The importance of noncapped viral genomic RNA to alphaviral infection and pathogenesis.

Alphaviruses are mosquito-borne arboviruses that have a positive sense, single-stranded RNA genome, which are capable of causing severe disease and thus pose a significant burden to public health. During alphaviral replication, significant quantities of viral genomic RNAs that lack the canonical alphaviral 5’ cap structure are produced and packaged into viral particles, despite the fact that these noncapped genomes cannot be translated and are essentially noninfectious. The production and packaging of the noncapped genomes has been found to be true for infections across multiple vertebrate and invertebrate cell lines and alphavirus species, although the proportion of ncgRNA produced differs. Despite knowing this, the importance of the noncapped genomic vRNA (ncgRNA) to alphaviral infection has yet to be fully characterized. In this dissertation, the importance of the ncgRNAs to alphaviral infection was assessed by using mutations of the nsP1 protein of Sindbis virus (SINV), which altered the synthesis of the ncgRNAs during infection by modulating the protein’s capping efficiency. It was found that decreasing ncgRNA production lead to decreased viral growth kinetics via decreased particle production; whereas increasing ncgRNA production led to wild type growth kinetics and particle production. In addition to being necessary for efficient viral replication in vitro, we also found that the noncapped genomic RNA was critical for alphaviral pathogenesis. Mice infected with the increased ncgRNA mutant exhibited wild type rates weight loss, and neurological symptoms, but exhibited a slight decrease in mortality. Conversely, the mice infected with the decreased ncgRNA mutant showed significantly reduced mortality and morbidity compared to mice infected with wild type virus. Interestingly, both mutants had modestly reduced viral titer in the brain compared to wild type virus. Importantly, examination of the brain tissue revealed that mice infected with the decreased ncgRNA mutant had significantly reduced cell death, immune cell infiltration, and expression of proinflammatory cytokines compared to the increased ncgRNA mutant and wild type virus. Collectively, these data indicate that the ncgRNA play an important role during alphaviral replication and pathogenesis by increasing the efficiency of particle production and modulating the host immune response.