Small RNA deep-sequencing identifies microRNAs and other small non-coding RNAs from human herpesvirus 6 (HHV-6B)

Roseolovirus, or human herpesvirus 6 (HHV-6) is a ubiquitous human pathogen infecting over 95% of the population by the age of two years. As with other herpesviruses, reactivation of HHV-6 can present with severe complications in immunocompromised individuals. Recent studies have highlighted the importance of herpesvirus-derived micro (mi)RNAs in modulating both cellular and viral gene expression. An initial report, which computed the likelihood of various viruses to encode for miRNAs, did not predict HHV-6 miRNAs. To experimentally screen for small HHV-6 encoded RNAs, we conducted large-scale sequencing of Sup-T-1 cells lytically infected with a laboratory strain of HHV-6B. This revealed an abundant 60-65 nucleotide RNA of unknown function derived from the lytic origin of replication (OriLyt) that gave rise to smaller RNA species of 18-19 nucleotides in length. In addition, we identified four pre-miRNAs, whose mature forms accumulated in Argonaute 2. In contrast to other beta-herpesviruses, HHV-6B miRNAs are expressed from direct repeat regions (DRL and DRR) located at either side of the genome. All miRNAs are conserved in the closely related HHV-6A variant, and one of them is a seed ortholog of the human miR-582-5p. Similar to alpha-herpesvirus miRNAs, they are expressed antisense to immediate early ORFs and thus have the potential to regulate key viral regulators. Overall design: Small RNA sequencing from total RNA or Ago2 associated small RNAs extracted from HHV-6 infected Sup-T-1 cells