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Blood, 1 August 2005, Vol. 106, No. 3, pp. 818-826.
Prepublished online as a Blood First Edition Paper on April 14, 2005; DOI 10.1182/blood-2004-10-3959.
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Submitted October 15, 2004
Accepted April 3, 2005
Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV
Sang-Kyung Lee, Derek M Dykxhoorn, Priti Kumar, Shahin Ranjbar, Erwei Song, Laura E Maliszewski, Vanessa Francois-Bongarcon, Anne Goldfeld, Manjunath N Swamy, Judy Lieberman, and Premlata Shankar*
The CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA, USA; Department of Pediatrics, Harvard Medical School, Boston, MA, USA
The CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA, USA
* Corresponding author; email: shankar{at}cbr.med.harvard.edu.
Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested three viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates and a vif sequence highly conserved across clades. Lentiviral expression of all three shRNAs inhibited replication of the homologous HIVIIIB strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2/5 clade B isolates. The gag sequence (conserved within clade B) protected 5/5 clade B isolates, but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although one or two mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.
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