Highresolution profiling and analysis of viral and host small RNAs during human cytomegalovirus infection

Human cytomegalovirus (HCMV) contributes its own set of microRNAs (miRNAs) during lytic infection of cells, likely fine-tuning conditions important for viral replication. To enhance our understanding of this component of the HCMV-host tran-scriptome, we have conducted deep-sequencing analysis of small RNAs (smRNA-seq) from infected human fibroblast cells. We found that HCMV-encodedmiRNAs accumulate to20 % of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of two novel HCMVmiRNAs, miR-US22 andmiR-US33as. Both of these miRNAs were capable of functionally repressing synthetic targets in transient transfection experiments. Additionally, through cross-linking and immunoprecipitation (CLIP) of Argonaute (Ago)-bound RNAs from infected cells, fol-lowed by high-throughput sequencing, we have obtained direct evidence for incorporation of all HCMVmiRNAs into the endoge-nous host silencing machinery. Surprisingly, three HCMVmiRNA precursors exhibited differential incorporation of their ma-ture miRNA arms between Ago2 and Ago1 complexes. Host miRNA abundances were also affected by HCMV infection, with significant upregulation observed for anmiRNA cluster containing miR-96, miR-182, andmiR-183. In addition to miRNAs, we also identified novel forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. Human cytomegalovirus (HCMV), a member of the herpesvi-rus family, is prevalent in the majority of the global popula-tion and the main viral cause of birth defects (19). HCMV is ca