Intrarectal transmission, systemic infection, and CD4+ T cell depletion in humanized mice infected with HIV-1

Intrarectal infection between men who have sex with men represents a predominant form of human immunodeficiency virus (HIV) transmission in developed countries. Currently there are no adequate small animal models that recapitulate intrarectal HIV transmission. Here we demonstrate that human lymphocytes generated in situ from hematopoietic stem cells reconstitute the gastrointestinal tract of humanized mice with human CD4+ T cells rendering them susceptible to intrarectal HIV transmission. HIV infection after a single intrarectal inoculation results in systemic infection with depletion of CD4+ T cells in gut-associated lymphoid tissue and other pathologic sequela that closely mimics those observed in HIV infected humans. This novel model provides the basis for the development and evaluation of novel approaches aimed at immune reconstitution of human gut-associated lymphoid tissue and for the development, testing, and implementation of microbicides to prevent intrarectal HIV-1 transmission

A Hydrophobic Binding Surface on the Human Immunodeficiency Virus Type 1 Nef Core Is Critical for Association with p21-Activated Kinase 2

The interaction of human immunodeficiency virus type 1 (HIV-1) Nef with p21-activated kinase 2 (Pak2) has been proposed to play an important role in T-cell activation and disease progression during viral infection. However, the mechanism by which Nef activates Pak2 is poorly understood. Mutations in most Nef motifs previously reported to be required for Pak2 activation (G(2), PxxP(72), and RR(105)) also affect other Nef functions, such as CD4 or major histocompatibility complex class I (MHC-I) downregulation. To better understand Nef interactions with Pak2, we performed mutational analysis of three primary HIV-1 Nef clones that exhibited similar capacities for downregulation of CD4 and MHC-I but variable abilities to associate with activated Pak2. Our results demonstrate that Nef amino acids at positions 85, 89, 187, 188, and 191 (L, H, S, R, and F in the clade B consensus, respectively) are critical for Pak2 association. Mutation of these Nef residues dramatically altered association with Pak2 without affecting Nef expression levels or CD4 and MHC-I downregulation. Furthermore, compensation occurred at positions 89 and 191 when both amino acids were substituted. Since residues 85, 89, 187, 188, and 191 cluster on the surface of the Nef core domain in a region distinct from the dimerization and SH3-binding domains, we propose that these Nef residues form part of a unique binding surface specifically involved in association with Pak2. This binding surface includes exposed and recessed hydrophobic residues and may participate in an as-yet-unidentified protein-protein interaction to facilitate Pak2 activation

Inhibition of Endosomal/Lysosomal Degradation Increases the Infectivity of Human Immunodeficiency Virus

Productive entry of human immunodeficiency virus type 1 (HIV-1) into a host cell is believed to proceed via fusion of the viral envelope with the host cell's plasma membrane. Interestingly, the majority of HIV-1 particles that bind to the cell surface are taken up by the host cell via endocytosis; however, this mode of internalization generally does not result in infection. Presumably, virus particles remain trapped in the endocytic pathway and are eventually degraded. Here, we demonstrate that treatment of cells with various pharmacological agents known to elevate the pH of endosomes and lysosomes allows HIV-1 to efficiently enter and infect the host cell. Pretreatment of cells with bafilomycin A1 results in up to a 50-fold increase in the infectivity of HIV-1(SF2). Similarly, pretreatment of target cells with amantadine, concanamycin A, concanamycin B, chloroquine, and ammonium chloride resulted in increases in HIV-1 infectivity ranging between 2- and 15-fold. Analysis of receptor and coreceptor expression, HIV-long terminal repeat (LTR) transactivation, and transduction with amphotropic-pseudotyped murine leukemia virus (MLV)-based vectors suggests that the increase in infectivity is not artifactual. The increased infectivity under these conditions appears to be due to the ability of HIV-1 and MLV particles to enter via the endocytic pathway when spared from degradation in the late endosomes and lysosomes. These results could have significant implications for the administration of current and future lysosmotropic agents to patients with HIV disease

