Decay of the VSV-G-pseudotyped HIV-1 in resting CD4 T cells.

<p>(A) Resting CD4 T cells were purified by negative depletion, rested overnight, and then pre-stimulated with anti-CD3/CD28 beads for 1 hour and infected with HIV-1(VSV-G) (552 ng p24 per million cells) (A1). Cells were also infected without pre-stimulation (A2 to A5) and then stimulated with anti-CD3/CD28 beads at 2 hours, day 1, day 3, or day 5 post infection to initiate viral replication. The p24 release was measured following anti-CD3/CD28 stimulation (marked as day “0” on the X-axis of each panel). (B) is a repeat of (A) in the same donor, using HIV-1_NL4-3 (Wt) (552 ng p24 per million cells).</p

Measurement of viral entry and DNA synthesis following HIV-1(VSV-G) infection of resting CD4 T cells.

<p>(A) Resting or pre-activated (CD3/CD28 beads, two beads per cell) CD4 T cells (1×10⁶) were infected with 200 ng of Nef-luciferase-tagged HIV-1_NL4-3 (Wt) or HIV-1(VSV-G) for 2 hours. Infected cells were washed three times and then used to measure luciferase activity in live cells. As a control, CEM-SS cells were identically infected with the Nef-luciferase-tagged HIV-1(VSV-G). Uninfected cells were identically treated and measured for luciferase activities. (B) Resting or pre-activated (overnight PHA plus IL-2 treatment) CD4 T cells (1×10⁶) were infected with 200 ng of HIV-1_NL4-3 (Wt) or HIV-1(VSV-G) in 0.5 ml for 2 hours. Following infection, cells were treated with TrypLE (Invitrogen) for 2 minutes at 37°C and then washed an additional three times with medium. Cells were pelleted and subsequently lysed for p24 ELISA. (C to E) Resting CD4 T cells were infected with an equal TCID₅₀ dose of HIV-1_NL4-3 (Wt) or HIV-1(VSV-G) (2.53×10⁵ TCID_50/Rev-CEM per million cells). Following infection for 2 hours, cell-free viruses were washed away. Cells were cultured for 5 days and then activated with anti-CD3/CD28 beads. (C) A measurement of p24 release confirmed that only the Wt virus but not HIV-1(VSV-G) replicated following CD3/CD28 stimulation. (D, E) Total cellular DNA from infected cells was extracted at different time points, and then amplified with real-time PCR for HIV late DNA (D) or 2-LTR-circles (E). (F, G) is a repeat of (D, E) on another donor using an equal p24 level of Wt and HIV-1(VSV-G) to infect resting T cells. Total cellular DNA was extracted from infected cells at different time points and PCR-amplified for HIV late DNA (F) or 1-LTR circles (G), along with the β-actin pseudogene as a control.</p

Comparison of viral DNA synthesis in CEM-SS cells infected with either HIV-1(VSV-G) or HIV-1_NL4-3.

<p>Cells were infected with an equal TCID₅₀ dose of HIV-1(VSV-G) or HIV-1_NL4-3 (Wt) (1.27×10⁵ TCID_50/Rev-CEM per million cells). Following infection for 2 hours, cell-free viruses were washed away. Total cellular DNA was extracted from cells, and then amplified by real-time PCR to measure the synthesis of full-length HIV-1 DNA (2 hours post infection) (A) and 2-LTR circles at later time points (6 and 12 hours post infection) (B). The relative ratios of 2-LTR circles at 12 hours and HIV-1 DNA at 2 hours were plotted (C).</p

Different effects of dynasore on the replication of HIV-1(VSV-G) and Wt in a human T cell, Rev-CEM.

<p>(A) Rev-CEM, a Rev-dependent GFP indicator cell, was pretreated for 30 minutes with 80 µM, 8 µM, or 0.8 µM dynasore, respectively, or treated with 0.1% DMSO as a control. Cells were subsequently infected with HIV-1(VSV-G) in the presence of dynasore for 2 hours. Following infection, cells were washed three times with medium, and then cultured in the absence of dynasore. Viral replication was monitored by flow cytometry analysis of HIV-dependent GFP expression at 48 hours (20,000 cells analyzed per sample). Propidum iodide (PI) was added into the cell suspension prior to flow cytometry. Viable cells were gated (R1) based on low PI staining and cell size (FSC). GFP expression within the viable cell population (R1) was measured. Both the GFP percentage (%) and mean intensity (M) were shown. (B) is an identical experiment using HIV-1_NL4-3 (Wt). For the 80 µM dynasore treatment, another three-independent infections with each virus were performed. The averages from the three-independent experiments are: 15.05%±0.21 (VSV-G), 10.11%±0.06 (VSV-G plus 80 µM dynasore), <i>p</i> = 0.0003; 2.94%±0.39 (Wt), 2.84%±0.30 (Wt plus 80 µM dynasore), <i>p</i> = 0.38.</p

The HIV envelope but not VSV-G is capable of mediating latent infection of resting CD4 T cells.

<p>Resting CD4 T cells were purified from the peripheral blood by negative depletion. Cells were unstimulated (A) or activated with magnetic beads conjugated with antibodies against the human CD3 and CD28 receptors (two beads per cell) for 1 day (B), and then analyzed for cell cycle progression using 7-AAD, PY staining to confirm sufficient T cell activation following stimulation. (C) In CD3/CD28 pre-stimulated CD4 T cells, a higher than Wt level of viral replication was observed in cells infected with HIV-1(VSV-G). One million cells were infected with 25 ng (p24) of HIV-1_NL4-3 (wt) or the VSV-G pseudotyped virus (VSV-G). (D) In resting CD4 T cells that were not pre-stimulated, only the Wt but not the VSV-G pseudotyped HIV-1 replicated following T cell activation at day 5. Cells were infected with 25 ng (p24) of both viruses, incubated for 5 days, and then activated with anti-CD3/CD28 beads. (E) and (F) were a repeat of (C) and (D) in another donor, with AZT (50 µM) added at day 1 and day 5 post infection, respectively, to limit viral replication to a single cycle.</p