Replication of recombinant HIV-1 encoding EGFP or DsRed.

<p>(A) Concentration of X4 HIV-1 (HIV_NL-432), X4 HIV-1 expressing EGFP (HIV_NL-E), X4 HIV-1 expressing DsRed (HIV_NL-D), or R5HIV-1 expressing DsRed (HIV_NLAD8-D) in PHA-stimulated PBMCs of two donors. Virus production was monitored by in-house p24 antigen ELISA. (B) FACS analysis of HIV-1-infected T cells expressing EGFP or DsRed at 7 dpi.</p

Genomic structure and co-receptor usage of recombinant HIV-1 encoding EGFP or DsRed.

<p>(A) The structure of provirus DNA encoding <i>EGFP</i> or <i>DsRed</i> designated as pNL-E and pNL-D for X4 HIV-1 and pNLAD8-D for R5 HIV-1. EGFP or DsRed is not expressed as a fusion protein of Env because of one base insertion after the Env stop codon. Nef is also independently expressed under the control of IRES. To confirm the coreceptor usage of these fluorescent HIV-1, 1G5 (B) cells, 1G5/CCR5 (C) cells were infected with HIV-1_NL432 (parent strain), HIV-1_NL-E, HIV-1_NL-D, or HIV-1_NLAD8-D. After 48 h post-infection, cell lysates were prepared and the Luc assay was performed. The data represents the averages ±SD of three independent experiments.</p

FACS-based analysis of dual infection in primary CD4⁺ T cells.

<p>(A) FACS analysis of primary CD4⁺ T cells (1) unstimulated, (2) stimulated with 5 µg/ml anti-CD3 and 10 µg/ml anti-CD28 for 24 h (strong activation), (3) stimulated with the same concentrations of anti-CD3 and anti-CD28 for 2 h (medium activation), or (4) stimulated with 10-fold lower concentrations of anti-CD3 and anti-CD28 for 2 h (weak activation). Cells were then infected with equal amounts of X4 and R5 HIV-1 and analyzed at 5 dpi. (B) Quantitative PCR analysis of CD4⁺T cells separately infected with either R5 or X4 HIV-1. R-U5 and U5-Gag was analyzed by qPCR in two donors. The amount of HIV-1–specific DNA per cell was normalized to β-globin gene expression. The data represents the average ±SD of three independent experiments. **, <i>P</i><0.005; ***, <i>P</i><0.0005.</p

Infectivity of HIV-1 in MDDCs.

<p>(A) Surface expression of CCR5 and CXCR4 in MDDCs. MDDCs were stained with anti-CCR5 (left) and anti-CXCR4 mAb (right), or with isotype control mAbs (dotted line). The reproducible representative of the FACS profiles of several individuals is depicted. (B) Quantitative PCR analysis of R5 (HIV_NLAD8-D) and X4 (HIV_NL-E) HIV-1-infected MDDCs. Data represent the average ±SD of three independent experiments.</p

Analysis of CD4⁺ T cell activation.

<p>(A) Primary CD4⁺ T cells were stimulated with anti-CD3 (5 µg/ml)+anti-CD28 (10 µg/ml) (black bold line), anti-CD3 (0.5 µg/ml)+anti-CD28 (1 µg/ml) (black line), or cocultured with allogeneic CD4⁺ T cells for 2 h (gray line). Isotype control mAbs were used as a negative staining control (shaded peak). (B) One-step qRT-PCR analysis of IFN-γ mRNA expression in primary CD4⁺ T cells. Primary CD4⁺ T cells were cocultured with allogeneic CD4⁺ T cells for 2 or 24 h and then stimulated with anti-CD3 (5 µg/ml)+anti-CD28 (10 µg/ml) (IL-2 strong), anti-CD3 (0.5 µg/ml)+anti CD28 (1 µg/ml) (IL-2 weak), or no TCR stimulation (IL-2) for 2 h. Total RNA was extracted and analyzed by qRT-PCR. The data was normalized to EF-1α and the relative amount of IFN-γ is shown. The data represents the average ±SD of three independent experiments.</p