Two murine cytomegalovirus microRNAs target the major viral immediate early 3 gene.

Human cytomegalovirus is responsible for morbidity and mortality in immune compromised patients and is the leading viral cause of congenital infection. Virus-encoded microRNAs (miRNAs) represent interesting targets for novel antiviral agents. While many cellular targets that augment productive infection have been identified in recent years, regulation of viral genes such as the major viral immediate early protein 72 (IE72) by hcmv-miR-UL112-1 may contribute to both the establishment and the maintenance of latent infection. We employed photoactivated ribonucleotide-enhanced individual nucleotide resolution crosslinking (PAR-iCLIP) to identify murine cytomegalovirus (MCMV) miRNA targets during lytic infection. While the PAR-iCLIP data were of insufficient quality to obtain a comprehensive list of cellular and viral miRNA targets, the most prominent PAR-iCLIP peak in the MCMV genome mapped to the 3' untranslated region of the major viral immediate early 3 (ie3) transcript. We show that this results from two closely positioned binding sites for the abundant MCMV miRNAs miR-M23-2-3p and miR-m01-2-3p. Their pre-expression significantly impaired viral plaque formation. However, mutation of the respective binding sites did not alter viral fitness during acute or subacute infection in vivo. Furthermore, no differences in the induction of virus-specific CD8+ T cells were observed. Future studies will probably need to go beyond studying immunocompetent laboratory mice housed in pathogen-free conditions to reveal the functional relevance of viral miRNA-mediated regulation of key viral immediate early genes.

Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic-latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30-p53-DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.