The CXCR4-Tropic Human Immunodeficiency Virus Envelope Promotes More-Efficient Gene Delivery to Resting CD4⁺ T Cells than the Vesicular Stomatitis Virus Glycoprotein G Envelope^{▿ †}

Cell lines, plasmids, and viruses.

The CD4⁺ T lymphoblastoid cell line CEMss (46), a subclonal cell line derived from CEM cells (14, 37), was maintained at 1 × 10⁵ to 5 × 10⁵ cells/ml in RPMI 1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; HyClone) and 100 μg/ml of penicillin-streptomycin (Mediatech Inc.). Viral supernatants were prepared by calcium-phosphate transfection of 293T cells using the following plasmids: pNL4-3 (1), pNL4-3.HSA.R+.E− (pNL4-3Δenv) (10, 19), pHIT (VSV-G) (16), a plasmid encoding the HIV envelope from the isolate LAI (kindly donated by Robert Doms), and pMM310 (carrying β-lactamase-conjugated HIV Vpr [BlaM-Vpr]) (54). The lentiviral vector VRX494, referred to as HIVvector in this paper, was pseudotyped with either VSV-G or HIV LAI envelopes and was donated by VIRxSYS (31).

Purification and stimulation of resting CD4⁺ T cells.

Unstimulated CD4⁺ T cells, purified from leukapheresis-enriched peripheral blood mononuclear cells (PBMCs) by RosetteSep (StemCell Technologies) (2), were obtained from the University of Pennsylvania's Immunology Core. To obtain pure resting CD4⁺ T cells, cells were stained with phycoerythrin-labeled antibodies against CD25, CD69, and HLA-DR (BD Biosciences) and antiphycoerythrin magnetic beads (Miltenyi Biotec), as recommended by the manufacturers. The labeled cells were then applied to a magnetic column to separate the cells that expressed activation markers from the unlabeled resting CD4⁺ T cells. This method generally yields >98% pure resting CD4⁺ T cells (2). In the cases where cells were activated, CD4⁺ T cells were stimulated with CD3/CD28 beads (Invitrogen) at 1 bead per cell for 72 h at 37°C.

Monitoring cell activation.

The level of cell activation was monitored with flow cytometry before and after infection by measuring the level of activation marker expression and by measuring the level of DNA/RNA staining using 7-aminoactinomycin D (7-AAD) and pyronin Y (24, 25, 53). PBMCs (10⁶ cells/ml) stimulated with phytohemagglutinin (PHA) (10 μg/ml) and interleukin-2 (10 U/ml) were treated with sodium butyrate (2.5 mM) (Sigma) or aphidicolin (10 μM) (Sigma) to serve as reference controls in order to distinguish cells entering the different stages of the cell cycle.

Inoculations.

Resting CD4⁺ T cells, stimulated CD4⁺ T cells, or CEMss cells were resuspended in viral supernatants at 1 × 10⁷ cells/ml and spinoculated at 1,200 × g for 2 h at 25°C (40). After inoculation, the cells were washed twice with phosphate-buffered saline (PBS) and 10% FBS to remove unbound virions. Following the washing step, cells were cultured at 37°C in RPMI 1640 medium supplemented with 10% FBS, 100 μg/ml of penicillin-streptomycin, and 1.25 μM saquinavir (Roche Pharmaceuticals). Selected samples were treated with a 1 μM concentration of the nonnucleoside reverse transcriptase inhibitor efavirenz (AIDS Reagents Program).

Measuring HIV DNA intermediates.

At selected time points postinoculation, total cellular DNA was purified using a QIAamp DNA Micro Kit (Qiagen). Early reverse transcription was measured by real-time PCR using primers and a fluorescent probe that detect the R-U5 region of the HIV long terminal repeat (2). Late reverse transcription was measured by real-time PCR using primers and a fluorescent probe that detects transcripts that have completed the second-strand transfer (SST) (52). Integration was measured by real-time Alu PCR as previously described (2) using the primers described by O'Doherty et al. (39). Cell numbers were estimated by measuring the number of β-globin copies per sample by real-time PCR (40).

Measuring viral fusion.

