Host Proteins Interact with the Hiv-1 Core to Facilitate and Restrict

Host cell proteins, termed restriction factors, which inhibit viral replication at various stages of the viral life cycle, determine the species-specific tropism of numerous retroviruses. Many members of the TRIM family of proteins act as viral restriction factors. One well-characterized example is the ability of TRIM5á from rhesus macaques (rhTRIM5á) to inhibit human immunodeficiency virus type-1 (HIV-1) soon after viral entry but prior to reverse transcription (RT). It is well established that the restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5á. Following the binding of the viral core, TRIM5á mediates an event or series of events that result in the abortive disassembly of the viral core in a manner that prevents the accumulation of reverse transcription (RT) products. When the proteasome was inhibited using pharmacological drugs, TRIM5á-mediated inhibition of RT products and abortive disassembly of the viral core were relieved without affecting the ability of TRIM5á to inhibit retroviral infection. Even though parts of the mechanism of TRIM5á-mediated restriction of HIV-1 were identified, the specific roles of individual molecules have yet to be examined. In AIM 1 I identify a direct interaction between TRIM5á and the proteasome complex. Furthermore, this interaction occurs during restriction of HIV-1. Additionally, in AIM 2 I demonstrate that SUMO-1 and SUMO interacting motifs (SIMs) are important for TRIM5á restriction of HIV-1 and TRIM5á stability. As mentioned before, the viral capsid is the determinant of TRIM5-mediated restriction. The capsid houses the viral RNA and other necessary proteins for a productive infection. However, the precise process of HIV-1 uncoating is still unknown. Studies suggest that the process of uncoating is modulated by viral and cellular factors. Previously, HIV-1 was shown to traffic on microtubules, in a dynein and kinesin dependent mechanism. However, key host proteins that mediate uncoating of the core are unknown. In AIM 3 I show that HIV-1 utilizes microtubules, and in particular dynein to facilitate uncoating of the core. This dissertation further establishes that HIV-1 core interacts with various proteins in the host during early events of the viral life cycle

Role of SUMO-1 and SUMO Interacting Motifs in Rhesus TRIM5α-mediated Restriction

Background TRIM5α is a member of the tripartite motif family of proteins that restricts retroviral infection in a species-specific manner. The restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5α. Previously, we determined that two SUMO interacting motifs (SIMs) present in the B30.2/SPRY domain of human TRIM5α (huTRIM5α) were important for the restriction of N-tropic Murine Leukemia Virus. Here, we examined whether SUMO expression and the SIM1 and SIM2 motifs in rhesus monkey TRIM5α (rhTRIM5α) are similarly important for Human Immunodeficiency Type 1 (HIV-) restriction. Results We found that mutation of SIM1 and SIM2 of rhTRIM5α abolished the restriction of HIV-1 virus. Further, knockdown of SUMO-1 in rhTRIM5α expressing cells abolished restriction of HIV-1. These results may be due, in part, to the ability of SUMO-1 to stabilize rhTRIM5α protein expression, as SUMO-1 knockdown increased rhTRIM5α turnover and the mutations in SIM1 and SIM2 led to more rapid degradation than the wild type protein. The NF-κB signaling ability of rhTRIM5α was also attenuated by SUMO-1 knockdown. Finally, upon inhibition of CRM1-dependent nuclear export with Leptomycin B (LMB), wild type rhTRIM5α localized to SUMO-1 bodies in the nucleus, while the SIM1 and SIM2 mutants did not localize to SUMO-1. Conclusions Our results suggest that the rhTRIM5α B30.2/SPRY domain is not only important for the recognition of the HIV-1 CA, but it is also important for its association with SUMO-1 or SUMO-1 modified proteins. These interactions help to maintain TRIM5α protein levels and its nuclear localization into specific nuclear bodies

TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions

<p>Abstract</p> <p>Background</p> <p>The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues.</p> <p>Results</p> <p>The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions.</p> <p>Conclusions</p> <p>Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.</p

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

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

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

Mesenchymal stem cells attenuate acute liver injury by altering ratio between interleukin 17 producing and regulatory natural killer T cells.

Mesenchymal stem cells (MSCs) are, due to immunomodulatory characteristics, considered as novel agents in the treatment of immune-mediated acute liver failure. Although it is known that MSCs can regulate activation of T lymphocytes, their capacity to modulate function of neutrophils and natural killer T (NKT) cells, major interleukin (IL) 17-producing cells in acute liver injury, is still unknown. By using 2 well-established murine models of neutrophil and NKT cell-mediated acute liver failure (induced by carbon tetrachloride and α-galactoceramide), we investigated molecular and cellular mechanisms involved in MSC-mediated modulation of IL17 signaling during acute liver injury. Single intravenous injection of MSCs attenuate acute hepatitis and hepatotoxicity of NKT cells in a paracrine, indoleamine 2,3-dioxygenase (IDO)-dependent manner. Decreased levels of inflammatory IL17 and increased levels of immunosuppressive IL10 in serum, reduced number of interleukin 17-producing natural killer T (NKT17) cells, and increased presence of forkhead box P3 + IL10-producing natural killer T regulatory cells (NKTregs) were noticed in the injured livers of MSC-treated mice. MSCs did not significantly alter the total number of IL17-producing neutrophils, CD4+, and CD8 + T lymphocytes in the injured livers. Injection of mesenchymal stem cell-conditioned medium (MSC-CM) resulted with an increased NKTreg/NKT17 ratio in the liver and attenuated hepatitis in vivo and significantly reduced hepatotoxicity of NKT cells in vitro. This phenomenon was completely abrogated in the presence of IDO inhibitor, 1-methyltryptophan. In conclusion, the capacity of MSCs to alter NKT17/NKTreg ratio and suppress hepatotoxicity of NKT cells in an IDO-dependent manner may be used as a new therapeutic approach in IL17-driven liver inflammation. Liver Transplantation 23 1040-1050 2017 AASLD