Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration.
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.
Nishitsuji
Hironori
H
Department of Immunotherapeutics, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.
Kohara
Michinori
M
Kannagi
Mari
M
Masuda
Takao
T
D002460:Cell Line; D015696:Gene Products, tat / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D015658:HIV Infections / Q000628:therapy; D019427:HIV Integrase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics; D014779:Virus Replication / Q000502:physiology*; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D034741:RNA, Small Interfering; D012367:RNA, Viral; D054322:tat Gene Products, Human Immunodeficiency Virus; D019427:HIV Integrase