JOURNAL OF VIROLOGY, Dec. 1996, p. 8993–8996 0022-538X/96/$04.0010 Copyright q 1996, American Society for Microbiology
Vol. 70, No. 12
Rapamycin Stimulates Viral Protein Synthesis and Augments the Shutoff of Host Protein Synthesis upon Picornavirus Infection LAURA BERETTA,† YURI V. SVITKIN,
AND
NAHUM SONENBERG*
Department of Biochemistry and McGill Cancer Center, McGill University, Montre´al, Que´bec H3G 1Y6, Canada Received 7 May 1996/Accepted 5 September 1996
The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1 in mammalian cells and yeast. We recently showed that rapamycin inhibits both in vitro and in vivo cap-dependent, but not capindependent, translation. This inhibition is causally related to reduced phosphorylation and consequent activation of 4E-BP1, a repressor of the function of the cap-binding protein, eIF4E. Two members of the picornavirus family, encephalomyocarditis virus and poliovirus, inhibit phosphorylation of 4E-BP1. Since translation of picornavirus mRNAs is cap independent, inhibition of phosphorylation of 4E-BP1 could contribute to the shutoff of host protein synthesis. Here, we show that rapamycin augments both the shutoff of host protein synthesis and the initial rate of synthesis of viral proteins in cells infected with encephalomyocarditis virus and poliovirus. 4E-BP1 for binding to eIF4E through a similar binding site (21). Thus, the interaction of 4E-BP1 with eIF4E results in the specific inhibition of cap-dependent, but not cap-independent, translation, both in vitro and in vivo (23). Recently, it was shown that EMCV and poliovirus infections inhibit phosphorylation of 4E-BP1 (7). The reduced phosphorylation of 4E-BP1 in EMCV-infected cells coincides with the shutoff of host protein synthesis. In contrast, reduction of phosphorylation of 4E-BP1 in poliovirus-infected cells lags behind the shutoff of host protein synthesis (7). Since rapamycin inhibits specifically cap-dependent translation by inhibition of phosphorylation of 4E-BP1, it is anticipated that it would accelerate the shutoff of host protein synthesis and enhance virus protein synthesis during EMCV and possibly poliovirus infection. To test this prediction, NIH 3T3 cells were infected with EMCV strain K-2 (31), in the presence or absence of rapamycin (Fig. 1). Metabolic labeling with [35S]methionine was performed at 2 to 5 h postinfection. Rapamycin alone only slightly reduced protein synthesis (less than 10% inhibition at 5 h of treatment) (Fig. 1A). Under the infection conditions used here in NIH 3T3 cells, there was only a moderate inhibition (10%) of host protein synthesis after 5 h of infection (Fig. 1B, lane 11). We showed earlier that in Krebs cells, the shutoff of host protein synthesis occurs 5 h after infection (7). Virus proteins first become visible after 4 h of infection (lane 8). However, in the presence of rapamycin, a comparable amount of viral protein is seen already after 3 h of infection (lane 6) and viral protein synthesis is maximal at 4 h of infection (lane 9). At 5 h of infection in the presence of rapamycin, the shutoff of host protein synthesis is more pronounced than without rapamycin (50% inhibition; compare lane 12 with lane 11). These results show that rapamycin accentuates the effect of EMCV infection on both host and viral protein synthesis. We also examined the phosphorylation state of 4E-BP1 during infection (Fig. 2). EMCV infection did cause a slight inhibition of phosphorylation of 4E-BP1 at 5 h postinfection consistent with the small inhibition of host protein synthesis (compare lane 11 with lane 2). However, rapamycin inhibited phosphorylation of 4E-BP1 already at the first time point studied (2 h) and inhibition of phosphorylation was augmented during the course of infection (compare lanes 3, 6, 9, and 12).
