Study On The Inhibition Of HIV-1by Multi-Target RNA Interference

Acquired immunodeficiency syndrome (AIDS) is caused by the Human immunodeficiency virus (HIV) and it has been a social problem over the past three decades. At present, there is no effective vaccine to prevent HIV infection. Antiviral therapy is the most effective way to treat AIDS. Meanwhile, the emergences of side effects and drug-resistant virus became a bundle for AIDS therapy. RNA interference (RNAi) was first discovered in1998, and it has been widely used in the field of gene silence and inhibition of many kinds of viruses (such as HIV). Due to the higher mutation of HIV-1, a long-term suppression can not be got only by a single siRNA. A long-term inhibition of HIV-1can be achieved by combination of multi-target RNAi which targets the highly conserved region in HIV-1genome. This combinationa of various anti-HIV siRNA can reduce the escape mutation, and get a long-term inhibiton to HIV-1. It’s important to launch RNAi for prevention and treatment of HIV-1.In this study, all of the experiments were based on RNAi. We performed a combined inhibition of HIV-1in TZM-bl cells, silencing of HIV-1gag gene from epidemic strains among men who have sex with men (MSM) in Tianjin area by short hairpin RNA(shRNA), and inhibition of HIV-1by multiple-targets RNAi.The vpu gene is a unique accessory gene to HIV-1and it encodes a small transmembrane protein Vpu. Tetherin protein can inhibits the HIV-1release from cell membrane. Vpu protein can counteract the effect of tetherin. In order to reduce the antagonism of Vpu and enhance the antiviral effect of tetherin, we performed a combined inhibition of HIV-1by the combination of Vpu-targeted siRNA and tetherin over-expressed retrovirus. The luciferase assay of TZM-bl cells infected by HIV-1showed that, the replication of HIV-1was effectively inhibited by vpu-RNAi retrovirus. The results showed that a single RNAi of vpu can achieve better inhibitory effect than the combined inhibition.There are many kinds of subtypes of HIV-1. The epidemic virus strains are different from other areas and population. After confirming the inhibitory effect of single vpu-RNAi in cell level, the samples derived from the MSM in Tianjin area was used in our study. Forty-four HIV-1gag genes were amplified using PCR. We designed five shRNAs based on the conserved sequences of gag genes. The gag-EGFP fusion transcript reporter system was used to select the shRNA in highest inhibition. Among the five candidates of shRNA, gag-shRNA-3represented a widely inhibition against all selected targets either in subtype B or subtype C HIV-1.The inhibition effect of vpu-RNAi and gag-RNAi was evaluated in our study. We performed a multi-targets RNAi by targeting four genes of HIV-1(gag, env, tat, vpu). In our study, a cassette that expresses four different siRNAs with one single human U6promoter is constructed. This cassette is denominated as double long hairpin RNA (dlhRNA). HIV-1genes EGFP fusion transcript reporter system was used to check the inhibiton efficiency. Four short hairpin RNAs (shRNAs), eight long hairpin RNAs (lhRNAs) and two double long hairpin RNAs (dlhRNAs) expressing plasmids were constructed and dlhRNA-VGTE was selected as the optimal one from those inhibitory RNAs. Considering the efficiency and persistence during the gene delivery, the dlhRNA expression cassette was subcloned into the lentiviral vector FG12to get the corresponding lentivirus. In the HIV-1replication assay, complete suppression as the negative control was achieved by FG12-VGTE at MOI of0.04. Moreover, there is no IFN response induced by dlhRNA cassette. Our results showed that expression of dlhRNA could provide highly effective protection against HIV-1infection for10days in VGTE-TZM-bl cells. In conclusion, the multi-target RNA interference strategy might be a promising therapeutic approach to anit-HIV in clinical.