection, consistent with the M protein mutant being a loss-of-function mutant that no longer delays eIF2{alpha} phosphorylation. The fact that eIF2{alpha} phosphorylation at late times after infection was decreased relative to cells infected with the rwt virus could also be caused by the loss of an important function in this M protein mutant, in that it no longer inhibits RNA synthesis and nuclear/cytoplasmic transport (29–31), stresses that could increase eIF2{alpha} phosphorylation. It is also possible that the ability of the cell to synthesize new proteins in the M51R virus infected cells leads to the expression of proteins that counteract eIF2{alpha} kinases, such as the GADD-34 protein, which has been shown to be induced by M51 mutant VSV by other researchers (32).

Our results have led to a new proposal of translation control during VSV infection. In this model (illustrated in Fig. 5), VSV infection results in the rapid modification of eIF4F, which is responsible for blocking host translation. A later phase of translation control is exerted by the host antiviral response, which induces the phosphorylation of eIF2{alpha} and blocks viral translation. This model predicts that the kinetics of eIF2{alpha} phosphorylation is important for viral replication. This prediction will be tested in future experiments.


   FOOTNOTES
 
* This work was supported by Public Service Health Service Grants AI052304 and AI32983 from the National Institute of Allergy and Infectious Diseases, NIH (to D.S.L). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by Signal Transduction Mechanisms and Cell Function training program grant CA09422 from NCI and Public Health Service Grant AI051805 from the National Institute of Allergy and Infectious Diseases, NIH. To whom correspondence should be addressed: Tel.: 336-716-2270; Fax: 336-716-9928; E-mail: jconnor{at}wfubmc.edu.

1 The abbreviations used are: VSV, vesicular stomatitis virus; PKR, protein kinase R; eIF2{alpha}, eukaryotic translation initiation factor 2{alpha}; wt, wild type; MEF, mouse embryonic fibroblast; rwt, recombinant wild-type; hpi, hours postinfection. Back


   ACKNOWLEDGMENTS
 
We thank Griffith Parks, Maryam Ahmed, and Zackary Whitlow for helpful advice and comments on the manuscript, and we thank the Cancer Center Analytical Imaging Facility for phosphorimaging. We also thank Donalyn Scheuner and Randall J. Kaufman for the kind donation of knock-in MEF cell lines.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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