title: Latent HIV-1 reactivation in transgenic mice requires cell cycle -dependent demethylation of CREB/ATF sites in the LTR.
abstract: OBJECTIVE
  We previously produced a line of transgenic mice that carried the HIV-1 genome deficient in the gene. Although the HIV-1 genome in the lymphocytes was dormant under normal physiological conditions, it could be reactivated by lipopolysaccharide (LPS) administration via induction of interleukin-1alpha/beta and tumour necrosis factor-alpha. In this report, we analysed further the reactivation mechanism of the latent HIV-1 using this transgenic mouse model.
  
  
  METHODS
  and methods: Possible involvement of CpG methylation in HIV-1 latency was examined by treating transgenic lymphocytes with a demethylating agent, 5'-azacytidine. CpG methylation in the HIV-1 long terminal repeat (LTR) was analysed using the bisulfite genomic sequencing method. As previous studies suggested that CpG demethylation depended on the cell cycle progression, we analysed the relation between cell cycle progression and LPS-induced reactivation of HIV-1 by labelling lymphocytes with an intracellular fluorescein, carboxyfluorescein diacetate succinimidyl ester.
  
  
  RESULTS
  We found that 5'-azacytidine enhanced HIV-1 expression ninefold compared to treatment with LPS alone. Furthermore, HIV-1 p24 induction by LPS was observed only in cells that had undergone cell division, while induction was prevented in cells in which cell cycle progression was blocked either by mimosine, aphidicolin, or nocodazole. LPS-induced HIV-1 reactivation was associated with demethylation of two CpG sites located in the CREB/ATF binding sites in the HIV-1 LTR in a cell cycle-dependent manner.
  
  
  CONCLUSIONS
  These observations indicate that cell cycle progression-dependent demethylation of the CREB/ATF sites in the LTR is crucial for the reactivation of latent HIV-1 genome in transgenic mice.
authors.affiliation: Center for Experimental Medicine and the Department of Cancer Research, Institute of Medical Science, University of Tokyo, Japan.
authors.forename: J
authors.initials: J
authors.lastname: Tanaka
authors.forename: T
authors.initials: T
authors.lastname: Ishida
authors.forename: B-I
authors.initials: BI
authors.lastname: Choi
authors.forename: J
authors.initials: J
authors.lastname: Yasuda
authors.forename: T
authors.initials: T
authors.lastname: Watanabe
authors.forename: Y
authors.initials: Y
authors.lastname: Iwakura
mesh_terms: D051696:Activating Transcription Factors; D000818:Animals; D001374:Azacitidine / Q000494:pharmacology; D001798:Blood Proteins / Q000235:genetics*; D002453:Cell Cycle; D018899:CpG Islands / Q000235:genetics; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D019175:DNA Methylation; D004279:DNA, Viral / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D015658:HIV Infections / Q000821:virology*; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D051379:Mice; D008809:Mice, Inbred C3H; D008822:Mice, Transgenic; D014157:Transcription Factors / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics; D017735:Virus Latency / Q000235:genetics
chemical_list: D051696:Activating Transcription Factors; D001798:Blood Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D004279:DNA, Viral; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D014157:Transcription Factors; D014764:Viral Proteins; D001374:Azacitidine