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Journal of Virology, March 2008, p. 2938-2951, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.02126-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Targets of Small Interfering RNA Restriction during Human Immunodeficiency Virus Type 1 Replication{triangledown}

Yong Gao,1 Michael A. Lobritz,1,2 Justin Roth,3 Measho Abreha,1 Kenneth N. Nelson,1 Immaculate Nankya,1,2 Dawn M. Moore-Dudley,1,2 Awet Abraha,1 Stanton L. Gerson,3 and Eric J. Arts1,2*

Division of Infectious Diseases, Department of Medicine,1 Department of Molecular Biology and Microbiology,2 Case Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Ave., Cleveland, Ohio 441063

Received 26 September 2007/ Accepted 4 January 2008

Small interfering RNAs (siRNAs) have been shown to effectively inhibit human immunodeficiency virus type 1 (HIV-1) replication in vitro. The mechanism(s) for this inhibition is poorly understood, as siRNAs may interact with multiple HIV-1 RNA species during different steps of the retroviral life cycle. To define susceptible HIV-1 RNA species, siRNAs were first designed to specifically inhibit two divergent primary HIV-1 isolates via env and gag gene targets. A self-inactivating lentiviral vector harboring these target sequences confirmed that siRNA cannot degrade incoming genomic RNA. Disruption of the incoming core structure by rhesus macaque TRIM5{alpha} did, however, provide siRNA-RNA-induced silencing complex access to HIV-1 genomic RNA and promoted degradation. In the absence of accelerated core disruption, only newly transcribed HIV-1 mRNA in the cytoplasm is sensitive to siRNA degradation. Inhibitors of HIV-1 mRNA nuclear export, such as leptomycin B and camptothecin, blocked siRNA restriction. All HIV-1 RNA regions and transcripts found 5' of the target sequence, including multiply spliced HIV-1 RNA, were degraded by unidirectional 3'-to-5' siRNA amplification and spreading. In contrast, HIV-1 RNA 3' of the target sequence was not susceptible to siRNA. Even in the presence of siRNA, full-length HIV-1 RNA is still encapsidated into newly assembled viruses. These findings suggest that siRNA can target only a relatively "naked" cytoplasmic HIV-1 RNA despite the involvement of viral RNA at nearly every step in the retroviral life cycle. Protection of HIV-1 RNA within the core following virus entry, during encapsidation/virus assembly, or within the nucleus may reflect virus evolution in response to siRNA, TRIM5{alpha}, or other host restriction factors.

* Corresponding author. Mailing address: Division of Infectious Diseases, BRB 1034, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106. Phone: (216) 368-8904. Fax: (216) 368-2034. E-mail: eja3{at}case.edu

{triangledown} Published ahead of print on 16 January 2008.

Journal of Virology, March 2008, p. 2938-2951, Vol. 82, No. 6
0022-538X/08/$08.00+0     doi:10.1128/JVI.02126-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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