Vpr Promotes Macrophage-Dependent HIV-1 Infection of CD4⁺ T Lymphocytes

<div><p>Vpr is a conserved primate lentiviral protein that promotes infection of T lymphocytes in vivo by an unknown mechanism. Here we demonstrate that Vpr and its cellular co-factor, DCAF1, are necessary for efficient cell-to-cell spread of HIV-1 from macrophages to CD4⁺ T lymphocytes when there is inadequate cell-free virus to support direct T lymphocyte infection. Remarkably, Vpr functioned to counteract a macrophage-specific intrinsic antiviral pathway that targeted Env-containing virions to LAMP1⁺ lysosomal compartments. This restriction of Env also impaired virological synapses formed through interactions between HIV-1 Env on infected macrophages and CD4 on T lymphocytes. Treatment of infected macrophages with exogenous interferon-alpha induced virion degradation and blocked synapse formation, overcoming the effects of Vpr. These results provide a mechanism that helps explain the in vivo requirement for Vpr and suggests that a macrophage-dependent stage of HIV-1 infection drives the evolutionary conservation of Vpr.</p></div

DCAF1 is required for Vpr-dependent HIV-1 spread from macrophages to CD4⁺ T lymphocytes.

<p>(A) Scatter-plot of Vpr-dependent cell cycle arrest in 293T cells (x-axis) versus relative infection frequency of CD4⁺ T lymphocytes co-cultured (“CC”) with infected MDM as outlined in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005054#ppat.1005054.g002" target="_blank">Fig 2A</a>. Infection frequency was assessed by flow cytometry and was normalized to that of wild type (y-axis). Best-fit curve from linear regression analysis, <i>R</i>^<i>2</i> = 0.99 (n = 4 donors). (B) Immunoblot of DCAF1 and GAPDH in MDM seven days after transduction with lentivirus encoding shRNA targeting luciferase (“control”) or DCAF1. (C) Summary graph showing relative infection frequency of T lymphocytes co-cultured (“CC” as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005054#ppat.1005054.g002" target="_blank">Fig 2A</a>) with MDM that had been treated with the indicated shRNA and infected with the indicated HIV-1 89.6 (n = 3 donors). (D) Immunoblot of HIV-1 89.6 Env and Gag in MDM-T lymphocyte co-culture whole-cell lysates diluted as indicated. Arrows denote lysates with comparable levels of Gag pr55 in the presence and absence of Vpr. (E) Summary graph of relative Env levels quantified by densitometry and normalized to Gag pr55 levels and to wild type (n = 4 donors). Error bars represent SEM. *p<0.05, ***p<0.001, “n.s.”p>0.05, student’s paired t-test.</p

Vpr enhances macrophage-dependent infection of CD4⁺ T lymphocytes.

<p>(A) Graphical outline of experimental setup as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005054#ppat.1005054.g001" target="_blank">Fig 1A</a>. (B) Summary graph of quantity of virions released into the supernatant of the indicated cultures after inoculation with wild type or <i>vpr</i>-null HIV-1 89.6 as indicated (n = 5 donors). (C) Summary graph of infected cell frequency in the indicated cultures (n = 11 donors for CD4⁺ T or 17 donors for MDM and CC) as measured by flow cytometry. (D) Diagram illustrating use of HIV-1 NL4-3 pseudotyped with YU-2 Env (red) to infect MDM for a single round and subsequent spread of wild type NL4-3 (blue) to T lymphocytes. (E) Summary graph of relative infected cell frequency in the indicated cell types after addition of HIV-1 YU-2 pseudo-NL4-3 as described in A. Infection frequency was measured by flow cytometry and normalized to infection frequency of wild type HIV-1 in MDM. The color of the X axis label of each summary graph corresponds to the culture condition shown in A, except that for “spin” condition, PHA-activated CD4⁺ primary T lymphocytes were centrifuged for 2500 RPM with 50μg HIV-1 NL4-3 in polybrene (n = 3 donors). Error bars represent SEM. *p<0.05, **p<0.01, ***p<0.001, student’s paired t-test.</p

Efficient HIV-1 infection of T lymphocytes requires contact with infected macrophages.

