title: Mutational inactivation of herpes simplex virus 1 microRNAs identifies viral mRNA targets and reveals phenotypic effects in culture.
abstract: Herpes simplex virus 1 (HSV-1), a ubiquitous human pathogen, expresses several viral microRNAs (miRNAs). These, along with the latency-associated transcript, represent the only viral RNAs detectable in latently infected neuronal cells. Here, for the first time, we analyze which HSV-1 miRNAs are loaded into the RNA-induced silencing complex (RISC), the key effector of miRNA function. Only 9 of the 17 reported HSV-1 miRNAs, i.e., miR-H1 to miR-H8 plus miR-H11, were found to actually load into the RISC. Surprisingly, this analysis also revealed that HSV-1 miRNAs loaded into the RISC with efficiencies that differed widely; <1% of the miR-H1-3p miRNA detectable in HSV-1-infected cells was loaded into the RISC. Analysis of HSV-1 mutants individually lacking the viral miR-H2, miR-H3, or miR-H4 miRNA revealed that loss of these miRNAs affected the rate of replication of HSV-1 in neuronal cells but not in fibroblasts. Analysis of mRNA and protein expression, as well as assays mapping viral miRNA binding sites in infected cells, showed that endogenous HSV-1 miR-H2 binds to viral ICP0 mRNA and inhibits its expression, while endogenous miR-H4 inhibits the expression of the viral ICP34.5 gene. In contrast, no viral mRNA target for miR-H3 could be detected, even though miR-H3, like miR-H4, is perfectly complementary to ICP34.5 mRNA. Together, these data demonstrate that endogenous HSV-1 miRNA expression can significantly alter viral replication in culture, and they also identify two viral mRNA targets for miR-H2 and miR-H4 that can partially explain this phenotype.
authors.affiliation: Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina, USA.
authors.forename: Omar
authors.initials: O
authors.lastname: Flores
authors.forename: Sanae
authors.initials: S
authors.lastname: Nakayama
authors.forename: Adam W
authors.initials: AW
authors.lastname: Whisnant
authors.forename: Hassan
authors.initials: H
authors.lastname: Javanbakht
authors.forename: Bryan R
authors.initials: BR
authors.lastname: Cullen
authors.forename: David C
authors.initials: DC
authors.lastname: Bloom
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D005347:Fibroblasts / Q000821:virology; D015967:Gene Expression Regulation, Viral; D057809:HEK293 Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009154:Mutation; D041681:NIH 3T3 Cells; D009474:Neurons / Q000821:virology; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex; D011817:Rabbits; D044767:Ubiquitin-Protein Ligases / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism; D014779:Virus Replication / Q000235:genetics* / Q000502:physiology
chemical_list: D017874:Immediate-Early Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; D012367:RNA, Viral; D034743:RNA-Induced Silencing Complex; D014764:Viral Proteins; C066469:gamma 34.5 protein, Human herpesvirus 1; D044767:Ubiquitin-Protein Ligases; C051804:Vmw110 protein, Human herpesvirus 1
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