Transfection viral supernatants of pNL4-3 or pNL4-3Δenv pseudotyped with VSV-G containing BlaM-Vpr were utilized to inoculate unstimulated and stimulated CD4⁺ T cells by spinoculation at 1,200 × g for 2 h at 4°C. After inoculation, the cells were washed twice with cold PBS plus 10% FBS to remove unbound virions. Cells were then resuspended in RPMI medium-10% FBS and incubated at 37°C for 30 min to induce viral fusion. After fusion, cells were washed twice with CO₂-independent medium (Invitrogen) without serum and without antibiotics. A stock CCF2-AM (Invitrogen) loading solution was prepared for staining the cells: 2 μl of CCF2 at 200 μg/ml, and 8 μl of 100 mg/ml Pluronic-F127 surfactant in dimethyl sulfoxide, and 0.1% acetic acid, diluted to a total of 1 ml with CO₂-independent medium without antibiotics or serum. One million cells were resuspended in 100 μl of CCF2 loading solution and incubated at room temperature for 1 h to allow dye uptake. Cells were subsequently washed with CO₂-independent medium, resuspended in CO₂-independent medium containing 10% FBS and 2.5 mM probenecid (Sigma-Aldrich), and incubated at 25°C in a CO₂-free atmosphere for ∼12 h. After incubation, the cells were washed once with fluorescence-activated cell sorting buffer (PBS-1% bovine serum albumin-2 mM EDTA) and fixed with 1% paraformaldehyde. Fixed cells were then analyzed by flow cytometry using a BD LSR II flow cytometer.

Measuring viral binding.

Viral binding was assessed by measuring the level of cell-associated p24^Gag in samples collected immediately after inoculation using a p24^Gag-specific enzyme-linked immunosorbent assay (ELISA) (Perkin Elmer). The approximate number of viral particles bound to cells was calculated as previously described (40).

A commonly used, HIV-based gene therapy vector fails to infect resting CD4⁺ T cells as assessed by reverse transcription.

Given the recent finding that resting CD4⁺ T cells can be directly transduced by HIV (2, 38, 41, 58), we sought to determine whether a commonly used lentiviral vector could also transduce these cells (Fig. 1 and 2). To do this, we used the HIV-based gene therapy vector HIVvector (VRX494) (Fig. 2A) (21, 31). This vector lacks all essential and auxiliary HIV proteins, encodes green fluorescent protein (GFP), and is pseudotyped with VSV-G [HIVvector(VSV-G)]. We inoculated unstimulated CD4⁺ T cells (Fig. 1) and the activated T-cell line CEMss with wild-type HIV (wtHIV) or HIVvector(VSV-G). CEMss cells have similar susceptibility to HIV infection as stimulated CD4⁺ T cells. Unstimulated cells were purified from enriched PBMCs by red blood cell Rosette, which negatively selects CD4⁺ T cells from other T-cell types, B cells, NK cells, granulocytes, and monocytes. Most unstimulated cells are considered to be in the G₀/G₁a stage of the cell cycle (Fig. 1C) (24), but a significant proportion of these cells express activation markers (Fig. 1A). We assayed reverse transcription by quantitative PCR using primers that detect the second-strand transfer (SST) step of reverse transcription (Fig. 2B and C) (52).

We found no detectable reverse transcription by HIVvector(VSV-G) in unstimulated CD4⁺ T cells while we detected increasing levels of reverse transcription over time with wtHIV (Fig. 2B). In contrast, the levels of reverse transcription of HIVvector(VSV-G) and wtHIV were similar in CEMss cells (Fig. 2C). Thus, an early step in the infection of unstimulated CD4⁺ T cells with HIVvector(VSV-G) is impaired. Importantly, the cells were >90% viable, with a minimal decrease of the absolute numbers of viable cells at the time points when reverse transcription was measured (see Supplement S1 in the supplemental material), indicating that the lack of infection is not due to cell death.

The viral envelope is the critical factor: VSV-G-pseudotyped virions do not infect unstimulated CD4⁺ T cells as assessed by reverse transcription.

One of the major differences between wtHIV and HIVvector(VSV-G) particles, in addition to the lack of auxiliary proteins, is that the HIVvector carries a deletion of HIV env and is pseudotyped with VSV-G. We set out to investigate whether this difference was responsible, at least in part, for the dramatic difference between wtHIV and HIVvector(VSV-G) reverse transcription in unstimulated cells (Fig. 2). To do this, we inoculated unstimulated CD4⁺ T cells and CD3/CD28 bead-stimulated primary CD4⁺ T cells with the following four viruses: HIVvector(VSV-G), wtHIV, wtHIV with a deletion of env and pseudotyped with VSV-G [HIVΔenv(VSV-G)], or wtHIV with a deletion of env and pseudotyped with X4-tropic HIV Env [HIVΔenv(HIV Env)]. We measured late reverse transcripts at 0, 24, and 48 h postinoculation.