<p>(A) Graphical outline of experimental setup depicting HIV-1 infection of MDM and co-cultivation with autologous, PHA-activated CD4⁺ T lymphocytes (CC) as detailed in Methods. (B) Summary graph of quantity of virions released into culture supernatant as measured by Gag CAp24 ELISA (n = 5 donors). (C) Summary graph of infected cell frequency in the indicated cultures as measured by flow cytometry (n = 11 donors for CD4⁺ T or 17 donors for MDM and CC). (D) Diagrammatic representation of virus-permeable transwell. (E) Summary graph of relative infected cell frequency in co-cultures prepared as shown in A in the presence or absence of transwell inserts (n = 4 donors). Infection frequency was determined by flow cytometry and values were normalized to MDM infection frequency without transwell insert. (F) Summary graph of relative infected cell frequency, as measured by flow cytometry and normalized to isotype (iso), in the indicated cultures prepared as shown in A. Neutralizing antibodies to HIV-1 Env gp120 (2G12, b12), gp41 (Z13E1) or CD4 (SIM.2) were added at the time of initial infection (MDM) or at the time of CD4⁺ T addition and co-cultivation (CC) at 1 μg/ml (1x) and/or 10 μg/ml (10x), as indicated. Error bars represent SEM. **p<0.01, ***p<0.001, student’s paired t-test. The color of the X axis label of each summary graph corresponds to the culture condition shown in part A.</p

Vpr promotes Env-dependent virological synapse formation between macrophages and CD4⁺ T lymphocytes.

<p>(A) Representative confocal micrographs of MDM infected for seven days and briefly co-cultured with CD4⁺ T lymphocytes pre-stained for surface CD4. Co-localization between HIV-1 Gag MAp17 (red) in MDM and surface CD4 (green) on T lymphocytes is indicated as virological synapses (VS). Merged images include phalloidin staining of actin (magenta) and DAPI staining of nuclei (blue). Inset depicts magnified VS from same image (top) or from a different representative image (bottom). Scale bar (white) represents 10 μm. (B) Summary graph of relative VS observed per ‘n’ number of Gag⁺ MDM from three donors infected by wild type or <i>vpr</i>-null HIV-1 89.6. (C) Summary graph of relative VS per ‘n’ number of Gag⁺ MDM, as in B, of MDM infected with YU-2 Env-pseudotyped <i>env</i>-null 89.6 (third column), wild type 89.6-infected MDM treated for two days prior to co-culture with interferon-α (IFNα, fourth column) or treated with 10 μg/ml (10x) anti-Env gp120 neutralizing antibody b12 during co-cultivation with CD4⁺ T lymphocytes (final column), (D) Immunoblot of DCAF1 and GAPDH in MDM from two donors after silencing of DCAF1 as outlined in Methods. (E) Summary graph of relative VS per ‘n’ number of Gag⁺ MDM, as in B, of MDM treated with control or DCAF1-specific shRNA and infected with the indicated HIV-1 89.6. <i>*</i>p<0.05 **p<0.01 ***p<0.001, Fisher’s exact test.</p

A Mechanistic Study of Tumor-Targeted Corrole Toxicity

HerGa is a self-assembled tumor-targeted particle that bears both tumor detection and elimination activities in a single, two-component complex (Agadjanian et al. <i>Proc. Natl. Acad. Sci. U.S.A.</i> <b>2009</b>, <i>106</i>, 6105–6110). Given its multifunctionality, HerGa (composed of the fluorescent cytotoxic corrole macrocycle, S2Ga, noncovalently bound to the tumor-targeted cell penetration protein, HerPBK10) has the potential for high clinical impact, but its mechanism of cell killing remains to be elucidated, and hence is the focus of the present study. Here we show that HerGa requires HerPBK10-mediated cell entry to induce toxicity. HerGa (but not HerPBK10 or S2Ga alone) induced mitochondrial membrane potential disruption and superoxide elevation, which were both prevented by endosomolytic-deficient mutants, indicating that cytosolic exposure is necessary for corrole-mediated cell death. A novel property discovered here is that corrole fluorescence lifetime acts as a pH indicator, broadcasting the intracellular microenvironmental pH during uptake in live cells. This feature in combination with two-photon imaging shows that HerGa undergoes early endosome escape during uptake, avoiding compartments of pH < 6.5. Cytoskeletal disruption accompanied HerGa-mediated mitochondrial changes whereas oxygen scavenging reduced both events. Paclitaxel treatment indicated that HerGa uptake requires dynamic microtubules. Unexpectedly, low pH is insufficient to induce release of the corrole from HerPBK10. Altogether, these studies identify a mechanistic pathway in which early endosomal escape enables HerGa-induced superoxide generation leading to cytoskeletal and mitochondrial damage, thus triggering downstream cell death