Gammaherpesvirus ncRNAs share conserved features of binding and virulence despite lack of sequence conservation

Abstract

Gammaherpesvirus (γHV) non-coding RNAs (ncRNAs) are integral modulators of viral infection. The γHVs engage RNA polymerase III (pol III)-dependent transcription of both host and viral ncRNAs, which contribute to viral establishment, gene expression, and pathogenesis. Viral ncRNAs, such as the EBV-encoded RNAs (EBERs), reportedly interact with multiple host RNA-binding proteins (RBPs) and contribute to inflammatory responses implicated in the development of malignancies. Here, we examined RBP interactions of the pol III-transcribed tRNA-miRNA encoded non-coding RNAs (TMERs) of murine γHV68, and the potential contributions of these and the related EBERs to in vivo pathogenesis. Using sequential enzymatic treatments, we found that several TMER1 forms retain a 5’-triphosphate, lending the possibility of recognition by the innate immune sensor RIG-I. We further examined the interactions of TMERs and EBERs with host RBPs, and found that multiple TMERs and EBERs interact with the La protein, though minimal interaction was detected with RIG-I during primary virus infection. Finally, we investigated the contributions of the TMERs and EBERs to disease in an immune-compromised mouse model with a series of viral recombinants. We found that expression of multiple single TMERs, or the EBERs expressed in place of the TMERs, was capable of restoring virulence to a viral recombinant lacking expression of all TMERs. Ultimately, these studies demonstrate that divergent pol III-transcribed γHV ncRNAs share interaction characteristics with two host RBPs and conserved contributions to disease, despite little to no significant sequence conservation. These findings support a model of convergent functions of the sequence-variable pol III-transcribed γHV ncRNAs.IMPORTANCEViruses manipulate the infected cell and host inflammatory responses through expression of coding and non-coding RNAs. The gammaherpesviruses are a subfamily of herpesviruses associated with chronic inflammatory diseases and malignancies, especially in immune-compromised individuals. Among these, the human Epstein-Barr virus and murine gammaherpesvirus 68 (γHV68) express highly abundant, RNA polymerase III-dependent, short non-coding RNAs. Whether these sequence-divergent ncRNAs have conserved functional properties is unknown. By using viral recombinants to allow direct comparison of these ncRNAs during primary infection, we find that the sequence-divergent ncRNAs of γHV68 and EBV share a conserved property to bind to the host RNA binding protein, La, and function interchangeably to facilitate in vivo pathogenesis. These studies demonstrate that abundant, RNA polymerase III-dependent viral ncRNAs can potently function to alter the host cell landscape and promote disease in a sequence-independent manner.