Inhibition of Lysosome and Proteasome Function Enhances Human Immunodeficiency Virus Type 1 Infection

We previously reported that inhibition of endosomal/lysosomal function can dramatically enhance human immunodeficiency virus type 1 (HIV-1) infectivity, suggesting that under these conditions productive HIV-1 infection can occur via the endocytic pathway. Here we further examined this effect with bafilomycin A1 (BFLA-1) and show that this enhancement of infectivity extends to all HIV-1 isolates tested regardless of coreceptor usage. However, isolate-specific differences were observed in the magnitude of the effect. This was particularly evident in the case of the weakly infectious HIV-1(SF2), for which we observed the greatest enhancement. Using reciprocal chimeric viruses, we were able to determine that both the disproportionate increase in the infectivity of HIV-1(SF2) in response to BFLA-1 and its weak infectivity in the absence of BFLA-1 mapped to its envelope gene. Further, we found HIV-1(SF2) to have lower fusion activity and to be 12-fold more sensitive to the fusion inhibitor T-20 than HIV-1(NL4-3). Proteasomal inhibitors also enhance HIV-1 infectivity, and we report that the combination of a lysosomal and a proteasomal inhibitor greatly enhanced infectivity of all isolates tested. Again, HIV-1(SF2) was unique in exhibiting a synergistic 400-fold increase in infectivity. We also determined that inhibition of proteasomal function increased the infectivity of HIV-1 pseudotyped with vesicular stomatitis virus G protein. The evidence presented here highlights the important role of the lysosomes/proteasomes in the destruction of infectious HIV-1(SF2) and could have implications for the development of novel antiviral agents that might take advantage of these innate defenses

Activation of p21-Activated Kinase 2 by Human Immunodeficiency Virus Type 1 Nef Induces Merlin Phosphorylation

The accessory human immunodeficiency virus type 1 (HIV-1) protein Nef activates the autophosphorylation activity of p21-activated kinase 2 (PAK2). Merlin, a cellular substrate of PAK2, is homologous to the ezrin-radixin-moesin family and plays a critical role in Rac signaling. To assess the possible impact on host cell metabolism of Nef-induced PAK2 activation, we investigated the phosphorylation of merlin in Nef expressing cells. Here we report that Nef induces merlin phosphorylation in multiple cell lines independently of protein kinase A. This intracellular phosphorylation of merlin directly correlates with in vitro assay of the autophosphorylation activity of Nef-activated PAK2. Importantly, merlin phosphorylation induced by Nef was also observed in human primary T cells. The finding that Nef induces phosphorylation of the key signaling molecule merlin suggests several possible roles for PAK2 activation in HIV pathogenesis

Reconstitution of the Female Reproductive Tract of Humanized BLT Mice with Human Hematopoietic Cells

<p>Immunohistochemical analysis of the vagina, ectocervix, endocervix, and uterus of female BLT mice for the presence of human hematopoietic lineages (brown cells) (bars indicate 25 μm). Robust reconstitution with cells relevant to HIV-1 infection, including human T cells, monocyte/macrophages, and dendritic cells, was observed in each compartment of the FRT of humanized BLT mice, demonstrating the efficient repopulation of these important mucosal sites.</p

Changes in CD4⁺CCR5⁺ and CD8⁺CCR5⁺ Human T Cell Levels Resulting from HIV-1 Infection

