title: Silencing of HIV-1 subtype C primary isolates by expressed small hairpin RNAs targeted to gag.
abstract: Discovery of sequence-specific silencing by activating the RNA interference (RNAi) pathway has led to exciting new strategies for treating infection with human immunodeficiency virus type 1 (HIV-1). Of the HIV-1 subtypes, C is especially common in areas of the world that are worst affected. Although prone to mutation, genome plasticity of this subtype is limited in functionally important regions. We identified conserved sequences within the HIV-1 subtype C gag open reading frame and assessed whether they are suitable targets for inhibition of viral replication by RNA Pol III-driven small hairpin RNAs (shRNAs). Initially, the efficacy of each of a panel of 10 shRNAs against HIV-1 was determined using a reporter assay. shRNAs A and B, which targeted the 5 end of gag, were most effective and were used to assess inhibition of replication in cultured cells of two R5 isolates (Du151 and Du422) and one X4 virus (SW7). These shRNAs diminished intracellular HIV-1 gag RNA and HIV-1 protein concentrations as well as p24 secretion by up to 80% without inducing an interferon response. However, shRNA-mediated knockdown efficacy against each of these viral isolates varied slightly. These data show successful activation of RNAi to inhibit the replication of biologically distinct HIV-1 subtype C isolates. The effector shRNAs described here are potential candidates for gene therapy applications against the most common global subtype of HIV-1.
authors.affiliation: HBV Research Unit, Department of Molecular Medicine and Haematology, University of the Witwatersrand, Wits 2050, Johannesburg, South Africa.
authors.forename: Eleanor
authors.initials: E
authors.lastname: Cave
authors.forename: Marc S
authors.initials: MS
authors.lastname: Weinberg
authors.forename: Tonie
authors.initials: T
authors.lastname: Cilliers
authors.forename: Sergio
authors.initials: S
authors.lastname: Carmona
authors.forename: Lynn
authors.initials: L
authors.lastname: Morris
authors.forename: Patrick
authors.initials: P
authors.lastname: Arbuthnot
mesh_terms: D002460:Cell Line; D020868:Gene Silencing; D018390:Gene Targeting; D017930:Genes, Reporter; D015750:Genes, gag; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015497:HIV-1 / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D034741:RNA, Small Interfering; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins