AEGiS-15IAC: In vitro HIV-1 integrase inhibition by RNA interference (RNAi).

15th International AIDS Conference

Bangkok, Thailand - July 11-16, 2004

In vitro HIV-1 integrase inhibition by RNA interference (RNAi).

Int Conf AIDS 2004 Jul 11-16; 15:(abstract no. WeOrA1312)

Lau TS, Wan CC
Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China

BACKGROUND: HIV-1 Integrase is an enzyme that mediates the integration of HIV-1 viral DNA to host genome. RNA interference (RNAi) was demonstrated to suppress specific protein expression through the induction of sequence-specific gene silencing. One of the RNAi approach is to introduce vectors to cells that encode 21-25 nucleotides (nt) so as to form hairpin siRNAs to trigger gene silencing. In this study, we examined the potential efficacy of this RNAi method to inhibit cellular HIV-1 integrase.

METHODS: HIV-1 integrase (HIV-IN) cDNA was cloned into mammalian expression vector pEGFP-C1 (Clontech). The RNAi vector, pSilencer 1.0-U6 (Ambion), was used for hairpin siRNA synthesis. For hairpin siRNA synthesis, four regions of the 21 nt sequences were chosen corresponding to the HIV-IN cDNA at positions, 19, 79, 158 and 495 (GenBank acc no: AF078150). Both the HIV-IN: GFP and RNAi vectors were co-transfected in HeLa cells. After 4 days of incubation, the expression of HIV-IN was analyzed by RT-PCR and Western blot.

RESULTS: By fluorescence confocal microscopy, the GFP-HIV-IN fusion protein was found to express in the nucleus of transfected HeLa cells. RT-PCR study indicated a decrease in HIV-IN mRNA levels in cells co-transfected with U6-HIV-IN RNAi vectors. Western blot analysis also showed the reduction in HIV-IN protein expression. There were significant variations in the degree of RNAi effect among the four RNAi vectors, with the order of potency: pos19>pos495>pos79>pos158. Over 80% of HIV-IN protein was suppressed in pos19 and pos495 co-transfected cells.

CONCLUSIONS: Cellular expression of HIV-1 integrase can be effectively suppressed by gene-specific gene silencing on HIV-IN gene sequence. siRNAs targeted on the 5'- and 3'-terminial of the HIV-IN cDNAs were found have more prominent RNAi effect. The result demonstrated that gene-specific HIV-IN silencing might be of potential clinical efficacy against AIDS.

Keywords: AEGIS, RNA Interference, HIV Integrase, RNA, Small Interfering, Green Fluorescent Proteins, HIV, Acquired Immunodeficiency Syndrome, HIV Seropositivity, Gene Silencing, HIV-1, HIV Infections, Genetic Vectors, Luminescent Proteins, Hela Cells, Blotting, Western, RNA, Messenger, Humans, In Vitro, genetics

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WeOrA1312