Infection of cells with most viruses causes a shutoff of host protein synthesis (reviewed in reference 22). The shutoff is exerted at the level of translation, since cellular mRNAs can be recovered from virus-infected cells in an intact and functionally active form (1, 11, 15, 18, 30). Translation initiation of most eukaryotic mRNAs is facilitated by the mRNA 59 cap structure m7GpppX (where X is any nucleotide). The multisubunit translation initiation factor eIF4F binds to the cap structure via the eIF4E subunit to promote ribosome binding (28). In contrast to cellular mRNAs, mRNAs of some viruses, such as picornaviruses, which include encephalomyocarditis virus (EMCV) and poliovirus, are uncapped and contain an internal ribosome entry site (IRES), which promotes translation by a cap- and eIF4E-independent mechanism (13, 14, 24). The immunosuppressant drug rapamycin blocks progression of the cell cycle at G1. Rapamycin forms a complex with the immunophilin protein FKBP (FK506-binding protein), which binds to a family of kinases named FRAP in humans, RAFT in rats, and TOR in yeast (5, 10, 16, 25, 26, 29). Rapamycin inhibits cap-dependent, but not cap-independent, translation (3). Inhibition of cap-dependent translation by rapamycin is also observed in yeast (2). The inhibition can be reproduced in vitro, as cap-dependent translation is inhibited in extracts from rapamycin-treated cells (3). Inhibition by rapamycin is causally related to the reduced phosphorylation and consequent activation of 4E-BP1 (eIF4E-binding protein 1) (3, 8, 20), a repressor of the function of the cap-binding protein, eIF4E (19, 23). 4E-BP1 (also termed PHAS-I) is a small heat- and acidstable protein whose activity is regulated by phosphorylation (12, 19, 23). Rapamycin inhibits phosphorylation of 4E-BP1 within 1 h after treatment (3). The underphosphorylated form of 4E-BP1 interacts strongly with eIF4E, while the phosphorylated form is unable to interact (19, 23). 4E-BP1 blocks the interaction between eIF4E and eIF4G (eIF4G associates with eIF4E in the cap-binding complex eIF4F) in vitro (9). This inhibition is explained by the competition between eIF4G and * Corresponding author. Phone: (514) 398-7274. Fax: (514) 3981287. Electronic mail address:
[email protected]. † Present address: INSERM U.365, Institut Curie, 75005 Paris, France. 8993
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FIG. 2. Phosphorylation of 4E-BP1. Cells infected with EMCV and treated with rapamycin as described in Fig. 1 were lysed in 20 mM Tris-HCl, pH 7.5, buffer containing 5 mM EDTA and 100 mM KCl. The homogenate was centrifuged at 9,000 rpm (Sorvall SS34 rotor) for 10 min, and the supernatant was collected. To analyze for 4E-BP1, 50 mg of protein was dissolved in Laemmli sample buffer (17) and subjected to electrophoresis on a sodium dodecyl sulfate– 15% polyacrylamide gel. Proteins were transferred to a 0.22-mm-pore-size nitrocellulose membrane, which was blocked in 5% milk for 2 h and then incubated for 2 h with rabbit polyclonal antiserum 11208 against 4E-BP1 (1:1,000) (3) in 10 mM Tris-HCl, pH 8.0, buffer containing 150 mM NaCl. The blot was subsequently incubated with 125I-protein A (Amersham) (1:1,000) and exposed against an X-ray film.
FIG. 1. Effect of rapamycin on EMCV infection in NIH 3T3 cells; time course of protein synthesis. NIH 3T3 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were infected with EMCV strain K-2 (31), at a multiplicity of infection of 100 PFU per cell, in serum-free medium in the presence or absence of rapamycin (20 ng/ml). Rapamycin and EMCV were added at the same time. At 2 to 5 h postinfection, cells were washed and incubated in methionine-free medium for 30 min with [35S]methionine (10 mCi/ml). Cells were lysed in buffer containing 0.5% Nonidet P-40, 140 mM NaCl, and 30 mM Tris-HCl, pH 7.5, and nuclei were removed by centrifugation. The supernatant from an equal number of cells was analyzed on a sodium dodecyl sulfate–12.5% polyacrylamide gel. The gel was dried and exposed against an X-ray film. Radiolabeled proteins were quantified with a Phosphorimager (Fuji; Bas 2000). Viral proteins are indicated with arrows. (A) Protein synthesis in mock-infected cells. (B) Protein synthesis in EMCV-infected cells.
These results, taken together with those in Fig. 1, suggest that rapamycin-induced inhibition of phosphorylation of 4E-BP1 facilitates the shutoff of host protein synthesis by EMCV. To extend this study to another member of the picornavirus family, we examined the effect of rapamycin on poliovirus infection. As for EMCV, translation initiation of poliovirus RNA is carried out by binding of ribosomes to an IRES element (6, 24). The shutoff of host protein synthesis following poliovirus infection is faster than that observed after EMCV
infection (6). Thus, we analyzed the effect of rapamycin at early times of infection (2 and 3.5 h). HeLa cells were infected with the Mahoney strain of poliovirus type I. Virus proteins were detected already after 2 h of infection, at a time when some inhibition of host protein synthesis occurred (Fig. 3; compare lane 2 with lane 1). In the presence of rapamycin, however, at 2 h after infection, the shutoff of host protein synthesis was strong (66% inhibition) and viral protein synthesis was significantly stimulated (threefold) (lane 3). At 3.5 h postinfection, the shutoff of host protein synthesis was complete, and rapamycin had no further effect on virus protein synthesis (lanes 4 to 6). Thus, the inhibition of host protein synthesis and the rate of synthesis of viral protein in poliovirusinfected cells were significantly enhanced by rapamycin. These results show that the effects of rapamycin are common for viruses whose translation is achieved by internal ribosome binding, in a cap-independent manner. To exclude the possibility that rapamycin affects virus RNA synthesis, we analyzed poliovirus RNA levels by Northern (RNA) blotting. We have not detected any effect of rapamycin on RNA levels (data not shown). A pertinent question is whether the effect of rapamycin on the shutoff of host protein synthesis and early detection of viral proteins is reflected in virus yield. We have performed several experiments with poliovirus and EMCV and observed an increase of approximately twofold in virus titer in the presence of rapamycin (29a). It is conceivable that, because at the late stage of infection virus protein synthesis is very efficient, virus yield is not strongly affected by rapamycin. As a control for a virus mRNA that translates by a capdependent mechanism, we used vesicular stomatitis virus (VSV) (4). NIH 3T3 cells were infected with VSV, and the
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FIG. 4. Effect of rapamycin on VSV infection; time course of protein synthesis. NIH 3T3 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were infected with VSV in serumfree medium in the presence or absence of rapamycin (20 ng/ml). Rapamycin and VSV were added at the same time. At several time points postinfection, the cells were incubated in methionine-free medium for 30 min with [35S]methionine (10 mCi/ml). Cells were lysed, and the supernatant was analyzed on a sodium dodecyl sulfate–12.5% polyacrylamide gel as described in the legend to Fig. 1. Viral proteins are indicated with arrows. FIG. 3. Effect of rapamycin on poliovirus infection; time course of protein synthesis. HeLa cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were infected with the Mahoney strain of poliovirus type I at a multiplicity of infection of 100 PFU per cell, in the presence or absence of rapamycin (20 ng/ml). Rapamycin and poliovirus were added at the same time. At 2 and 3.5 h postinfection, cells were washed and incubated in methionine-free medium for 30 min with [35S]methionine (10 mCi/ ml). Cells were lysed, and the supernatant was analyzed on a sodium dodecyl sulfate–12.5% polyacrylamide gel as described in the legend to Fig. 1. Viral proteins are indicated by arrows.
