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.

Figure 1. Schematic representation of packaging constructs (A) and gene transfer vectors (B).

The packaging constructs (A) were created by inserting the gag/pro-pol coding region from pNL4-3 in pCDNA3 between the human cytomegalovirus immediate early promoter and the bovine growth hormone poly A signal (BGHpA). The plasmid pGP/HIV-1 350 RRE contains the HIV-1 RRE; while pGP/1-4xCTE contains either 1-, 2- or 4-copies of MPMV CTE, respectively. The RRE or CTE sequences were inserted between the gag/pro-pol coding sequence and the BGHpA signal. The gene transfer vectors (B) are derivatives of pN-GIT72 or pN- EF1α-EGFP-WPRE. They were modified to contain 1-, 2- or 4- copies of CTE. The HIV-1 RRE in pN- EF1α-EGFP-WPRE was replaced with the 1045 nt SIVmac239 RRE to create pN- EF1α-EGFP-1x-4x CTE/SIV RRE. The vector pN- EF1α-EGFP-2A-M10- 4xCTE /SIV RRE is similar to pN- EF1α-EGFP-4xCTE/SIV RRE, but contains EGFP-2A-M10 fusion protein instead of only EGFP. FS: frame-shift mutation; 5′ss: 5′ splice site; 3′ss: 3′ splice site, ΔΨ : deletion in encapsidation signal; IRES: encephalomyocarditis virus internal ribosome entry site; 2A: foot and mouth disease virus 2A protease cleavage factor; CPPT/CTS: central polypurine tract/central termination sequence.

Figure 2. Particle production by packaging constructs with RRE or CTE HEK 293T were transfected with packaging plasmids pGP/HIV-1 350 RRE, pGP/1xCTE, pGP/2xCTE or pGP/4xCTE.

The transfections with pGP/HIV-1 350 RRE, received either pCI-Neo or pCI-HIV Rev. The supernatants were harvested 72 hours post-transfection and assayed for HIV-1 capsid protein (p24) by ELISA and SEAP activity using a commercial kit. Mean SEAP-adjusted p24 levels are shown. Each experiment was carried out in duplicate. Error bar = 1 SD.

Figure 3. Virus stock production by various combinations of packaging and gene transfer vectors containing either HIV-1 RRE or one or more modules of MPMV CTE.

HEK 293T cells were transfected with indicated packaging and gene transfer vectors. One set of transfections received pCI-Neo (speckled bars) and a parallel set of transfections received pCI-HIV Rev (cross-hatched bars). All transfections received VSV-G envelope expression construct and a SEAP expression construct. The supernatants were harvested 72 h post-transfection and used for infection of naïve HEK 293T cells. The virus titers (top panel) were determined by flow cytometry of infected cells as described in Materials and Methods and normalized for transfection efficiency against SEAP activity. The supernatants were also assayed for virus particle content by ELISA for HIV-1 capsid protein (p24). The SEAP-adjusted p24 values are shown in the bottom panel. Discordant results between the titers and the p24 levels are indicated by asterisks (*) above the corresponding bars. Each experiment was carried out in duplicate. Error bar = 1 SD.

Figure 4. Effect of Rev M10 coexpression on vector production by packaging systems regulated by HIV-1 RRE, CTE or combinations thereof.

Four different combinations of packaging and gene transfer vectors (RRE/RRE; RRE/CTE; CTE/RRE; CTE/CTE) were tested for production of vector stocks. The plasmid pCI-Rev M10 was used at the indicated amounts during vector stock production. The total amount of plasmid added was kept constant by using pCI-Neo as a filler plasmid. All transfections also received a VSV-G expression construct (pMD.G) and pCI-HIV Rev except for the packaging system (CTE/CTE) that used CTE in both packaging and gene transfer vector constructs. The vector titers were determined by infection of Jurkat T-cells. For each kind of packaging system, the titer obtained in the absence of pCI-Rev M10 was used for normalization. The different combinations were designated based on the transport element in the packaging construct and gene transfer vector. Each experiment was carried out in duplicate. Error bar = 1 SD. RRE/RRE: pGP/HIV 350 RRE and pN-GIT72; RRE/CTE: pGP/HIV 350 RRE and pN-GIT72-2xCTE; CTE/RRE: pGP/4xCTE an d pN-GIT72; CTE/CTE: pGP/4xCTE and pN-GIT72-2xCTE.

Figure 5. Packaging of gene transfer vectors containing CTE or both CTE and SIVmac239 RRE by pGP-4xCTE.

The vectors pN- EF1α-EGFP-WPRE, pN- EF1α-EGFP-1xCTE, pN- EF1α-EGFP-2xCTE, and pN- EF1α-EGFP-4xCTE (A and C) and their corresponding SIV RRE containing vectors (B and D) were individually packaged using pGP/4xCTE in HEK 293T cells as described in Materials and Methods. One set of transfections received pCI-Neo (speckled bars) and a parallel set received pCI-HIV Rev (cross-hatched bars) The SEAP-adjusted titers are shown in panels A and B while the SEAP-adjusted p24 levels are shown in panels C and D. Each experiment was carried out in duplicate. Error bar = 1 SD.

Figure 6. Flow cytometry analysis of Jurkat T-cells transduced by HIV-1 vectors encoding EGFP or EGFP-2A-Rev M10.

The histograms show cell number along the Y-axis and EGFP expression along the X-axis. The packaging constructs used are indicated above the histograms, while the transgene and the transport elements in the gene transfer vectors are shown below. The same packaging construct was used for each pair of gene transfer vectors. The percentage EGFP positive population, as determined by marker/gate M1, is also shown. Representative data from an experiment carried out in duplicate.

Figure 7. Comparison of packaging systems for delivery of Rev M10 into Jurkat T-cells.

Packaging plasmids pGP/HIV-1 350 RRE, pGP/SIV 1045 RRE and pGP/4xCTE were used to produce virus stocks with indicated gene transfer vectors. The virus stocks were used for infection of Jurkat T-cells and vector titer was estimated. The titer of each Rev M10 encoding vector, was normalized to that obtained with the corresponding control vector expressing only EGFP. The SEAP-adjusted titers for the control vectors are indicated above the corresponding bars. The Rev M10 encoding vectors were tested at three different amounts as shown. Each experiment was carried out in duplicate. Error bar = 1 SD.

Figure 8. Virus particle production by gene modified Jurkat T-cells challenged with replication defective HIV-1.

Jurkat T-cells, either unmodified or transduced with the indicated vectors encoding either EGFP (light grey) or EGFP-2A-M10 (dark grey), were challenged with replication defective HIV-1 pseudotyped with VSV.G as described in Materials and Methods. The HIV-1 capsid protein released into the supernatant of the infected cells was measured by p24 ELISA. The results were normalized to the p24 released from infected but unmodified control cells. Each experiment was carried out in duplicate. Error bar = 1 SD.