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Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

Neznanov N, Dragunsky EM, Chumakov KM, Neznanova L, Wek RC, Gudkov AV, Banerjee AK - PLoS ONE (2008)

Bottom Line: We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication.We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation.Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America. neznann@ccf.org

ABSTRACT
Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

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Inhibition of VSV replication in MG132 treated fibroblasts depends on GCN2.(A) Attenuation of translation in MG132- (MG), and Bortezomib (Bort) -treated cells is GCN2-dependent. Control wt GCN2+/+ MEF and GCN2−/− MEF, or cells treated with proteasome inhibitors for 4 h were incubated with S35-methionine/cysteine for 30 min. Protein synthesis was estimated by electrophoresis and autoradiography. (B) Western immunoblotting analysis of GCN2-dependent phosphorylation of eIF2α in response to MG132. 10 µg of protein extracts from control and MG132 treated cells were analyzed with indicated antibodies. Efficiency's fold of eIF2α phosphorylation (Phosp(x)) was estimated with ImageJ software. (C, D) Replication of VSV was not affected by proteasome inhibitors in GCN2−/− MEF. Proteasome inhibitors were added 1 h after infection with VSV (MOI = 1) and cells were incubated over night. Replication of VSV was estimated by titration in two experiments (C), or by Western immunoblotting with anti P-VSV protein Abs (D). Tubulin (tub) is a protein loading control.
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pone-0001887-g010: Inhibition of VSV replication in MG132 treated fibroblasts depends on GCN2.(A) Attenuation of translation in MG132- (MG), and Bortezomib (Bort) -treated cells is GCN2-dependent. Control wt GCN2+/+ MEF and GCN2−/− MEF, or cells treated with proteasome inhibitors for 4 h were incubated with S35-methionine/cysteine for 30 min. Protein synthesis was estimated by electrophoresis and autoradiography. (B) Western immunoblotting analysis of GCN2-dependent phosphorylation of eIF2α in response to MG132. 10 µg of protein extracts from control and MG132 treated cells were analyzed with indicated antibodies. Efficiency's fold of eIF2α phosphorylation (Phosp(x)) was estimated with ImageJ software. (C, D) Replication of VSV was not affected by proteasome inhibitors in GCN2−/− MEF. Proteasome inhibitors were added 1 h after infection with VSV (MOI = 1) and cells were incubated over night. Replication of VSV was estimated by titration in two experiments (C), or by Western immunoblotting with anti P-VSV protein Abs (D). Tubulin (tub) is a protein loading control.

Mentions: GCN2 is a protein kinase responsible for eIF2α phosphorylation in response to amino acid starvation and some other stresses [44], [47]. Proteasome inhibitors' induced stress and attenuated translation is GCN2-dependent [44], [47]. To prove the role of stress induced by proteasome inhibition in suppression of VSV replication, we used wild type (wt) GCN2+/+ MEF and GCN2−/− MEF for VSV infection. Inhibition of proteasome activity attenuated general translation in wt GCN2+/+ MEF, but did not have effect on protein synthesis in GCN2−/− cells (Fig. 10A). MG132-specific phosphorylation of eIF2α factor was less efficient in GCN2−/− than in wt GCN2+/+ MEF cells (Fig. 10B). In agreement with these data, VSV replication was affected by inhibitors of proteasomes in wt GCN2+/+ MEF, but was not changed in GCN2−/− cells (Fig. 10 C, D). Surprisingly, GCN2 activity suppressed VSV replication in MEF without additional treatment with proteasome inhibitors (Fig. 10 C, D).


Different effect of proteasome inhibition on vesicular stomatitis virus and poliovirus replication.

