Sindbis virus usurps the cellular HUR protein to stabilize its transcripts and promote infections of mammalian and mosquito cells

Abstract

Members of the genus Alphavirus are recognized as significant human pathogens. Infection of vertebrate hosts often results in febrile illness and occasionally severe encephalitis. The archetypical alphavirus is Sindbis virus, which we have utilized in these studies. The genomic and subgenomic RNAs of Sindbis virus strongly resemble cellular mRNAs as they are capped at their 5’ ends and polyadenylated at their 3’ termini. These features allow the viral RNAs to act like cellular mRNAs and make them prime substrates for the cellular mRNA decay machinery. Sindbis virus RNAs are indeed subject to degradation by the cellular mRNA decay machinery in cell culture models of infection. Nevertheless, they decay by a mechanism that is different from the majority of cellular mRNAs as the decay of Sindbis virus transcripts is predominantly deadenylation-independent. As cellular mRNAs are often regulated by elements present in their 3’ untranslated regions (UTR), we hypothesized that these viral 3’UTR elements were functioning similarly to cellular mRNA stability elements resulting in the enhancement of viral infection. The primary goal of the research described in this dissertation was to characterize in mechanistic detail how the Sindbis virus 3’UTR represses deadenylation. To this end we used both cell free extracts and tissue culture systems to assay the effects of the viral 3’UTR on transcript stability. Interestingly, multiple elements were found to be independently repressing deadenylation in mosquito cytoplasmic extracts. Further examination revealed that a major stability determinant was the U-rich element (URE) observed in the 3’UTR of many alphaviruses. The ability to repress deadenylation in our cell free extract system was similarly observed with the UREs of Venezuelan equine, eastern equine, western equine and Semliki Forest viruses. Taken together, these data strongly assert that the repression of deadenylation via the URE is evolutionarily conserved. Prior to this study, the URE had no ascribed function. The repression of deadenylation imparted by the URE correlated with the binding of a cellular 38kDa factor. This 38kDa factor was determined to be the cellular HuR protein. Both the human and mosquito HuR proteins were found to bind with high affinity to the Sindbis virus 3’UTR. Reduction of cellular HuR protein levels using RNAi resulted in an increase in the rate of viral RNA decay. Furthermore, a significant decrease in the titer of progeny virus was observed. A similar effect on viral titer was observed when the predominant HuR binding site, the URE, was deleted from the viral 3’UTR. Taken together these observations identify a novel Alphavirus/ host interface that significantly impacts viral biology. Furthermore these studies have confirmed our hypothesis that the members of genus Alphavirus have indeed evolved RNA stability elements that resemble cellular mRNA stability elements for the purpose of enhancing viral infection. Furthermore these studies identify a potential therapeutic anti-viral target - the cellular HuR protein.