436. shRNAs Target HIV-1 Vif Gene Coding for SOCS-Box Motif Prevent Virus Escape from RNA Interference-Mediated Inhibition

Abstract

RNA interference (RNAi) is powerful cellular mechanism in which double-stranded small interfering RNAs (siRNAs) induce sequence-specific degradation of homologous RNA molecules. Recent studies have shown that human immunodeficiency virus (HIV) replication can be effectively inhibited by intracellularly expressed siRNA or shRNA for short term. Since RNAi is highly sequence specific and HIV mutates rapidly, long-term inhibition of HIV replication is still a challenge for RNAi mediated virus gene therapy. Several groups report that HIV can escape RNAi inhibition as early as 21 days post challenge by target site point or deletion mutations. In this study, we developed a novel strategy to target the HIV Vif protein SOCS-box motif coding region using stably expressed shRNAs. HIV-1 Vif (viral infectivity factor) protein overcomes the antiviral activity of the host cellular DNA deaminase APOBEC3G by acting through Cul5-ElonginB-ElonginC E3 ubiquitin ligase. Sequence alignment of the Vif protein with other well-studied E3 ubiquitin complex binding proteins shows Vif contains the highly conserved SOCS-box motif that is the important binding domain to Cul5 complex. Mutations in this motif impeded the protein's ability to bind to this complex. For this reason, we designed two shRNAs targeting the highly conserved Vif SOCS-box motif-coding regions. Wild type virus will be directly inhibited by shRNA mediated RNAi. Although the virus may mutate and escape from RNAi via mutations in the target sites, the mutated SOCS-box motif will lose its ability to direct APOBEC3G to the proteasome, thereby allowing its incorporation into the virion, which will lead to defective virus. Our preliminary data shows the two stably expressed shRNAs can effectively inhibit HIV replication in the short term and reduce virus infectivity over long-term culture. There are no virus mutations with the shRNA target sites that we could detect up to one month after the initial challenge with HIV-1 IIIB. However, extended incubation shows one of the two target sites acquired a single point mutation after two months in culture. This point mutation is considerably delayed in comparison comparing with other reported escape mutations. The amino acid encoded by this mutation resulted in a very conservative change from Leu to Ile. The function of Vif with this amino acid changed mutation needs to be further studied. Nevertheless, we have yet to detect a mutation at the other conserved site in Vif. Thus, our results provide a novel strategy in targeting a highly conserved motif in an essential viral protein coding region which directly or indirectly can prevent virus escape from RNAi inhibition.