pattern of protein synthesis was examined (Fig. 4). Partial shutoff of host protein synthesis and some synthesis of viral proteins were already observed 2 h after infection (lane 2). Rapamycin had no effect on either VSV or host protein synthesis (lane 3). The shutoff of host protein synthesis and synthesis of viral proteins were maximal 4 h after infection. At 4 and 5 h of infection, rapamycin treatment slightly decreased viral protein synthesis (lanes 9 and 12). These results are consistent with the conclusion that rapamycin specifically stimulates cap-independent translation during virus infection. We also investigated the phosphorylation state of 4E-BP1. Whereas the shutoff of protein synthesis was observed as early as 2 h after infection and was complete after 4 h of infection, VSV infection did not reduce the phosphorylation of 4E-BP1 (data not shown). Rapamycin specifically inhibits cap-dependent translation both in vivo (NIH 3T3 cells) and in vitro (3). However, this inhibition is only partial (50% inhibition after 24 h of treatment) (3). Consistent with this, rapamycin exerted only a minor effect on translation under the conditions used here. However, addition of rapamycin to cells infected with EMCV or poliovirus enhanced virus-mediated inhibition of host protein synthesis. This effect was not observed in cells infected with VSV. Therefore, rapamycin in combination with EMCV or poliovirus displays a synergistic inhibitory effect on host protein synthesis. Inhibition of phosphorylation of 4E-BP1 upon
EMCV infection coincides with the shutoff of protein synthesis (7), and rapamycin inhibits phosphorylation of 4E-BP1 within 1 h (3). Therefore, the early reduction of phosphorylation of 4E-BP1 by rapamycin might explain the synergistic effects observed here. These results thus reinforce the role of 4E-BP1 in the shutoff of host protein synthesis in EMCV infection. We have observed only a small increase in virus yield in the presence of rapamycin. However, poliovirus and EMCV are two of the fastest replicating viruses in tissue culture cells. We have recently found that rapamycin exhibited a dramatic effect on the replication of a debilitated EMCV (29a). It would be therefore of great interest to test the effect of rapamycin on slow-replicating viruses such as hepatitis A and C viruses, which initiate translation by using an IRES element. The finding that rapamycin, an immunosuppressant that is evaluated in clinical studies, can stimulate the translation of viral mRNAs that initiate by internal ribosome binding is of clinical importance because hepatitis C virus also translates by this mechanism and hepatitis C virus is the leading cause of chronic viral hepatitis in the United States (18a). We thank A.-C. Gingras for the gift of anti-4E-BP1 antibody. This work was supported by a grant from the Medical Research Council of Canada to N.S. L.B. was supported by the Institut National de la Sante´ et de la Recherche Me´dicale (INSERM). REFERENCES 1. Abreu, S. L., and J. Lucas-Lenard. 1976. Cellular protein synthesis shut-off by mengovirus: translation of nonviral and viral mRNA’s in extracts from uninfected and infected Ehrlich ascites tumor cells. J. Virol. 18:182–194. 2. Barbet, N. C., U. Schneider, S. B. Helliwell, I. Stansfield, M. F. Tuite, and M. N. Hall. 1996. TOR controls translation initiation and early G1 progression in yeast. Mol. Biol. Cell 7:25–42. 3. Beretta, L., A. C. Gingras, Y. V. Svitkin, M. N. Hall, and N. Sonenberg. 1996. Rapamycin blocks the phosphorylation of 4E-BP1 and inhibits cap-dependent initiation of translation. EMBO J. 15:658–664. 4. Both, G. W., A. K. Banerjee, and A. J. Shatkin. 1975. Methylation-dependent
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