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Journal of Virology, March 2008, p. 2895-2903, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.02035-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Human Immunodeficiency Virus Type 1 Escape Is Restricted When Conserved Genome Sequences Are Targeted by RNA Interference

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, K3-110, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

Received 14 September 2007/ Accepted 5 December 2007

RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but—in contrast to what is observed with nef—no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.

Published ahead of print on 12 December 2007.