A Cyclophilin A-Independent Capsid Motif Renders HIV-1 Derived Vectors More Efficient at Transducing Human Cells.

Abstract We recently identified naturally occurring HIV-1 capsid (CA) motifs, which evade both cyclophilin A (CypA)-dependence in human cells and TRIM-5α-CypA (TRIM-Cyp) restriction in owl monkey kidney (OMK) cells (Chatterji, et al. JBC, In press). These HIV-1 capsid motifs contained either one (G89V) or four (V86P/H87Q/I91V/M96I) amino acid substitutions within the CypA binding site, but only the four amino acid mutant was able to bind CypA, yet does not require CypA incorporation to infect human cells. OMK cells are naturally resistant to HIV-1 infection due to TRIM-Cyp, a negative host factor, which efficiently targets incoming wild type HIV-1 capsid and thereby prevents the proper uncoating of the CA core. However, addition of cyclosporine A, which blocks the interaction of CypA and CA, or mutations within the CypA-CA binding site (G89V) yields OMK cells permissive to HIV-1. Interestingly, HIV-1 infection with the V86P/H87Q/I91V/M96I capsid mutant was able to achieve similar levels of infection in OMK cells as does wild type HIV-1 with the addition of cyclosporine A, emphasizing that this motif allows infection despite TRIM-Cyp-CA interactions. Alternatively, the interaction between HIV-1 CA and CypA, is considered as a positive host factor in human cells. It has been proposed that CypA either “protects” the virus from endogenous restriction factors or allows proper CA core uncoating. Human cells lacking CypA or treated with cyclosporine A are highly resistant to infection by wild type HIV-1. Virus is blocked during the early stage of infection, at the pre-reverse transcription step. An important goal of human gene therapy is to efficiently deliver genes to non-dividing or quiescent cells such as hematopoietic stem cells and T-cells, respectively. Improvements on vector design such as self-inactivating LTRs, VSV-G envelope pseudotyping, various viral capsids, and enhancer elements have improved both the safety and efficiency of gene delivery. To identify capsid motifs that enhance transduction, we assessed whether the G89V and V86P/H87Q/I91V/M96I capsid motifs increased transduction efficacy in primary human cells as compared to vectors packaged with wild type HIV-1 gag/pol. Preliminary studies, at low levels of transduction (2000 pg p24 input ~1 MOI) in 293T cells, showed a 3-fold increase in GFP expression using the V86P/H87Q/I91V/M96I-gag/pol packaged vectors as compared to wild type or G89V-gag/pol vectors. Studies with primary PBMC and CD34+ hematopoietic stem cells are underway and will address whether the V86P/H87Q/I91V/M96I capsid motif will allow for greater transduction efficiency for non-cycling, quiescent and/or partially activated cells. Providing a means to enhance uniform vector transduction efficacy should allow for lower levels of vector input during transduction, thereby reducing the probability for insertional mutagenesis.