Increasing the Capping Efficiency of the Sindbis Virus nsP1 Protein Negatively Affects Viral Infection

Abstract

Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe disease and are a significant burden to public health. Alphaviral replication results in the production of both capped and noncapped viral genomic RNAs, which are packaged into virions during the infections of vertebrate and invertebrate cells. However, the roles that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the proteins capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased capping efficiency, while a point mutant at D355A increased the capping efficiency of the SINV genomic RNA during genuine viral infection. Viral growth kinetics were significantly reduced for the D355A mutant relative to wild type infection, whereas the Y286A and N376A mutants showed modest decreases in growth kinetics. Overall genomic translation and nonstructural protein accumulation was found to correlate with increases and decreases in capping efficiency. However, genomic, minus strand, and subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral capping activity. In addition, translation of the subgenomic vRNA was found to be unimpacted by changes in capping efficiency. The mechanism by which decreased presence of ncgRNAs reduced viral growth kinetics was through the impaired production of viral particles. Collectively, these data illustrate the importance of ncgRNAs to viral infection and suggests that they play in integral role in the production of viral progeny.\n\nImportanceAlphaviruses have been the cause of both localized outbreaks and large epidemics of severe disease. Currently, there are no strategies or vaccines which are either safe or effective for preventing alphaviral infection or treating alphaviral disease. This deficit of viable therapeutics highlights the need to better understand the mechanisms behind alphaviral infection in order to develop novel antiviral strategies for alphaviral disease. In particular, this study details a previously uncharacterized aspect of the alphaviral life cycle, the importance of noncapped genomic viral RNAs to alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.