The Research On RNA Interference Targeting HIV-1 Vpr Gene

Vpr, one of the four accessory gene products of human immunodeficiency virustype 1(HIV-1), is the only non-structural protein being packaged into nascent virions.As a multi-functional protein, Vpr plays important roles in HIV pathogenesis, suchas being involved in long terminal repeat (LTR) activation, mediating the activenuclear import of preintegration complexes (PIC), inducing arrest of the cell cycle atthe G2 phase, and promoting apoptosis. Because of the significant role Vpr plays inHIV-1 life cycle,Vpr becomes the novel target in anti-HIV reseach.In this study, RNAi technology was applied to interfere the expression of HIV-1vpr gene. Two fusion protein expression vectors with fluorescent reporter gene, 13shRNA expression vectors and 4 miRNA expression vectors targeting to distinct sitesof vpr gene sequence were constructed. Inverted fluorescent microscope and flowcytometry were used to screen the gene silencing effects. It showed that shRNA4and miR2 had manifested significant inhibitive effect on the expression of GFP-Vprand Vpr-DsRed fusion protein. ShRNA14, shRNA7 and shRNA5 also had inhibitiveeffect but not as vigorously as shRNA4 and miR2. However, shRNA11, shRNA9,shRNA13, shRNA10, miR4 and miR3 did not exhibit inhibitive effect. Real-timePCR has been applied to relatively quantify the number of vpr mRNA copies. Theresults showed that the 2-△△Ct value of shRNA4 and miR2 is only 0.0094 and 0.2088respectively, indicating significant degradation of vpr mRNA. There existed adose-responsive effect for shRNA4, as the gene silencing effect increased with thehigher dose of shRNA4. Studies using pseudovirus system revealed that shRNA4and miR2 could influence the viral packaging and replication. In addition,expression of miRNA using type II promoter showed more significantly inhibitiveeffects than expression of shRNA using typeⅢpromoter, although their target wassame and conducted under the same conditions.