Entry and transcription as key determinants of differences in CD4 T-cell permissiveness to human immunodeficiency virus type 1 infection

Abstract

Isolated primary human cells from different donors vary in their permissiveness-the ability of cells to be infected and sustain the replication of human immunodeficiency virus type 1 (HIV-1). We used replicating HIV-1 and single-cycle lentivirus vectors in a population approach to identify polymorphic steps during viral replication. We found that phytohemagglutinin-stimulated CD4(+) CD45RO(+) CD57(-) T cells from healthy blood donors (n = 128) exhibited a 5.2-log-unit range in virus production. For 20 selected donors representing the spectrum of CD4 T-cell permissiveness, we could attribute up to 42% of the total variance in virus production to entry factors and 48% to postentry steps. Efficacy at key intracellular steps of the replicative cycle (reverse transcription, integration, transcription and splicing, translation, and budding and release) varied from 0.71 to 1.45 log units among donors. However, interindividual differences in transcription efficiency alone accounted for 64 to 83% of the total variance in virus production that was attributable to postentry factors. While vesicular stomatitis virus G protein-mediated fusion was more efficacious than CCR5/CD4 entry, the latter resulted in greater transcriptional activity per proviral copy. The phenotype of provirus transcription was stable over time, indicating that it represents a genetic trait.

FIG. 1.

Replication kinetics for HIV-1 in native or expanded CD4 T cells. (A) Cells isolated from 128 healthy blood donors were stimulated in vitro for 2 days with PHA and infected with 1,000 pg of p24 of R5-tropic NL4-3BaLenv. Infection was monitored as the p24 antigen level in the supernatant. (B) CD4 T-cell populations (n = 20) representative of the spectrum of permissiveness to HIV-1 were expanded twice in vitro with irradiated PBMCs, PHA, and IL-2 and infected. Error bars indicate SEMs. Shown are hyperpermissive (red), normally permissive (black), and hypopermissive (green) cells.

FIG. 2.

Immunological characterization of expanded CD4 T-cell pools. The expression of selected cytokines and chemokines (A) and surface activation markers (B) was analyzed by flow cytometry. CD4 T-lymphocyte pools represented a >97% pure population of CD45RO⁺ CD57⁻ T cells. All correlations (R²) were <0.15, and P values were >0.1. TNF, tumor necrosis factor; IFN, interferon.

FIG. 3.

Contributions of entry and nonentry host factors to cell permissiveness. (A) CCR5 receptor density, expressed as mean fluorescence intensity (MFI), correlated with p24 antigen production measured 7 days after infection with R5-tropic NL4-3BaLenv (R², 0.42; P, <0.0001). Similar estimates were obtained for percentages of CCR5⁺ cells. (B) Assessment of entry-independent factors influencing cell permissiveness. Viral vectors differing only in the mechanism of entry—HIVΔEnv pseudotyped with the R5-tropic JRFL envelope (10⁵ pg of p24) and with the VSV G protein envelope (10⁴ pg of p24)—were used to infect 6 × 10⁵ CD4 T cells. The correlation (R², 0.48; P, <0.0014) represents the shared contributions of intracellular steps to the cell permissiveness phenotype, measured as the amount of p24 in the supernatant 4 days posttransduction. Not shown in this analysis are values for cells from two donors homozygous for CCR5Δ32. Each point represents the mean of triplicate values.

FIG. 4.

Variations in intracellular steps. (A) Single-cycle experiments with HIVΔEnv pseudotyped with the R5-tropic JRFL envelope or with the VSV G protein envelope. Expanded CD4 T cells (6 × 10⁵ cells) were infected with 10⁵ pg of p24 of HIVΔEnv/R5 or 10⁴ pg of p24 of HIVΔEnv/VSV. DNA samples were taken at 12 h and 4 days (4d) postinfection, and HIV-1 gag copies were normalized to the cyclophilin A copy number to quantify the efficiencies of reverse transcription and integration per cell. RNA samples were taken at 4 days postinfection, reverse transcribed with random hexamers, and assessed for HIV-1 mRNA copies normalized to β-actin mRNA. Cell-associated p24 and extracellular p24 were assessed at 4 days postinfection by an ELISA. (B) Reporting postintegration events relative to the number of proviral copies underscored the higher efficiency of transcription after CD4/CCR5 entry than after VSV G protein-mediated fusion. Not shown for R5 values are the results for two donors homozygous for CCR5Δ32. Each point represents the mean of triplicate values for one donor.

FIG. 5.

Contributions of intracellular steps of the viral life cycle to cell permissiveness. Shown are pairwise correlation plots of measurements of 12-h viral cDNA (cDNA), proviral DNA (DNA), viral mRNA (RNA), cell-associated p24 (cap24), and extracellular p24 (ep24). The plots below and above the diagonal correspond to HIVΔEnv/VSV (red dots; B1 to B10) and HIVΔEnv/R5 (blue dots; C1 to C10), respectively. The diagonal plots (green dots; A1 to A5) represent the concordance between the viral constructs, but with the results for two donors homozygous for CCR5Δ32 excluded. Each point represents the mean of triplicate values for each donor. The correlations are displayed in the plots. Single, double, and triple asterisks indicate P values of <0.05, <0.01, and <0.001, respectively.

FIG. 6.

Temporal trends in permissiveness. CD4 T cells were isolated from three donors (1, blue; 2, green; 3, red) at three different times (days 0, 21, and 42). Cells were transduced with HIVΔEnv/VSV (broken lines; 10⁴ pg of p24) and HIVΔEnv/R5 (solid lines; 10⁵ pg of p24) to infect 6 × 10⁵ CD4 T cells. At 4 days postinfection, p24 antigen was measured in the supernatant (A), and real-time PCR was used to determine transcription per integrated provirus (B).