1. Lentiviral delivery of short hairpin RNAs protects CD4 T cells from multiple clades and primary isolates of HIV
- Author
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Sang-Kyung Lee, Anne E. Goldfeld, Vanessa François-Bongarçon, Judy Lieberman, Shahin Ranjbar, N. Manjunath Swamy, Laura E. Maliszewski, Erwei Song, Premlata Shankar, Derek M. Dykxhoorn, and Priti Kumar
- Subjects
CD4-Positive T-Lymphocytes ,Small interfering RNA ,viruses ,Immunology ,HIV Infections ,Transfection ,Antiviral Agents ,Biochemistry ,Virus ,Cell Line ,Conserved sequence ,Small hairpin RNA ,RNA interference ,Humans ,Gene silencing ,RNA, Small Interfering ,Clade ,Conserved Sequence ,Genes, vif ,Genetics ,biology ,Lentivirus ,Gene Therapy ,Cell Biology ,Hematology ,biology.organism_classification ,Genes, gag ,Virology ,Biological Therapy ,Genes, rev ,Mutation ,HIV-1 ,RNA Interference - Abstract
Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 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 3 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 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 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 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.
- Published
- 2005