Abstract
BackgroundWest Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed. Recent studies have suggested that RNA interference (RNAi) has therapeutic potential as a sequence specific inhibitor of viral infection. Here, we examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of West Nile virus in human cells.ResultsWNV replication and infection was greatly reduced when siRNA were introduced by cytoplasmic-targeted transfection prior to but not after the establishment of viral replication. WNV appeared to evade rather than actively block the RNAi machinery, as sequence-specific reduction in protein expression of a heterologous transgene was still observed in WNV-infected cells. However, sequence-specific decreases in WNV RNA were observed in cells undergoing active viral replication when siRNA was transfected by an alternate method, electroporation.ConclusionOur results suggest that actively replicating WNV RNA may not be exposed to the cytoplasmic RNAi machinery. Thus, conventional lipid-based siRNA delivery systems may not be adequate for therapy against enveloped RNA viruses that replicate in specialized membrane compartments.
Highlights
West Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed
In vitro generated small interfering RNAs (siRNAs) inhibits West Nile virus (WNV) infection in cells We have previously demonstrated that plasmid expressed hairpin siRNA efficiently inhibited infection of WNV in mouse and human cell lines [21]
Pretreatment of Huh7.5 cells with Cap siRNA resulted in approximately 1 log reduction of WNV RNA, whereas pretreatment of Huh7.5 cells with Cap-Mut siRNA showed no significant reduction of WNV RNA
Summary
West Nile virus is an emerging human pathogen for which specific antiviral therapy has not been developed. We examine the ability of exogenous small interfering RNAs (siRNAs) to block the replication of West Nile virus in human cells. West Nile virus (WNV) is a significant human and veterinary mosquito-borne pathogen that has rapidly spread across North America. WNV is an enveloped virus with an 11-kilobase positive strand RNA genome. It is translated directly from the genomic RNA as a single polyprotein and cleaved by cellular and viral proteases into ten mature proteins, three structural (C, M, and E) and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins [2,3]. Genomic viral RNA traffics to the endoplasmic reticulum (ER), where WNV protein (page number not for citation purposes)
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