Study of a tumor virus unveils a novel function for the miRNA biogenesis machinery

Abstract

Kaposi’s Sarcoma-associated Herpes Virus (KSHV) is a human herpesvirus associated with cancers. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. This work presented here is a novel approach to profile small RNAs from populations of cells undergoing predominantly lytic infection. Using two different next generation sequencing platforms, I cloned and sequenced both pre-microRNAs and derivative microRNAs (miRNAs). This analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5 prime end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. I report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miRs-K8 and K12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. I identify several novel small RNAs of low abundance, including viral microRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. This work also shows that much of the KSHV genome is transcribed in both the top and bottom strand orientations during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, I observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs). From the small RNA deep-sequencing, I also detected a low abundant small RNA fragment (23 nt) that maps to a putative hairpin structure (named hairpin K) within the KSHV PAN transcript. I demonstrate that hairpin K is a cis-negative regulatory element in PAN. It is well-appreciated that viruses utilize host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often utilize host-encoded factors to optimize replication. This work shows that Drosha, an endoribonuclease best known for its role in the biogenesis of miRNAs, can also function to directly regulate viral gene expression. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. I demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses.