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. 2002 Oct;76(20):10177-87.
doi: 10.1128/jvi.76.20.10177-10187.2002.

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

John H Connor  1 , Douglas S Lyles
Affiliations

Vesicular stomatitis virus infection alters the eIF4F translation initiation complex and causes dephosphorylation of the eIF4E binding protein 4E-BP1

John H Connor et al. J Virol. 2002 Oct.

Abstract

Vesicular stomatitis virus (VSV) modulates protein synthesis in infected cells in a way that allows the translation of its own 5'-capped mRNA but inhibits the translation of host mRNA. Previous data have shown that inactivation of eIF2alpha is important for VSV-induced inhibition of host protein synthesis. We tested whether there is a role for eIF4F in this inhibition. The multisubunit eIF4F complex is involved in the regulation of protein synthesis via phosphorylation of cap-binding protein eIF4E, a subunit of eIF4F. Translation of host mRNA is significantly reduced under conditions in which eIF4E is dephosphorylated. To determine whether VSV infection alters the eIF4F complex, we analyzed eIF4E phosphorylation and the association of eIF4E with other translation initiation factors, such as eIF4G and the translation inhibitor 4E-BP1. VSV infection of HeLa cells resulted in the dephosphorylation of eIF4E at serine 209 between 3 and 6 h postinfection. This time course corresponded well to that of the inhibition of host protein synthesis induced by VSV infection. Cells infected with a VSV mutant that is delayed in the ability to inhibit host protein synthesis were also delayed in dephosphorylation of eIF4E. In addition to decreasing eIF4E phosphorylation, VSV infection also resulted in the dephosphorylation and activation of eIF4E-binding protein 4E-BP1 between 3 and 6 h postinfection. Analysis of cap-binding complexes showed that VSV infection reduced the association of eIF4E with the eIF4G scaffolding subunit at the same time as its association with 4E-BP1 increased and that these time courses correlated with the dephosphorylation of eIF4E. These changes in the eIF4F complex occurred over the same time period as the onset of viral protein synthesis, suggesting that activation of 4E-BP1 does not inhibit translation of viral mRNAs. In support of this idea, VSV protein synthesis was not affected by the presence of rapamycin, a drug that blocks 4E-BP1 phosphorylation. These data show that VSV infection results in modifications of the eIF4F complex that are correlated with the inhibition of host protein synthesis and that translation of VSV mRNAs occurs despite lowered concentrations of the active cap-binding eIF4F complex. This is the first noted modification of both eIF4E and 4E-BP1 phosphorylation levels among viruses that produce capped mRNA for protein translation.

