“
“Recent in vitro proteomics screens revealed that many host proteins could interact with the replication proteins of Tomato bushy stunt virus (TBSV), which is a small, plus-stranded RNA virus (Z. Li, D. Barajas, T. Panavas, D. A. Herbst, and P. D. Nagy, J. Virol. 82: 6911-6926, 2008). To further Foretinib ic50 our understanding of the roles of host factors in TBSV replication, we have tested the effect of Rsp5p, which is a member of the Nedd4 family of E3 ubiquitin ligases. The full-length Rsp5p, via its WW domain, is shown to interact with

p33 and the central portion of p92(pol) replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided

degradation of p92(pol), while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92(pol). We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, CHIR-99021 cell line involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92(pol) reduces the stability of p92(pol), with consequent inhibition of TBSV replicase activity.”
“Superinfection exclusion is the ability of an established viral infection to interfere with a second viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells

suppress subsequent WNV infection. The WNV replicon also suppresses superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV superinfection occurs at the step of RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and peptide 2K V9M). Mutagenesis Bcl-w analysis showed that the mutations in structural genes nonselectively enhance viral infection in both naive and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naive cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation ( and, to a lesser extent, the NS4a mutation) functions through the enhancement of viral RNA synthesis.