4 Short Hairpin RNA Lentiviral Vector-Based System for TRIM5α Knockdown in Cynomolgus Monkey Embryonic Fibroblast Cells Against HIV Infection

Abstract Background: The restriction factor, TRIM5α is known to limit retroviral infection at an early post-entry stage in a species-specific manner. The current study was designed to assess the efficacy of TRIM5α in restricting the human immunodeficiency virus (HIV) using a pseudo-viral infection model in cynomolgus monkey embryonic fibroblast cells (CMEFs down the TRIM5α expression using the lentivirus short hairpin RNA (shRNA) knockdown vector system. Methods: Three shRNAs against the TRIM5α gene were designed and cloned into lentiviral vectors expressing the green fluorescent protein (GFP) and red fluorescent protein (RFP) reporter genes. The stable knockdown of TRIM5α gene expression was confirmed by both real-time RT-PCR as well as Western blot. The lentiviral inducible shRNA vector was then transduced into the CMEFs infected with HIV Pseudo-virus. Results: We successfully designed and cloned three shRNAs targets into lentiviral vectors to knock down the TRIM5α gene expression. Further, transduction of the shRNAs into the pseudo-viral HIV infected CMEFs showed greater susceptibility to the infection. Conclusion: Here, for the first time, we successfully knocked down the TRIM5α gene using RNA interference (RNAi) technology and found TRIM5α-null CMEF cells to be susceptible to HIV pseudo-virus infection. Our research provides an important strategy for the establishment of shRNA based lentiviral vector system for gene knockdown in the cynomolgus monkey animal model against HIV infection.