We found that VSV-G envelope is the critical factor that prevented efficient infection of unstimulated cells (Fig. 3). Reverse transcription of HIVvector(VSV-G) or HIVΔenv(VSV-G) in unstimulated cells was barely detectable while reverse transcription of HIVΔenv(HIV Env) and wtHIV increased over time (Fig. 3A). In contrast, all four viruses reverse transcribed in bead-stimulated primary CD4⁺ T cells (Fig. 3B). The kinetics of reverse transcription in unstimulated cells are not the kinetics of quiescent or activated cells but follow, rather, an intermediate kinetic profile. This likely indicates that unstimulated cells are in a range of activation states. In addition, we note that other groups of investigators report higher levels of infection of activated cells than resting cells (58), compared to our results in the present report. This may be due to variable activation of the cells or to donor-to-donor variation. Nevertheless, our findings indicate that HIV particles bearing VSV-G in place of HIV Env have impaired infection at or before reverse transcription in unstimulated CD4⁺ T cells but not in stimulated CD4⁺ T cells. Thus, the lack of infection of unstimulated cells by HIVvector(VSV-G) (Fig. 2B) resulted from substituting VSV-G for the X4-tropic HIV Env on the HIVvector particles.

Viral fusion is the critical step: VSV-G-pseudotyped viral particles do not fuse to unstimulated CD4⁺ T cells, as assessed by a BlaM-Vpr-based fusion assay.

Since the envelope appeared to be the critical factor, we tested if virus-cell fusion was the critical step of infection affected by VSV-G. To test this, we employed a BlaM-Vpr virus-to-cell fusion assay (7, 54). In this assay, the dye CCF2, a β-lactamase substrate that fluoresces green, is added to cells after viral particles that carry β-lactamase (covalently linked to Vpr [BlaM-Vpr]) have fused. The β-lactamase then cleaves CCF2, and CCF2 fluorescence changes from green to blue. The extent of viral fusion can be monitored by flow cytometry as the percentage of cells that convert from green to blue fluorescence. We prepared BlaM-Vpr-containing wtHIV and HIVΔenv(VSV-G) particles and inoculated unstimulated and stimulated CD4⁺ T cells.

We found that viral fusion is the critical step of infection affected by VSV-G (Fig. 4). We found that fusion of HIVΔenv(VSV-G) particles to unstimulated CD4⁺ T cells was undetectable (Fig. 4A) while wtHIV fused efficiently to unstimulated CD4⁺ T cells (Fig. 4B). In contrast, we found that both HIVΔenv(VSV-G) and wtHIV particles fused to stimulated CD4⁺ T cells (Fig. 4C and D, respectively). We speculate that high levels of cell activation are required for efficient VSV-G-mediated fusion since VSV-G-mediated fusion does not occur with unstimulated cells, which are in a range from low to intermediate levels of activation (Fig. 1A and C). Furthermore, we find that VSV-G-mediated fusion occurs most efficiently with CEMss cells and with lesser efficiency with CD3 and CD28 bead-stimulated primary cells. Thus, even though the viral inocula are matched for fusion to CEMss cells, HIV Env mediates fusion slightly more efficiently than VSV-G when we measure fusion to stimulated primary cells. Nevertheless, these findings indicate that VSV-G can mediate fusion to activated, but not unstimulated, CD4⁺ T cells. In addition, our BlaM-Vpr fusion data are consistent with the very limited amount of early reverse transcription detected over time in unstimulated cells inoculated with HIVvector(VSV-G) (see Supplement S3 in the supplemental material).

VSV-G-pseudotyped virions bind less efficiently to resting CD4⁺ T cells than HIV Env-pseudotyped virions.

Although the cellular receptor of VSV-G is still unknown (9, 49), we considered whether impaired fusion of VSV-G-pseudotyped particles to resting CD4⁺ T cells could be due to lack of VSV-G binding. To examine this, we assessed viral binding to resting and stimulated CD4⁺ T cells by measuring the level of cell-associated p24^Gag immediately after inoculation. Pure resting CD4⁺ T cells (CD25, CD69, and HLA-DR-negative cells) (Fig. 1A) were spinoculated with HIVvector(VSV-G) or HIVvector(HIV Env). Following inoculation, the cells were washed to remove unbound virions. The level of cell-associated p24^Gag was measured by ELISA. The number of viral particles bound per cell was calculated based on the prediction that there are ∼15,800 viral particles per picogram of p24^Gag (60). For example, in activated T cells, we found a p24^Gag value for HIVΔenv(VSV-G) of 61 pg/ml, which translated to 0.005 pg/cell and to 78 virions/cell (Fig. 5).

