Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

Abstract

The use of RNA transport elements from different viruses can provide novel attributes to HIV-1-based gene delivery systems such as improved safety or Rev independence. We previously described an HIV-1 based gene delivery system that utilized the simian immunodeficiency virus Rev-response element (RRE) in place of the HIV-1 RRE. Despite the use of Rev for the production of vector stocks, we showed the utility of this system for delivery of Rev M10, a dominant-negative mutant of HIV-1 Rev, into T-cells. Here, we investigated the use of RNA transport elements from Mason-Pfizer monkey virus or MPMV for the creation of high-titered Rev-free HIV-1-based packaging systems. The HIV-1 gag/pol expression constructs containing one or more copies of MPMV constitutive RNA transport element (CTE) were used to package similarly modified gene-transfer vectors in the presence or absence of Rev. An inverse correlation between the number of CTE modules and Rev dependency was noted for vector stock production. While packaging systems containing multiple CTEs were resistant to exogenously expressed Rev M10, the titers of vectors encoding Rev M10 were nevertheless reduced in comparison to vectors encoding only green fluorescent protein (GFP). In contrast, a gene transfer vector encoding the Rev M10 transgene and containing both RNA transport elements exhibited almost no loss in titer in comparison to a corresponding vector encoding only GFP. The optimized Rev-independent gene delivery system was used for delivery of Rev M10 transgene into T-lymphocytes. Upon challenge in single round infection assays with HIV-1, the modified T-cells produced fewer virus particles than control cells expressing GFP. This Rev-free packaging system may prove useful for targeting the Rev-RRE-Crm1 nucleocytoplasmic RNA transport pathway for inhibiting HIV replication.

Peer Review #1 of "RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety (v0.1)"

“…Rev and RRE have also been implicated to play a role in encapsidation of genomic RNA (Brandt et al 2007;Cockrell et al 2011) and this can also decrease mobilization. In contradistinction to this obervation, we have reported high-titered Rev-free HIV-1 based gene delivery systems (Srinivasakumar 2011). This would suggest that the effect of Rev and RRE on encapsidation may depend on the precise configuration of the gene-transfer vector.…”

RRE-deleting self-inactivating and self-activating HIV-1 vectors for improved safety

“…Rev and RRE have also been implicated to play a role in encapsidation of genomic RNA (Brandt et al, 2007; Cockrell et al, 2011) and this can also decrease mobilization. In contradistinction to this observation, we have reported high-titered Rev-free HIV-1 based gene delivery systems (Srinivasakumar, 2011). This would suggest that the effect of Rev and RRE on encapsidation may depend on the precise configuration of the gene-transfer vector.…”

The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

“…Multiple Rev molecules oligomerize cooperatively onto the RRE through several contacts onto the RRE from each Rev to generate a Rev-RRE ribonucleoprotein [ 2 , 3 ]. Targeting of the RRE has been attempted but proven unsuccessful [ 4 , 5 , 6 , 7 ]. Targeting the binding of CRM1 to Rev also has been unsuccessful due to lack of detailed structural information [ 8 , 9 ].…”