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Journal of Virology, August 2006, p. 7658-7666, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.00078-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Effective Suppression of Human Immunodeficiency Virus Type 1 through a Combination of Short- or Long-Hairpin RNAs Targeting Essential Sequences for Retroviral Integration

Hironori Nishitsuji,1 Michinori Kohara,2 Mari Kannagi,1 and Takao Masuda1*

Department of Immunotherapeutics, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan,1 Department of Microbiology and Cell Physiology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan2

Received 11 January 2006/ Accepted 16 May 2006

Small interfering RNA (siRNA) could provide a new therapeutic approach to treating human immunodeficiency virus type 1 (HIV-1) infection. For long-term suppression of HIV-1, emergence of siRNA escape variants must be controlled. Here, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNA) corresponding to conserved target sequences within the integrase (int) and the attachment site (att) genes, both of which are essential for HIV-1 integration. Compared to shRNA targeting of the HIV-1 transcription factor tat (shTat), shRNA against int (shIN) or the U3 region of att (shU3) showed a more potent inhibitory effect on HIV-1 replication in human CD4+ T cells. Infection with a high dose of HIV-1 resulted in the emergence of escape mutants during long-term culture. Of note, limited genetic variation was observed in the viruses resistant to shIN. A combination of shINs against wild-type and escape mutant sequences had a negative effect on their antiviral activities, indicating a potentially detrimental effect when administering multiple shRNA targeting the same region to combat HIV-1 variants. The combination of shIN and shU3 att exhibited the strongest anti-HIV-1 activity, as seen by complete abrogation of viral DNA synthesis and viral integration. In addition, a modified long-hairpin RNA spanning the 50 nucleotides in the shIN target region effectively suppressed wild-type and shIN-resistant mutant HIV-1. These results suggest that targeting of incoming viral RNA before proviral DNA formation occurs through the use of nonoverlapping multiple siRNAs is a potent approach to achieving sustained, efficient suppression of highly mutable viruses, such as HIV-1.

* Corresponding author. Mailing address: Department of Immunotherapeutics, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81-3-5803-5799. Fax: 81-3-5803-0235. E-mail: tmasu.impt{at}tmd.ac.jp.

Journal of Virology, August 2006, p. 7658-7666, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.00078-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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