Different Effect of Proteasome Inhibition on Vesicular Stomatitis Virus and Poliovirus Replication

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 eIF2α, 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

RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence.

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) was used to study sequence heterogeneity and stability in attenuated poliovirus type 3 at positions in which the vaccine virus differs from its wild-type progenitor. Of seven genomic positions tested, only two (positions 472 and 2493) show nucleotide heterogeneity. Propagation of the vaccine virus in cell cultures leads to rapid selection of virus with reversions at these two positions of the genome. The relative abundance of reversions at position 472 correlates with the results of monkey neurovirulence tests, while the mutation at position 2493 is not directly associated with neurovirulence of the virus in monkeys. Instead, the abundance of mutations at the latter position correlates with the source of the seed virus and its passage level. These results further indicate that MAPREC at position 472 can be used to assess the quality of poliovirus type 3 vaccine

MicroRNAs as potential biomarkers for VERO cell tumorigenicity

AbstractMicroRNA expression appears to capture the process of neoplastic development in vitro in the VERO line of African green monkey kidney (AGMK) cells (Teferedegne et al. PLoS One 2010;5(12):e14416). In that study, specific miRNA signatures were correlated with the transition, during serial tissue-culture passage, of low-density passaged 10–87 VERO cells from a non-tumorigenic phenotype at passage (p) 148 to a tumorigenic phenotype at p256. In the present study, six miRNAs (miR-376a, miR-654-3p, miR-543, miR-299-3p, miR-134 and miR-369-3p) were chosen from the identified signature miRNAs for evaluation of their use as potential biomarkers to track the progression of neoplastic development in VERO cells. Cells from the 10–87 VERO cell line at passage levels from p148 to p256 were inoculated into newborn and adult athymic nude mice. No tumors were observed in animals inoculated with cells from p148 to p186. In contrast, tumor incidences of 20% developed only in newborn mice that received 10–87 VERO cells at p194, p234 and p256. By qPCR profiling of the signature miRNAs of 10–87 VERO cells from these cell banks, we identified p194 as the level at which signature miRNAs elevated concurrently with the acquisition of tumorigenic phenotype with similar levels expressed beyond this passage. In wound-healing assays at 10-passage intervals between p150 to p250, the cells displayed a progressive increase in migration from p165 to p186; beginning at p194 and higher passages thereafter, the cells exhibited the highest rates of migration. By qPCR analysis, the same signature miRNAs were overexpressed with concomitant acquisition of the tumorigenic phenotype in another lineage of 10–87 VERO cells passaged independently at high density. Correlation between the passages at which the cells expressed a tumorigenic phenotype and the passages representing peaks in expression levels of signature miRNAs indicates that these miRNAs are potential biomarkers for the expression of the VERO cell tumorigenic phenotype

Different proteasome inhibitors affect VSV replication.

<p>(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.</p

Proteolytic cleavage of p65-RelA and eIF4G occurred later during poliovirus infection of the cells with inhibited proteasome activity.

<p>HeLa cells and MG 132 2 h pretreated HeLa cells were infected with poliovirus (MOI = 5) for 1 h. After change of medium, total protein extracts were collected every hour and tested with anti-p65-RelA C-terminus specific Abs (A) or with anti eIF4G N-terminus specific Abs (B). 10 µg of protein were tested in Western blotting experiments.</p

Inhibition of VSV replication in MG132 treated fibroblasts depends on GCN2.

<p>(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.</p

Different activation of eIF2α phosphorylation by VSV and poliovirus infections.

<p>HeLa cells were infected with VSV for 4 h, infected with poliovirus for 4 h, or treated with 1 µM of thapsigargin for 1 hour. Cytoplasmic protein extracts from these and control cells were analyzed with Abs against eIF2α and phosphorylated form of eIF2α (panel A). Same membrane was analyzed with Abs against VSV P- protein and poliovirus capsid proteins (panel B).</p