Arthropod-borne viruses, such as the members of genus , are a significant concern to global public health. As obligate intracellular pathogens, RNA viruses must interact with the host cell machinery to establish, and complete, their viral lifecycles. Despite considerable efforts to define the host/pathogen interactions essential for alphaviral replication, an unbiased and inclusive assessment of alphaviral RNA:protein interactions has not been undertaken. Moreover, the biological and molecular importance of these interactions, in the full context of their molecular function as RNA-binding proteins, has not been fully realized. The data presented here introduces a robust viral RNA:protein discovery method to elucidate the Sindbis virus (SINV) RNA:Protein host interface. Cross-Link Assisted mRNP Purification (CLAMP) assessment reveals an extensive array of host/pathogen interactions centered on the viral RNAs (vRNAs). After prioritization of the host proteins associated with the vRNAs, we identified the site of Protein:vRNA interaction via a CLIP-seq approach and assessed the consequences of the RNA:protein binding event of hnRNP K, hnRNP I, and hnRNP M in regards to viral infection. Herein we demonstrate that mutation of the prioritized hnRNP:vRNA interaction sites effectively disrupted the hnRNP:vRNA interaction. Correlating with disrupted hnRNP:vRNA binding, SINV growth kinetics were reduced relative to wild type parental viral infections in a vertebrate and invertebrate tissue culture models of infection. The molecular mechanism leading to reduced viral growth kinetics were found to be dysregulated structural gene expression. Collectively, this study further defines the scope and importance of the alphavirus host/pathogen vRNA:protein interactions. Members of the genus Alphavirus are widely recognized for their potential to cause severe disease. Despite this recognition, there are no antiviral therapeutics, or safe and effective vaccines, currently available to treat alphaviral infection. Alphaviruses utilize the host cell machinery to efficiently establish and complete their viral lifecycle. However, the extent, and importance, of host/pathogen RNA:protein interactions is woefully under characterized. The efforts detailed in this study fulfill this critical gap; and the significance of this research is three-fold. First, the data presented here fundamentally expands the scope and understanding of alphavirus host/pathogen interactions. Secondly, this study identifies the site of interactions for several prioritized interactions and defines the contribution of the RNA:protein interaction at the molecular level. Finally, these studies build a strategy by which the importance of given host/pathogen interactions may be assessed, in the future, using a mouse model of infection.

Alphaviruses 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 treatment of alphaviral disease. In particular, this report details a previously uncharacterized aspect of the alphaviral life cycle: the importance of noncapped genomic viral RNAs for alphaviral infection. This offers new insights into the mechanisms of alphaviral replication and the impact of the noncapped genomic RNAs on viral packaging.

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.

25Alphaviruses are arthropod-borne RNA viruses that are capable of causing severe 26 disease and are a significant burden to public health. Alphaviral replication results in the 27 production of both capped and noncapped viral genomic RNAs, which are packaged 28 into virions during the infections of vertebrate and invertebrate cells. However, the roles 29 that the noncapped genomic RNAs (ncgRNAs) play during alphaviral infection have yet 30 to be exhaustively characterized. Here, the importance of the ncgRNAs to alphaviral 31 infection was assessed by using mutants of the nsP1 protein of Sindbis virus (SINV), 32 which altered the synthesis of the ncgRNAs during infection by modulating the protein's 33 capping efficiency. Specifically, point mutants at residues Y286A and N376A decreased 34 capping efficiency, while a point mutant at D355A increased the capping efficiency of 35 the SINV genomic RNA during genuine viral infection. Viral growth kinetics were 36 significantly reduced for the D355A mutant relative to wild type infection, whereas the 37 Y286A and N376A mutants showed modest decreases in growth kinetics. Overall 38 genomic translation and nonstructural protein accumulation was found to correlate with 39 increases and decreases in capping efficiency. However, genomic, minus strand, and 40 subgenomic viral RNA synthesis was largely unaffected by the modulation of alphaviral 41 capping activity. In addition, translation of the subgenomic vRNA was found to be 42 unimpacted by changes in capping efficiency. The mechanism by which decreased 43 presence of ncgRNAs reduced viral growth kinetics was through the impaired 44 production of viral particles. Collectively, these data illustrate the importance of 45 ncgRNAs to viral infection and suggests that they play in integral role in the production 46 of viral progeny. 47 94 5' termini of the alphaviral genomic and subgenomic vRNAs, which provided the first 95 evidence for independent promoter initiation for the two positive-sense vRNA species 96 (9). While these studies were able to identify the presence of the 5' type-0 cap structure, 97 they were, by the nature of their design and technological limitations, unable to 98 determine the relative frequency with which the positive sense vRNAs were capped. 99Recently, we reported findings that indicated that the alphaviral genomic vRNAs 100 are not ubiquitously capped, and that a significant proportion of the genomic vRNAs 101 produced during SINV and RRV infection lack the 5' type-0 cap structure (18). 102 Furthermore, analyses of infectious and noninfectious viral particles demonstrated that 103 both the capped and noncapped genomic vRNAs are packaged into viral particles 104 throughout the course of infection. Through the use of tissue culture models of 105 alphaviral infection, the presence of the noncapped vRNAs were found to correlate with 106 the activation of a type-I IFN response. Moreover, an attenuated RRV mutant was found 107to produce fewer noncapped genomic RNAs relative to wild type virulent RRV (18...

Alphaviruses are positive-sense RNA arboviruses that are capable of causing severe disease in otherwise healthy individuals. There are many aspects of viral infection that determine pathogenesis and major efforts regarding the identification and characterization of virulence determinants have largely focused on the roles of the nonstructural and structural proteins. Nonetheless, the viral RNAs of the alphaviruses themselves play important roles in regard to virulence and pathogenesis. In particular, many sequences and secondary structures within the viral RNAs play an important part in the development of disease and may be considered important determinants of virulence. In this review article, we summarize the known RNA-based virulence traits and host:RNA interactions that influence alphaviral pathogenesis for each of the viral RNA species produced during infection. Overall, the viral RNAs produced during infection are important contributors to alphaviral pathogenesis and more research is needed to fully understand how each RNA species impacts the host response to infection as well as the development of disease.