Gammaherpesvirus Small Noncoding RNAs Are Bifunctional Elements That Regulate Infection and Contribute to Virulence In Vivo

Abstract

Many viruses express noncoding RNAs (ncRNAs). The gammaherpesviruses (γHVs), including Epstein-Barr virus, Kaposi’s sarcoma-associated herpesvirus, and murine γHV68, each contain multiple ncRNA genes, including microRNAs (miRNAs). While these ncRNAs can regulate multiple host and viral processes in vitro, the genetic contribution of these RNAs to infection and pathogenesis remains largely unknown. To study the functional contribution of these RNAs to γHV infection, we have used γHV68, a small-animal model of γHV pathogenesis. γHV68 encodes eight small hybrid ncRNAs that contain both tRNA-like elements and functional miRNAs. These genes are transcribed by RNA polymerase III and are referred to as the γHV68 TMERs (tRNA-miRNA-encoded RNAs). To determine the total concerted genetic contribution of these ncRNAs to γHV acute infection and pathogenesis, we generated and characterized a recombinant γHV68 strain devoid of all eight TMERs. TMER-deficient γHV68 has wild-type levels of lytic replication in vitro and normal establishment of latency in B cells early following acute infection in vivo. In contrast, during acute infection of immunodeficient mice, TMER-deficient γHV68 has reduced virulence in a model of viral pneumonia, despite having an enhanced frequency of virus-infected cells. Strikingly, expression of a single viral tRNA-like molecule, in the absence of all other virus-encoded TMERs and miRNAs, reverses both attenuation in virulence and enhanced frequency of infected cells. These data show that γHV ncRNAs play critical roles in acute infection and virulence in immunocompromised hosts and identify these RNAs as a new potential target to modulate γHV-induced infection and pathogenesis.

The small noncoding RNAs (sncRNAs) of murine gammaherpesvirus 68 (MHV-68) are involved in regulating the latent-to-lytic switch in vivo

“…A practical consequence is that it might be inherently difficult to separate potential functions of the vtRNAs and miRNAs from each other. A first attempt in this direction was recently undertaken by Diebel et al 46,. suggesting an independent functional role of the vtRNAs in acute infection and pathogenesis.…”

MicroRNAs in large herpesvirus DNA genomes: recent advances

“…A recent study performed in pigs revealed that deletion of nine of the 11 miRNAs encoded in the large latency transcript of pseudorabies virus (PRV, suid herpesvirus 1) had little effect on latency establishment in the trigeminal ganglia (95). Recent studies have investigated the potential function of MuHV-4-encoded miRNAs in mice (96)(97)(98). Using MuHV-4 recombinant strains specifically deficient for the expression of viral miRNAs, the efficiency of B cell infection in vivo was only slightly affected.…”

“…These results suggest that ncRNAs, not only miRNAs, play major roles during infection in vivo and highlight the major importance of using genetically engineered recombinant viruses in animal models to address their roles in specific settings. Indeed, while the absence of MuHV-4 miRNAs did not significantly affect the colonization and latency in immunocompetent mice, impairment of MuHV-4 miRNA expression reduced the virulence of MuHV-4 infection in immunocompromised IFN-γ-deficient mice, suggesting a role in promoting gammaherpesvirus-associated disease (96,97). In addition, research conducted on malignant catarrhal fever (MCF), a T-cell lymphoproliferative cattle disease induced by OvHV-2 or alcelaphine herpesvirus 1 (AlHV-1) revealed the expression of a very high number of miRNAs after propagation of LCLs and in vivo (73,74).…”

Small RNA deep sequencing identifies viral microRNAs during malignant catarrhal fever induced by alcelaphine herpesvirus 1

“…Interestingly, a small cluster of EBV-encoded miRNAs has been shown to promote its transforming properties (Feederle et al, 2011). Although very little is known on the actual functions of c-HV-encoded miRNAs in vivo, two recent studies suggested that murid herpesvirus 4 (MuHV-4)-encoded miRNAs are not essential for viral replication or latency establishment in immunocompetent mice but potentiate the acute pathology induced by MuHV-4 infection in IFN-c-deficient mice (Diebel et al, 2015;Feldman et al, 2014).…”