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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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Different proteasome inhibitors affect VSV replication.(A) Proteasome inhibitor 1 and Bortezomib decreased VSV replication. Titration of VSV from the medium of overnight infected HeLa cells. VSV infection (MOI = 1) for one hour was substituted by the regular medium with indicated concentration of proteasome inhibitors. VSV was titrated by plaque assay after overnight growth. (B) Analysis of P-protein synthesis in the cells treated with proteasome inhibitor 1. HeLa cells were infected with VSV (MOI = 5) for 4 h and treated with proteasome inhibitor 1 (PI) or MG132 (MG) at a time of VSV infection. The total protein extracts (5 µg) from these cells were analyzed by Western blotting with anti-P-protein Abs. The concentrations of proteasome inhibitors varied from 5 to 20 µM. Keratin 18 (K18) was a protein loading control. (C) Bortezomib suppressed VSV replication. HeLa cells were infected with VSV, treated with Bortezomib (100 nM) and MG132 (5 µM), and analyzed as described in panel B. K18 was a protein loading control.
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pone-0001887-g003: Different proteasome inhibitors affect VSV replication.(A) Proteasome inhibitor 1 and Bortezomib decreased VSV replication. Titration of VSV from the medium of overnight infected HeLa cells. VSV infection (MOI = 1) for one hour was substituted by the regular medium with indicated concentration of proteasome inhibitors. VSV was titrated by plaque assay after overnight growth. (B) Analysis of P-protein synthesis in the cells treated with proteasome inhibitor 1. HeLa cells were infected with VSV (MOI = 5) for 4 h and treated with proteasome inhibitor 1 (PI) or MG132 (MG) at a time of VSV infection. The total protein extracts (5 µg) from these cells were analyzed by Western blotting with anti-P-protein Abs. The concentrations of proteasome inhibitors varied from 5 to 20 µM. Keratin 18 (K18) was a protein loading control. (C) Bortezomib suppressed VSV replication. HeLa cells were infected with VSV, treated with Bortezomib (100 nM) and MG132 (5 µM), and analyzed as described in panel B. K18 was a protein loading control.

Mentions: To confirm that inhibitory effect of MG132 is due to its suppression of proteasome activity, we tested different proteasome inhibitors. Proteasome inhibitor 1 is a modified tri-peptide with a structure different from MG132. It proteasome-inhibiting activity requires higher concentrations than MG132. In these experiments MG132 served as a positive control. Both proteasome inhibitors affected VSV replication in HeLa cells in a similar manner (Fig. 3 panels A and B). Bortezomib (PS341) is a specific inhibitor of proteasomes, approved by FDA as anti-cancer drug [35]. Its structure and mechanism of action is different from MG132 and proteasome inhibitor 1. It is more active than MG132 and was used at concentration as low as 100 nM. Bortezomib, like other proteasome inhibitors, suppressed VSV replication (Fig. 3 panels A and C).


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)

Different proteasome inhibitors affect VSV replication.(A) Proteasome inhibitor 1 and Bortezomib decreased VSV replication. Titration of VSV from the medium of overnight infected HeLa cells. VSV infection (MOI = 1) for one hour was substituted by the regular medium with indicated concentration of proteasome inhibitors. VSV was titrated by plaque assay after overnight growth. (B) Analysis of P-protein synthesis in the cells treated with proteasome inhibitor 1. HeLa cells were infected with VSV (MOI = 5) for 4 h and treated with proteasome inhibitor 1 (PI) or MG132 (MG) at a time of VSV infection. The total protein extracts (5 µg) from these cells were analyzed by Western blotting with anti-P-protein Abs. The concentrations of proteasome inhibitors varied from 5 to 20 µM. Keratin 18 (K18) was a protein loading control. (C) Bortezomib suppressed VSV replication. HeLa cells were infected with VSV, treated with Bortezomib (100 nM) and MG132 (5 µM), and analyzed as described in panel B. K18 was a protein loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2268745&req=5

pone-0001887-g003: Different proteasome inhibitors affect VSV replication.(A) Proteasome inhibitor 1 and Bortezomib decreased VSV replication. Titration of VSV from the medium of overnight infected HeLa cells. VSV infection (MOI = 1) for one hour was substituted by the regular medium with indicated concentration of proteasome inhibitors. VSV was titrated by plaque assay after overnight growth. (B) Analysis of P-protein synthesis in the cells treated with proteasome inhibitor 1. HeLa cells were infected with VSV (MOI = 5) for 4 h and treated with proteasome inhibitor 1 (PI) or MG132 (MG) at a time of VSV infection. The total protein extracts (5 µg) from these cells were analyzed by Western blotting with anti-P-protein Abs. The concentrations of proteasome inhibitors varied from 5 to 20 µM. Keratin 18 (K18) was a protein loading control. (C) Bortezomib suppressed VSV replication. HeLa cells were infected with VSV, treated with Bortezomib (100 nM) and MG132 (5 µM), and analyzed as described in panel B. K18 was a protein loading control.
Mentions: To confirm that inhibitory effect of MG132 is due to its suppression of proteasome activity, we tested different proteasome inhibitors. Proteasome inhibitor 1 is a modified tri-peptide with a structure different from MG132. It proteasome-inhibiting activity requires higher concentrations than MG132. In these experiments MG132 served as a positive control. Both proteasome inhibitors affected VSV replication in HeLa cells in a similar manner (Fig. 3 panels A and B). Bortezomib (PS341) is a specific inhibitor of proteasomes, approved by FDA as anti-cancer drug [35]. Its structure and mechanism of action is different from MG132 and proteasome inhibitor 1. It is more active than MG132 and was used at concentration as low as 100 nM. Bortezomib, like other proteasome inhibitors, suppressed VSV replication (Fig. 3 panels A and C).

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