Downloads and Tools

•  Article PDF
  3.64 MB PDF document

Supplementary Material

•  Supplementary File
  3.92 MB PDF document

This is an open access article.
Print this page Email this page

Recipient's Email:

Download Citation

---

Share on Facebook

Authors: Kazuo Suzuki, Takaomi Ishida, Makoto Yamagishi, Chantelle Ahlenstiel, Sanjay Swaminathan, Katharine Marks, Daniel Murray, Erin M McCartney, Michael R Beard, Marina Alexander, Damian F.J. Purcell, David A Cooper, Toshiki Watanabe and Anthony D Kelleher

Kazuo Suzuki

Corresponding author: k.suzuki@amr.org.au
Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Takaomi Ishida

Research Center for Asian Infectious Disease, Institute of Medical Science, University of Tokyo; Tokyo, Japan

Makoto Yamagishi

Laboratory of Tumour Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo; Tokyo, Japan

Chantelle Ahlenstiel

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Sanjay Swaminathan

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Katharine Marks

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Daniel Murray

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Erin M McCartney

School of Molecular and Biomedical Science, The University of Adelaide & SA Pathology; Adelaide, Australia

Michael R Beard

School of Molecular and Biomedical Science, The University of Adelaide & SA Pathology; Adelaide, Australia

Marina Alexander

Department of Microbiology and Immunology, University of Melbourne; Parkville, VIC Australia

Damian F.J. Purcell

Department of Microbiology and Immunology, University of Melbourne; Parkville, VIC Australia

David A Cooper

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Toshiki Watanabe

Research Center for Asian Infectious Disease, Institute of Medical Science, University of Tokyo; Tokyo, Japan

Anthony D Kelleher

Immunovirology Laboratory, St Vincent's Centre for Applied Medical Research; Darlinghurst, NSW Australia

Preview

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.