title: Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAs.
abstract: BACKGROUND
  RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences.
  
  
  METHODS
  In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay.
  
  
  RESULTS
  Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study.
  
  
  CONCLUSIONS
  These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs.
authors.affiliation: Laboratory of Experimental Virology, Department of Medical Microbiology and Centre for Infection and Immunity Amsterdam (CINIMA), Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Karin J
authors.initials: KJ
authors.lastname: von Eije
authors.forename: Olivier
authors.initials: O
authors.lastname: ter Brake
authors.forename: Ben
authors.initials: B
authors.lastname: Berkhout
mesh_terms: D002460:Cell Line; D002478:Cells, Cultured; D017930:Genes, Reporter; D015658:HIV Infections / Q000235:genetics / Q000821:virology; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry*; D012367:RNA, Viral / Q000737:chemistry; D014162:Transfection; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering; D012367:RNA, Viral