Neznanov N, Dragunsky EM, Chumakov KM, Neznanova L, Wek RC, Gudkov AV, Banerjee AK - PLoS ONE (2008)

Inhibition of VSV replication in MG132 treated fibroblasts depends on GCN2.(A) Attenuation of translation in MG132- (MG), and Bortezomib (Bort) -treated cells is GCN2-dependent. Control wt GCN2+/+ MEF and GCN2−/− MEF, or cells treated with proteasome inhibitors for 4 h were incubated with S35-methionine/cysteine for 30 min. Protein synthesis was estimated by electrophoresis and autoradiography. (B) Western immunoblotting analysis of GCN2-dependent phosphorylation of eIF2α in response to MG132. 10 µg of protein extracts from control and MG132 treated cells were analyzed with indicated antibodies. Efficiency's fold of eIF2α phosphorylation (Phosp(x)) was estimated with ImageJ software. (C, D) Replication of VSV was not affected by proteasome inhibitors in GCN2−/− MEF. Proteasome inhibitors were added 1 h after infection with VSV (MOI = 1) and cells were incubated over night. Replication of VSV was estimated by titration in two experiments (C), or by Western immunoblotting with anti P-VSV protein Abs (D). Tubulin (tub) is a protein loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2268745&req=5

pone-0001887-g010: Inhibition of VSV replication in MG132 treated fibroblasts depends on GCN2.(A) Attenuation of translation in MG132- (MG), and Bortezomib (Bort) -treated cells is GCN2-dependent. Control wt GCN2+/+ MEF and GCN2−/− MEF, or cells treated with proteasome inhibitors for 4 h were incubated with S35-methionine/cysteine for 30 min. Protein synthesis was estimated by electrophoresis and autoradiography. (B) Western immunoblotting analysis of GCN2-dependent phosphorylation of eIF2α in response to MG132. 10 µg of protein extracts from control and MG132 treated cells were analyzed with indicated antibodies. Efficiency's fold of eIF2α phosphorylation (Phosp(x)) was estimated with ImageJ software. (C, D) Replication of VSV was not affected by proteasome inhibitors in GCN2−/− MEF. Proteasome inhibitors were added 1 h after infection with VSV (MOI = 1) and cells were incubated over night. Replication of VSV was estimated by titration in two experiments (C), or by Western immunoblotting with anti P-VSV protein Abs (D). Tubulin (tub) is a protein loading control.
Mentions: GCN2 is a protein kinase responsible for eIF2α phosphorylation in response to amino acid starvation and some other stresses [44], [47]. Proteasome inhibitors' induced stress and attenuated translation is GCN2-dependent [44], [47]. To prove the role of stress induced by proteasome inhibition in suppression of VSV replication, we used wild type (wt) GCN2+/+ MEF and GCN2−/− MEF for VSV infection. Inhibition of proteasome activity attenuated general translation in wt GCN2+/+ MEF, but did not have effect on protein synthesis in GCN2−/− cells (Fig. 10A). MG132-specific phosphorylation of eIF2α factor was less efficient in GCN2−/− than in wt GCN2+/+ MEF cells (Fig. 10B). In agreement with these data, VSV replication was affected by inhibitors of proteasomes in wt GCN2+/+ MEF, but was not changed in GCN2−/− cells (Fig. 10 C, D). Surprisingly, GCN2 activity suppressed VSV replication in MEF without additional treatment with proteasome inhibitors (Fig. 10 C, D).

Bottom Line: We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication.We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation.Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio, United States of America. neznann@ccf.org

ABSTRACT
Proteasome activity is an important part of viral replication. In this study, we examined the effect of proteasome inhibitors on the replication of vesicular stomatitis virus (VSV) and poliovirus. We found that the proteasome inhibitors significantly suppressed VSV protein synthesis, virus accumulation, and protected infected cells from toxic effect of VSV replication. In contrast, poliovirus replication was delayed, but not diminished in the presence of the proteasome inhibitors MG132 and Bortezomib. We also found that inhibition of proteasomes stimulated stress-related processes, such as accumulation of chaperone hsp70, phosphorylation of eIF2alpha, and overall inhibition of translation. VSV replication was sensitive to this stress with significant decline in replication process. Poliovirus growth was less sensitive with only delay in replication. Inhibition of proteasome activity suppressed cellular and VSV protein synthesis, but did not reduce poliovirus protein synthesis. Protein kinase GCN2 supported the ability of proteasome inhibitors to attenuate general translation and to suppress VSV replication. We propose that different mechanisms of translational initiation by VSV and poliovirus determine their sensitivity to stress induced by the inhibition of proteasomes. To our knowledge, this is the first study that connects the effect of stress induced by proteasome inhibition with the efficiency of viral infection.

Show MeSH
Related in: MedlinePlus