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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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(A) The accumulation of poliovirus RNA was delayed but not abolished in MG132 treated poliovirus-infected cells.Northern blot hybridization of 5 µg of total RNA from poliovirus infected cells with poliovirus protein 3C hybridization probe. Hybridization with GAPDH gene was a RNA loading control. (B) The inhibition of proteasome activity does not affect the entrance of poliovirus into the cells. MG treated and control HeLa cells were pre-incubated with S35-labeled poliovirus (MOI = 100) for 1 h at 4°C. To estimate adsorption background, cells (ad) were washed with cold PBS. Virus internalization (in) was estimated by accumulation of S35-labeled poliovirus capsid proteins during additional 1 h incubation at 37°C. S35-labeled proteins were analyzed by electrophoresis and autoradiography. (C) Poliovirus capsid proteins accumulate slower in MG132 pretreated cells. The extracts from poliovirus-infected cells were analyzed with anti-poliovirus capsid Abs. Control or MG132 2 h pretreated cells were incubated with poliovirus (MOI = 5) for 1 h. Virus containing medium was washed out and cells were incubated for indicated time. 10 µg of protein from infected cells were analyzed by Western blotting with anti-poliovirus capsid Abs.
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pone-0001887-g005: (A) The accumulation of poliovirus RNA was delayed but not abolished in MG132 treated poliovirus-infected cells.Northern blot hybridization of 5 µg of total RNA from poliovirus infected cells with poliovirus protein 3C hybridization probe. Hybridization with GAPDH gene was a RNA loading control. (B) The inhibition of proteasome activity does not affect the entrance of poliovirus into the cells. MG treated and control HeLa cells were pre-incubated with S35-labeled poliovirus (MOI = 100) for 1 h at 4°C. To estimate adsorption background, cells (ad) were washed with cold PBS. Virus internalization (in) was estimated by accumulation of S35-labeled poliovirus capsid proteins during additional 1 h incubation at 37°C. S35-labeled proteins were analyzed by electrophoresis and autoradiography. (C) Poliovirus capsid proteins accumulate slower in MG132 pretreated cells. The extracts from poliovirus-infected cells were analyzed with anti-poliovirus capsid Abs. Control or MG132 2 h pretreated cells were incubated with poliovirus (MOI = 5) for 1 h. Virus containing medium was washed out and cells were incubated for indicated time. 10 µg of protein from infected cells were analyzed by Western blotting with anti-poliovirus capsid Abs.

Mentions: To understand the possible role of proteasomes in poliovirus replication, we studied kinetics of virus infection by titration infectious virus released into the medium of poliovirus-infected HeLa cells with and without two-hour MG132 pre-treatment. Although virus titers during late phases of viral infection (5–6 h) were similar in control cells and in cells pre-treated with MG132, virus accumulation was noticeably delayed between 3 and 4 h in cells, in which proteasome activity was suppressed with MG132 (Fig. 4A). The efficiency of proteasome inhibitor was confirmed by observing stabilization of IκBα in TNF-treated HeLa cells (Fig. 4B) [36]. As an independent confirmation of the changes in the kinetic of poliovirus accumulation in cells pre-treated with proteasome inhibitor, we found that the appearance and accumulation of poliovirus capsid proteins was similarly delayed in cells pre-treated with MG132 (Fig. 4C). In the same way, the appearance of viral non-structural proteins 3C and 3A/3AB was also delayed by one hour in MG132 treated poliovirus-infected cells (Fig. 4D). Bortezomib had similar effect on poliovirus replication (Fig. 4E). In agreement with the immunoblotting data, as assessed by Northern blot there was a delay in the accumulation of viral genomic RNA in MG132 pretreated cells (Fig. 5A).


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)

(A) The accumulation of poliovirus RNA was delayed but not abolished in MG132 treated poliovirus-infected cells.Northern blot hybridization of 5 µg of total RNA from poliovirus infected cells with poliovirus protein 3C hybridization probe. Hybridization with GAPDH gene was a RNA loading control. (B) The inhibition of proteasome activity does not affect the entrance of poliovirus into the cells. MG treated and control HeLa cells were pre-incubated with S35-labeled poliovirus (MOI = 100) for 1 h at 4°C. To estimate adsorption background, cells (ad) were washed with cold PBS. Virus internalization (in) was estimated by accumulation of S35-labeled poliovirus capsid proteins during additional 1 h incubation at 37°C. S35-labeled proteins were analyzed by electrophoresis and autoradiography. (C) Poliovirus capsid proteins accumulate slower in MG132 pretreated cells. The extracts from poliovirus-infected cells were analyzed with anti-poliovirus capsid Abs. Control or MG132 2 h pretreated cells were incubated with poliovirus (MOI = 5) for 1 h. Virus containing medium was washed out and cells were incubated for indicated time. 10 µg of protein from infected cells were analyzed by Western blotting with anti-poliovirus capsid Abs.
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Related In: Results  -  Collection

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pone-0001887-g005: (A) The accumulation of poliovirus RNA was delayed but not abolished in MG132 treated poliovirus-infected cells.Northern blot hybridization of 5 µg of total RNA from poliovirus infected cells with poliovirus protein 3C hybridization probe. Hybridization with GAPDH gene was a RNA loading control. (B) The inhibition of proteasome activity does not affect the entrance of poliovirus into the cells. MG treated and control HeLa cells were pre-incubated with S35-labeled poliovirus (MOI = 100) for 1 h at 4°C. To estimate adsorption background, cells (ad) were washed with cold PBS. Virus internalization (in) was estimated by accumulation of S35-labeled poliovirus capsid proteins during additional 1 h incubation at 37°C. S35-labeled proteins were analyzed by electrophoresis and autoradiography. (C) Poliovirus capsid proteins accumulate slower in MG132 pretreated cells. The extracts from poliovirus-infected cells were analyzed with anti-poliovirus capsid Abs. Control or MG132 2 h pretreated cells were incubated with poliovirus (MOI = 5) for 1 h. Virus containing medium was washed out and cells were incubated for indicated time. 10 µg of protein from infected cells were analyzed by Western blotting with anti-poliovirus capsid Abs.
Mentions: To understand the possible role of proteasomes in poliovirus replication, we studied kinetics of virus infection by titration infectious virus released into the medium of poliovirus-infected HeLa cells with and without two-hour MG132 pre-treatment. Although virus titers during late phases of viral infection (5–6 h) were similar in control cells and in cells pre-treated with MG132, virus accumulation was noticeably delayed between 3 and 4 h in cells, in which proteasome activity was suppressed with MG132 (Fig. 4A). The efficiency of proteasome inhibitor was confirmed by observing stabilization of IκBα in TNF-treated HeLa cells (Fig. 4B) [36]. As an independent confirmation of the changes in the kinetic of poliovirus accumulation in cells pre-treated with proteasome inhibitor, we found that the appearance and accumulation of poliovirus capsid proteins was similarly delayed in cells pre-treated with MG132 (Fig. 4C). In the same way, the appearance of viral non-structural proteins 3C and 3A/3AB was also delayed by one hour in MG132 treated poliovirus-infected cells (Fig. 4D). Bortezomib had similar effect on poliovirus replication (Fig. 4E). In agreement with the immunoblotting data, as assessed by Northern blot there was a delay in the accumulation of viral genomic RNA in MG132 pretreated cells (Fig. 5A).

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