<div><p>(A) Comparison of the levels of human CD4⁺CCR5⁺ T cells in the indicated tissues in a representative naive BLT mouse, an HIV-1–infected, and an HIV-1–exposed BLT mouse that received FTC/TDF for pre-exposure prophylaxis. Liver and lung were the examined tissues with the greatest constitutive CCR5 expression, and they both showed significant loss of CD4⁺CCR5⁺ T cells due to HIV-1 infection.</p> <p>(B) Box plot depicting the levels of CD8⁺CCR5⁺ T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice.</p> <p>(C) Comparison of the levels of human CD8⁺CCR5⁺ T cells in the indicated tissues in representative naive, HIV-1 infected and FTC/TDF treated BLT mice. All tissues examined showed increases in CD8⁺CCR5⁺ T cells resulting from HIV-1 infection of BLT mice.</p> <p>(D) Box plot depicting the levels of CD8⁺CCR5⁺ T cells in the indicated tissues for naive (green), HIV-1–infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In the box plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Naive (<i>n</i> = 5), HIV-1 infected (<i>n</i> = 4), and FTC/TDF + HIV-1 (<i>n</i> = 3). Gating strategy for flow cytometric analysis: live cells → human CD45 → human CD3 → human CD4 or CD8 → CCR5.</p> <p>BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid.</p></div

Systemic CD4⁺ T Cell Loss Resulting from Intravaginal HIV-1 Infection in Humanized BLT Mice

<div><p>(A) Comparison of the levels of CD4⁺ or CD8⁺ human T cells in the indicated tissues in representative BLT mice that were either naive, HIV-1 infected, or that received FTC/TDF for pre-exposure prophylaxis prior to exposure to HIV-1. Note the HIV-1 induced reduction in the double-positive CD4⁺CD8⁺ thymocytes.</p> <p>(B and C) Box plots depicting the levels of CD4⁺ (B) or CD8⁺ (C) T cells in the indicated tissues for naive (green), HIV-1 infected (white), and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. In these plots, the boxes extend from the first to the third quartiles, enclosing the middle 50% of the data. The middle line within each box indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Data from naive, HIV-1-, or FTC/TDF-treated plus HIV-1–exposed mice were not collected on the same day. Naive (<i>n</i> = 5), HIV-1 infected (<i>n</i> = 4), and FTC/TDF + HIV-1 (<i>n</i> = 3). Flow cytometry gating for this figure was performed as described for <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050016#pmed-0050016-g002" target="_blank">Figure 2</a>.</p> <p>BM, bone marrow; LN, lymph node; PB, peripheral blood; Thymic Org., implanted thymic organoid.</p></div

Pre-exposure FTC/TDF Treatment Resulted in Complete Protection of Humanized BLT Mice from Intravaginal HIV-1 Transmission

<div><p>(A) Box plot depicting real-time PCR levels of HIV-1 viral DNA in the indicated tissues for HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. (Viral DNA copies per million CD4⁺ T cells shown.) HIV-1 infected (<i>n</i> = 2) and FTC/TDF + HIV-1 (<i>n</i> = 4).</p> <p>(B) Box plot depicting virus rescue results from HIV-1–infected (white) and FTC/TDF-treated plus HIV-1–exposed (red) BLT mice. Virus rescue data expressed as pg/ml of p24 per 1 × 10⁵ CD4⁺ T cells cocultured with PHA/IL2-activated peripheral blood lymphocyte from a seronegative donor. HIV-1 infected (<i>n</i> = 2) and FTC/TDF + HIV-1 (<i>n</i> = 4). In the box plots, the middle line indicates the median of the data, whereas the vertical line extends from lowest to the highest values. Dashed lines indicate the limit of detection for each assay.</p> <p>(C) In situ hybridization analysis to determine the presence of productively HIV-1–infected cells in the indicated tissues from HIV-1–infected or FTC/TDF-treated BLT mice (bars indicate 50 μm). Note the lack of HIV-1 in the BLT mice that received pre-exposure prophylaxis with FTC/TDF. HIV-1 infected (<i>n</i> = 4) and FTC/TDF + HIV-1 (<i>n</i> = 3).</p> <p>BM, bone marrow; LPL, lamina propria lymphocytes; Thymic Org., implanted thymic organoid; SI, small intestine.</p></div