Production of Noncapped Genomic RNAs Is Critical to Sindbis Virus Disease and Pathogenicity.

Autumn T. LaPointe,V Douglas Landers,Kevin J. Sokoloski,Claire E. Westcott,John T. Patton

doi:10.1128/mbio.02675-20

Abstract

Alphaviruses are positive-sense RNA viruses that utilize a 5' cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Nonetheless, significant quantities of viral genomic RNAs that lack a canonical 5' cap structure are produced during alphaviral replication and packaged into viral particles. However, the role/impact of the noncapped genomic RNA (ncgRNA) during alphaviral infection in vivo has yet to be characterized. To determine the importance of the ncgRNA in vivo, the previously described D355A and N376A nsP1 mutations, which increase or decrease nsP1 capping activity, respectively, were incorporated into the neurovirulent AR86 strain of Sindbis virus to enable characterization of the impact of altered capping efficiency in a murine model of infection. Mice infected with the N376A nsP1 mutant exhibited slightly decreased rates of mortality and delayed weight loss and neurological symptoms, although levels of inflammation in the brain were similar to those of wild-type infection. Although the D355A mutation resulted in decreased antiviral gene expression and increased resistance to interferon in vitro, mice infected with the D355A mutant showed significantly reduced mortality and morbidity compared to mice infected with wild-type virus. Interestingly, expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. Collectively, these data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis.IMPORTANCE Mosquito-transmitted alphaviruses have been the cause of widespread outbreaks of disease that can range from mild illness to lethal encephalitis or severe polyarthritis. There are currently no safe and effective vaccines or therapeutics with which to prevent or treat alphaviral disease, highlighting the need to better understand alphaviral pathogenesis to develop novel antiviral strategies. This report reveals production of noncapped genomic RNAs (ncgRNAs) to be a novel determinant of alphaviral virulence and offers insight into the importance of inflammation to pathogenesis. Taken together, the findings reported here suggest that the ncgRNAs contribute to alphaviral pathogenesis through the sensing of the ncgRNAs during alphaviral infection and are necessary for the development of severe disease.

Highlights

Alphaviruses are positive-sense RNA viruses that utilize a 5= cap structure to facilitate translation of viral proteins and to protect the viral RNA genome
We have shown that altering capping efficiency impacts how much IFN is produced, but does altering Sindbis virus (SINV) capping efficiency affect the virus’ capacity to resist IFN treatment when the cells are treated with equal amounts of type I IFN? To determine whether changes in noncapped genomic RNA (ncgRNA) production affected the sensitivity of the nsP1 mutant viruses to exogenous type I IFN, IFN-competent L929 cells were infected with either the wild type or a SINV nsP1 mutant virus, recombinant type I IFN was added at 0, 1, 2, 3, or 4 h postinfection, and viral titer was measured at 24 hpi (Fig. 5A to C) [23]
When capping efficiency was increased with the D355A mutant, nonstructural gene expression was found to be increased, the magnitude was not as drastic as that previously seen with the Toto1101-derived background (Fig. 2)

Summary

Introduction

Alphaviruses are positive-sense RNA viruses that utilize a 5= cap structure to facilitate translation of viral proteins and to protect the viral RNA genome. Expression of proinflammatory cytokines was found to be significantly decreased in mice infected with the D355A mutant, suggesting that capping efficiency and the production of ncgRNA are vital to eliciting pathogenic levels of inflammation. These data indicate that the ncgRNA have important roles during alphaviral infection and suggest a novel mechanism by which noncapped viral RNAs aid in viral pathogenesis. Alphaviruses produce three RNA species during infection: the genomic strand, which encodes the nonstructural proteins; the minus-strand RNA template; and the subgenomic RNA, which encodes the structural proteins Both the genomic and subgenomic RNAs have a type 0 cap structure added to their 5= ends to facilitate translation and protect the 5= end of the transcripts [6,7,8]. The m7GMP moiety is transferred to the 5= end of the vRNA molecule by the guanylyltransferase activities of nsP1, resulting in the 7meGppA type 0 cap structure [11]

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