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Figures

FIG. 1.
FIG. 1.
Viral and host protein synthesis following VSV infection. (A) HeLa cells were mock infected (M) or infected with VSV for the indicated times and then labeled with [35S]methionine for 10 min. Protein lysates (10 μl) were electrophoresed on a 12% gel, a phosphorescence image of which is shown. Viral proteins are indicated to the right of the image. (B) Total RNA was prepared from HeLa cells mock infected (M) or infected with VSV for the indicated times. RNA was separated on a 1% gel and transferred to nitrocellulose, and viral mRNA was detected with a 32P-labeled probe against the M protein mRNA. A phosphorescence image of a representative gel is shown. (C) Quantification of viral protein synthesis inhibition (closed triangles) and M protein mRNA (open triangles). The rate of viral protein synthesis was determined from experiments like that shown in panel A by quantitation of the radioactivity in the viral L and G bands and the N/P and M bands. The amount of mRNA present was determined by densitometry of M protein mRNA in Northern blots like that in panel B. The data shown are means ± standard deviations of three separate experiments
FIG. 2.
FIG. 2.
(A) Extracts from mock- and VSV-infected cells were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting with antibodies against eIF4E, eIF4E-P phosphorylated at serine 209 [eIF4E-P (Ser 209)], or the VSV M protein. Shown is a single blot reprobed with the three different antibodies. M, mock infection. (B) Quantification of host protein synthesis inhibition (closed squares) and eIF4E phosphorylation (closed circles). The rate of host protein synthesis was determined from experiments like that shown in Fig. 1 by quantitation of the radioactivity between the viral L and G bands, between the P and M bands, and in the region below the M band. The levels of eIF4E phosphorylation were determined by densitometry of phospho-eIF4E-specific Western blots and quantitated as a ratio to total eIF4E. The data shown are means ± standard deviations of three experiments.
FIG. 3.
FIG. 3.
VSV infection does not decrease the levels of eIF4G or phosphorylated Mnk1. Extracts from HeLa cells that were mock infected (M) or infected with VSV for the indicated time were separated on a 6% gel (for eIF4G) or a 12% gel and then analyzed by Western blotting with antibodies against eIF4G, Mnk1 phosphorylated at Thr 197 and Thr 202, and eIF4E.
FIG. 4.
FIG. 4.
Isolation of eIF4E and eIF4E-binding proteins from VSV-infected cells. (A) HeLa cells were mock infected (M) or infected with VSV for the indicated times. Cell extracts were then incubated with m7-GTP Sepharose to enrich for eIF4E and eIF4E-binding proteins. Proteins bound to eIF4E were determined by Western blotting following electrophoresis on a 4 to 20% gradient gel with antibodies against eIF4E, eIF4G, and 4E-BP1. Shown is a single blot reprobed with the three different antibodies. (B) Quantitation of eIF4G (closed triangles) and 4E-BP1 (open triangles) bound to eIF4E during VSV infection. eIF4G was quantitated as a ratio of eIF4G signal to eIF4E signal and expressed as a percentage of the mock-infected control. eIF4E-BP1 was also quantitated as a ratio to eIF4E and was expressed as a percentage of the value at 6 h postinfection. The data shown are averages ± standard deviations of three separate experiments.
FIG. 5.
FIG. 5.
4E-BP1 phosphorylation in VSV-infected cells. Cells were mock infected (M) or infected with VSV; cell extracts were separated on a 15% gel and analyzed by Western blotting with antibodies to 4E-BP1 phosphorylated at Ser 65 (A) or total 4E-BP1 (B). Protein loading was analyzed by blotting with an antibody against actin. Quantitation of the relative amounts of phospho 4E-BP1 (compared to mock infection) is shown directly below the bands.
FIG. 6.
FIG. 6.
Effect of tsO82 mutant virus on host cell protein synthesis and eIF4E phosphorylation. (A) HeLa cells were mock infected (M) or infected with tsO82 virus for the indicated times and then labeled with [35S]methionine for 10 min. Lysates (10 μl) were electrophoresed on a 12% gel, a phosphorescence image of which is shown. Viral proteins are indicated to the right of the image. (B) Quantification of host protein synthesis inhibition following tsO82 virus or VSV infection. The rate of host protein synthesis was determined from experiments like that shown in panel A by quantitation of the radioactivity between the viral L and G bands, between the P and M bands, and in the region below the M band. Cells infected with wt VSV are represented by filled squares, and cells infected with tsO82 virus are represented by open squares. (C) Extracts from mock- and VSV-infected cells were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by Western blotting with antibodies to eIF4E phosphorylated at serine 209 [eIF4E-P (Ser 209)]. (D) Quantification of eIF4E phosphorylation following VSV or tsO82 infection. The levels of eIF4E phosphorylation were determined by densitometry of phospho-eIF4E-specific Western blots and normalized to total eIF4E. Cells infected with wt VSV are represented by filled circles, and cells infected with tsO82 virus are represented by open circles. The data shown are averages ± standard deviations of three separate experiments.
FIG. 7.
FIG. 7.
4E-BP1 phosphorylation and isolation of eIF4E-binding proteins in tsO82 virus-infected cells. (A) Extracts from mock-, wt VSV-, or tsO82 virus-infected cells were incubated with m7-GTP Sepharose to enrich for eIF4E and eIF4E-binding proteins. Proteins bound to eIF4E were detected by Western blotting following separation on a 4 to 20% gel. The ratio of eIF4E to eIF4E-BP1 was quantified as described in Materials and Methods. (B) Extracts from mock-, wt VSV- or tsO82 virus-infected cells were separated on a 15% gel and analyzed by Western blotting with antibodies to total eIF4E-BP1.
FIG. 8.
FIG. 8.
Effect of rapamycin on host and viral protein synthesis in VSV-infected cells. (A) Western blot analysis of total 4E-BP1 following treatment of HeLa cells with 25 nM rapamycin for the indicated times. (B) HeLa cells were either left untreated (−) or treated (+) with 25 nM rapamycin (Rapa) for 1 h. These cells were then mock infected (−) or infected with VSV (+) for the indicated times and labeled with [35S]methionine for 10 min. Lysates were electrophoresed on a 12% gel, a phosphorescence image of which is shown. (C) Quantitation of host protein synthesis in untreated (closed circles) or rapamycin-treated (open circles) cells infected with VSV. Rapamycin inhibition of protein synthesis is indicated as a dotted line. Results from four independent experiments were quantified and are expressed as a percentage of the untreated, mock-infected control. (D) VSV protein synthesis in rapamycin-treated (dark bars) or untreated (white bars) cells. Results from four independent experiments were quantified and expressed as a percentage of the level at 6 h postinfection in untreated cells.
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