title: Transcriptional gene silencing of HIV-1 through promoter targeted RNA is highly specific.
abstract: We have previously reported induction of transcriptional gene silencing (TGS) of HIV-1 by short hairpin RNA (shRNA) expressed in MOLT-4 cells. The shRNA (termed shPromA) targets the highly conserved tandem NF-kB binding sequences of the HIV-1 promoter. Recent articles have reported that TGS mediated by promoter-targeted siRNAs was exclusively the result of sequence non-specific off-target effects. Specifically, several mismatched siRNAs to the target promoter sequences were reported to also induce significant TGS, suggesting TGS was a consequence of off-target effects. Here, following extensive investigation, we report that shPromA induces sequence specific transcriptional silencing in HIV-1 infection in MOLT4 cells, while four shRNA variants, mismatched by 2-3 nucleotides, fail to suppress viral replication. We confirm similar levels of shRNA expression from the U6 promoter and the presence of processed/cleaved siRNAs for each construct in transduced MOLT-4 cells. HIV-1 sequence specific shPromA does not suppress HIV-2, which has an alternate NF-kB binding sequence. As a result of the unique sequence targeted, shPromA does not induce down-regulation of other NF-kB driven genes, either at the mRNA or protein level. Furthermore, we confirmed shPromA does not have sequence non-specific off-target effects through unaltered expression of CD4, CXCR4, and CCR5, which are used for viral entry. Additionally, shPromA does not alter PKR, IFN levels, and three downstream mediators of IFN-a response genes. Our data clearly shows that shPromA achieved highly specific TGS of HIV-1, demonstrating that effective TGS can be induced with minimal off-target effects.
authors.affiliation: Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia. k.suzuki@amr.org.au
authors.forename: Kazuo
authors.initials: K
authors.lastname: Suzuki
authors.forename: Takaomi
authors.initials: T
authors.lastname: Ishida
authors.forename: Makoto
authors.initials: M
authors.lastname: Yamagishi
authors.forename: Chantelle
authors.initials: C
authors.lastname: Ahlenstiel
authors.forename: Sanjay
authors.initials: S
authors.lastname: Swaminathan
authors.forename: Katharine
authors.initials: K
authors.lastname: Marks
authors.forename: Daniel
authors.initials: D
authors.lastname: Murray
authors.forename: Erin M
authors.initials: EM
authors.lastname: McCartney
authors.forename: Michael R
authors.initials: MR
authors.lastname: Beard
authors.forename: Marina
authors.initials: M
authors.lastname: Alexander
authors.forename: Damian F J
authors.initials: DF
authors.lastname: Purcell
authors.forename: David A
authors.initials: DA
authors.lastname: Cooper
authors.forename: Toshiki
authors.initials: T
authors.lastname: Watanabe
authors.forename: Anthony D
authors.initials: AD
authors.lastname: Kelleher
mesh_terms: D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015704:CD4 Antigens / Q000276:immunology / Q000378:metabolism; D045744:Cell Line, Tumor; D005092:Exonucleases / Q000235:genetics; D005095:Exoribonucleases; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D020868:Gene Silencing; D015497:HIV-1 / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000235:genetics / Q000276:immunology; D006801:Humans; D007938:Leukemia; D008969:Molecular Sequence Data; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology / Q000821:virology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D019713:Receptors, CCR5 / Q000276:immunology / Q000378:metabolism; D019718:Receptors, CXCR4 / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D019892:eIF-2 Kinase / Q000378:metabolism
chemical_list: D015704:CD4 Antigens; C527276:CXCR4 protein, human; D034741:RNA, Small Interfering; D019713:Receptors, CCR5; D019718:Receptors, CXCR4; D019892:eIF-2 Kinase; D005092:Exonucleases; D005095:Exoribonucleases; C112346:ISG20 protein, human