title: Long-term suppression of HIV-1C virus production in human peripheral blood mononuclear cells by LTR heterochromatization with a short double-stranded RNA.
abstract: OBJECTIVE
  A region in the conserved 5' long terminal repeat (LTR) promoter of the integrated HIV-1C provirus was identified for effective targeting by a short double-stranded RNA (dsRNA) to cause heterochromatization leading to a long-lasting decrease in viral transcription, replication and subsequent productive infection in human host cells.
  
  
  METHODS
  Small interfering RNAs (siRNAs) were transfected into siHa cells containing integrated LTR-luciferase reporter constructs and screened for efficiency of inducing transcriptional gene silencing (TGS). TGS was assessed by a dual luciferase assay and real-time PCR. Chromatin modification at the targeted region was also studied. The efficacy of potent siRNA was then checked for effectiveness in TZM-bl cells and human peripheral blood mononuclear cells (PBMCs) infected with HIV-1C virus. Viral Gag-p24 antigen levels were determined by ELISA.
  
  
  RESULTS
  One HIV-1C LTR-specific siRNA significantly decreased luciferase activity and its mRNA expression with no such effect on HIV-1B LTR. This siRNA-mediated TGS was induced by histone methylation, which leads to heterochromatization of the targeted LTR region. The same siRNA also substantially suppressed viral replication in TZM-bl cells and human PBMCs infected with various HIV-1C clinical isolates for ≥3 weeks after a single transfection, even of a strain that had a mismatch in the target region.
  
  
  CONCLUSIONS
  We have identified a potent dsRNA that causes long-term suppression of HIV-1C virus production in vitro and ex vivo by heritable epigenetic modification at the targeted C-LTR region. This dsRNA has promising therapeutic potential in HIV-1C infection, the clade responsible for more than half of AIDS cases worldwide.
authors.affiliation: Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, 110029, India.
authors.forename: Anand
authors.initials: A
authors.lastname: Singh
authors.forename: Jayanth K
authors.initials: JK
authors.lastname: Palanichamy
authors.forename: Pradeep
authors.initials: P
authors.lastname: Ramalingam
authors.forename: Muzaffer A
authors.initials: MA
authors.lastname: Kassab
authors.forename: Mohita
authors.initials: M
authors.lastname: Bhagat
authors.forename: Raiees
authors.initials: R
authors.lastname: Andrabi
authors.forename: Kalpana
authors.initials: K
authors.lastname: Luthra
authors.forename: Subrata
authors.initials: S
authors.lastname: Sinha
authors.forename: Parthaprasad
authors.initials: P
authors.lastname: Chattopadhyay
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D020868:Gene Silencing / Q000502:physiology; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000502:physiology* / Q000821:virology*; D011533:Proviruses / Q000235:genetics* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics*; D014775:Virus Activation / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
chemical_list: D012330:RNA, Double-Stranded
keywords: HIV-1; RNAi; epigenetics; small interfering RNA; transcriptional gene silencing