We found that VSV-G-pseudotyped particles bound less efficiently than HIV Env-pseudotyped particles to resting CD4⁺ T cells while both vectors bound to stimulated cells at similar levels (Fig. 5). This difference in viral binding to resting CD4⁺ T cells between HIVvector(VSV-G) and HIVvector(HIV Env) corresponded to approximately sevenfold (Fig. 5). In agreement with our current results, we previously demonstrated that under spinoculation conditions, HIV viral particles bind about two- to fourfold less to resting CD4⁺ T cells than to stimulated cells (40). We attribute this to differences in the surface area exposed to viral particles during spinoculation. However, in the case of VSV-G-mediated binding, we found that particles bound ∼21-fold less efficiently to resting cells than to stimulated cells. Therefore, the difference in binding to resting cells between VSV-G and HIV Env is significant and cannot be attributed to differences in surface area between resting and stimulated cells (Fig. 5). These results were confirmed by measuring the level of cell-associated viral RNA after inoculation by reverse transcriptase PCR (not shown). Based on these observations, we conclude that viral particles pseudotyped with HIV Env bind better to resting cells than viral particles pseudotyped with VSV-G. The lower binding may result in slightly lower fusion, but the reduced binding is unlikely to explain the nearly complete absence of fusion of VSV-G-pseudotyped particles to resting CD4⁺ T cells (Fig. 4).

HIV Env rescues the ability of the lentiviral vector to transduce resting CD4⁺ T cells.

Among CD4⁺ T cells, memory and naïve cells in the G₀ stage of the cell cycle have the longest life span (34, 55). Given that HIVvector(VSV-G) did not efficiently infect unstimulated CD4⁺ T cells while wtHIV did, we decided to test whether pseudotyping HIVvector particles with X4-tropic HIV Env would allow integration of the vector into pure G₀ resting CD4⁺ T cells and whether this transduction would lead to cellular activation. To do this, we purified CD25, CD69, and HLA-DR-negative cells from unstimulated CD4⁺ T cells (Fig. 1A and C) and inoculated them with HIVvector(HIV Env) or HIVvector(VSV-G). We then measured integration using our standard Alu PCR assay (2, 39) at 0, 24, 48, and 72 h postinoculation. We found that HIVvector(HIV Env) integrated into both resting and stimulated CD4⁺ T cells (Fig. 6A and B) and that integration occurred without significant activation of the cells during and after the inoculation process (Fig. 7), confirming our prior findings with wtHIV (52). Cell activation was assessed by monitoring the level of surface expression of CD25, CD69, and HLA-DR (Fig. 7A) and by monitoring the level of DNA/RNA staining (25) (Fig. 7B) before spinoculation and at 0, 4, and 24 h postspinoculation. In contrast to our findings with HIVvector(HIV Env), we did not detect integration of HIVvector(VSV-G) to resting CD4⁺ T cells but detected integration to activated CD4⁺ T cells (Fig. 6A and B). Specifically, the level of transduction by HIVvector(HIV Env) is 100 times higher than HIVvector(VSV-G) in resting CD4⁺ T cells while HIVvector(VSV-G) integrates ∼1,000 times more effectively in stimulated cells than in resting cells. Notably, this large (∼1,000-fold) difference in susceptibility between resting and stimulated cells did not occur when HIV envelope was used. Instead, a 10-fold difference in integration was observed between resting and stimulated cells inoculated with HIVvector(HIV Env).

For efficient gene therapy, higher levels of transduction than those shown in Fig. 6A would be necessary. We therefore tested whether a less diluted virus stock would yield higher levels of integration (Fig. 6C). We found that using fivefold higher levels of the same viral stock increased the level of integration in resting CD4⁺ T cells fivefold such that on average 0.5 provirus was detected per resting CD4⁺ T cell, which is within the range necessary for effective gene therapy. The data shown in Fig. 6C are also consistent with our previous observations with wtHIV that a linear relationship exists between virus dilution and the level of integration in resting CD4⁺ T cells (2). We conclude that pseudotyping HIVvector with HIV Env instead of VSV-G permits transduction of pure resting CD4⁺ T cells. Our results suggest that X4-tropic HIV Env is a better gene therapy envelope than VSV-G when the gene therapy target is resting CD4⁺ T cells.