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. 2008 Jun 5:5:11.
doi: 10.1186/1742-6405-5-11.

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes

Narasimhachar Srinivasakumar  1
Affiliations

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes

Narasimhachar Srinivasakumar. AIDS Res Ther. .

Abstract

Background: Human immunodeficiency virus type 1 (HIV-1)-based gene delivery systems are popular due to their superior efficiency of transduction of primary cells. However, these systems cannot be readily used for delivery of anti-HIV-1 genes that target constituents of the packaging system itself due to inimical effects on vector titer. Here we describe HIV-1-based packaging systems containing the Rev-response element (RRE), of simian immunodeficiency virus (SIV) in place of the HIV-1 RRE. The SIV RRE-containing packaging systems were used to deliver the anti-Rev gene, Rev M10, into HIV-1 susceptible target cells.

Results: An HIV-1 based packaging system was created using either a 272- or 1045-nucleotide long RRE derived from the molecular clone SIVmac239. The 1045-nucleotide SIV RRE-containing HIV-1 packaging system provided titers comparable to that of the HIV-1 RRE-based one. Moreover, despite the use of HIV-1 Rev for production of vector stocks, this packaging system was found to be relatively refractory to the inhibitory effects of Rev M10. Correspondingly, the SIV RRE-based packaging system provided 34- to 130-fold higher titers than the HIV-1 RRE one when used for packaging a gene transfer vector encoding Rev-M10. Jurkat T-cells, gene modified with Rev M10 encoding HIV-1 vectors, upon challenge with replication defective HIV-1 in single-round infection experiments, showed diminished production of virus particles.

Conclusion: A simple modification of an HIV-1 gene delivery system, namely, replacement of HIV-1 RRE with that of SIV, allowed efficient delivery of Rev M10 transgene into T-cell lines for intracellular immunization against HIV-1 replication.

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Figures

Figure 1
Figure 1
Schematic representation of HIV-1 packaging and gene transfer vector constructs containing HIV-1 or SIV RRE. A) The packaging constructs contain Gag/Pol coding sequence derived from pNL4-3 inserted downstream of the human cytomegalovirus (CMV) immediate early promoter in pCDNA3. The RRE from HIV-1 (350 nt) or from SIVmac239 (272 or 1045 nt) was positioned downstream of the Gag/Pol coding sequence. Polyadenylyation sequence in pCDNA3 is derived from bovine growth hormone gene (BGHpA). B) The gene transfer vectors were derived from pNL4-3 and contain a transgene expression cassette consisting of Elongation factor 1 alpha promoter/enhancer elements (EF1α) driving the enhanced green fluorescent protein (EGFP) or a fusion protein consisting of EGFP-2A-Rev M10. Woodchuck post-transcriptional regulatory element (WPRE) was positioned downstream of the transgene. HIV-1 or SIV RRE was present upstream of the transgene expression cassette. Δψ: Deletion in the HIV-1 encapsidation signal between nt 751 and nt 779 of pNL4-3; LTR: HIV-1 long terminal repeat; FS: Frame-shift mutation in gag; CPPT/CTS: Central polypurine tract/central termination sequence; 2A: Foot and mouth disease virus 2A cleavage factor; M10: Rev M10; 5'ss: 5' splice site; 3'ss: 3' splice site.
Figure 2
Figure 2
Effect of Rev and Rex expression constructs on virus stock production by packaging and gene-transfer vectors containing the HIV-1 or SIV RREs. Vector stocks were produced using various combinations of packaging and gene transfer vectors containing either HIV-1 or SIV RREs as shown. SIV 1045 or SIV 272 refers to the length of SIV RRE present in the packaging construct or gene transfer vector. The effect of expression of Rev-like proteins from HIV-1 (pCI-HIV-Rev), SIV (pCI-SIV-Rev) and HTLV-1 Rex (pBCRex-1) on vector stock production was tested with each of the combinations. A control vector, pCI-Neo, was used in parallel. The mean vector titers, shown in the top panel, were calculated from % GFP positivity determined by flow cytometry. The bottom panel depicts mean p24 levels in the vector containing supernatants. The titers and p24 levels were normalized to SEAP activity present in the vector stock. Error bar = 1 SD. IU: infectious units. '*' denotes relatively high p24 levels with respect to titer (described in greater detail in the text).
Figure 3
Figure 3
Determination of optimal amounts of pCI-HIV-1 Rev for production of vector stocks with the SIV RRE-based HIV-1 packaging system. Vector stocks were produced in 293T cells using pGP/SIV 1045 RRE and pN-EF1α-EGFP-WPRE/SIV RRE with indicated amounts of pCI-HIV Rev or pCI-SIV Rev. The transfections also included pCMVtat, pMD.G and a SEAP expression construct. The vector stocks were used for infection of 293T cells and the resultant SEAP-adjusted vector titers are shown. Error bar = 1 SD.
Figure 4
Figure 4
Effect of increasing amounts of Rev M10-encoding plasmid (pCI-Rev M10) on titer of vector stocks produced with an HIV-1 packaging system containing either HIV-1 or SIV RRE. The HIV-1 RRE-based packaging system (HIV-1 RRE system) consisted of the packaging plasmid pGP/HIV-1 350 RRE and the gene transfer vector pN-EF1α-EGFP/HIV-1 RRE. The SIV RRE-based packaging system (SIV RRE system) consisted of the packaging plasmid pGP/SIV 1045 RRE and the gene transfer vector pN-EF1α-EGFP/SIV RRE. For production of virus stocks with the SIV RRE-based packaging system either HIV-1 Rev (pCI-HIV Rev) or SIV Rev (pCI-SIV Rev) expression construct was used, as indicated. All transfections also received a VSV-G envelope expression construct (pMD.G) and a HIV-1 Tat (pCMVtat) expression construct. The titers of the vector stocks were determined as described in Materials and Methods. The % of GFP + cells in the absence of pCI-Rev M10 (0 μg) was considered as 100% (Y-axis) for a given packaging system to which other titers obtained at each amount of pCI-Rev M10 were normalized. Increasing amounts of pCI-Rev M10 are depicted on the X-axis. For each transfection, the indicated amount of pCI-Neo was used as a 'filler', to keep the total amount of DNA added at 1.0 μg. The results shown are representative of two independent experiments.
Figure 5
Figure 5
Virus particle production in Jurkat T-cells transduced with different HIV-1 vectors upon challenge with a replication defective HIV-1. Jurkat T-cells were separately transduced with each of the indicated vectors (X-axis) and sorted to greater than 95% purity. Each population was either mock-infected or infected with VSV-G pseudotyped pNL4-3.HSA. R-E-. The supernatants from mock or virus-infected cells were obtained on days 1, 4 and 7 and assayed for HIV-1 p24 capsid protein using a commercial ELISA kit. The Y-axis shows mean p24 levels produced by each of the different cell populations on days 4 and 7 normalized to the p24 produced by infected but untransduced Jurkat cells (which was set at 100%). The results shown are from three independent experiments. Error bar = 1 SD.
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