title: HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period.
abstract: BACKGROUND
  Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection.
  
  
  RESULTS
  Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4-3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4-3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3' LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication.
  
  
  CONCLUSIONS
  The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4-3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle.
authors.affiliation: Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 1088639, Japan.
authors.forename: Mie
authors.initials: M
authors.lastname: Kobayashi-Ishihara
authors.forename: Makoto
authors.initials: M
authors.lastname: Yamagishi
authors.forename: Takuma
authors.initials: T
authors.lastname: Hara
authors.forename: Yuka
authors.initials: Y
authors.lastname: Matsuda
authors.forename: Ryutaro
authors.initials: R
authors.lastname: Takahashi
authors.forename: Ariko
authors.initials: A
authors.lastname: Miyake
authors.forename: Kazumi
authors.initials: K
authors.lastname: Nakano
authors.forename: Tadanori
authors.initials: T
authors.lastname: Yamochi
authors.forename: Takaomi
authors.initials: T
authors.lastname: Ishida
authors.forename: Toshiki
authors.initials: T
authors.lastname: Watanabe
mesh_terms: D002467:Cell Nucleus / Q000821:virology; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D057809:HEK293 Cells; D015658:HIV Infections / Q000821:virology; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D009154:Mutation; D016328:NF-kappa B / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011533:Proviruses / Q000235:genetics; D034622:RNA Interference; D016372:RNA, Antisense / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics*; D048348:Reverse Transcription; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication
chemical_list: D016328:NF-kappa B; D016372:RNA, Antisense; D012333:RNA, Messenger; D012367:RNA, Viral