Investigating Mechanisms of Immune Evasion by HIV-1 Vpr.

Human immunodeficiency virus (HIV) causes significant morbidity and mortality worldwide, yet several HIV proteins have not been fully characterized. The development of cell culture models to study retroviruses has accelerated discovery of basic steps in the HIV lifecycle. Examination of the HIV proteins reverse transcriptase, integrase, protease, Gag and Env in cell lines has led to life-saving pharmacological therapies. In contrast, lentiviral accessory proteins such as viral infectivity factor (Vif) and viral protein R (Vpr) are not yet targeted therapeutically, and are not required for replication in many cell lines. However, accessory proteins are required for HIV to counteract the innate immune system in vivo. Vpr is conserved among lentiviruses, but its function is unknown. In this dissertation, we examine the role of Vpr during HIV-1 infection of primary macrophages, primary CD4+ T cells and cell lines. We report that uracilation of HIV DNA by the host cytidine deaminase APOBEC3G (A3G) leads to upregulation of natural killer (NK) cell-activating ligands in CD4+ T cells. HIV limits NK cell-activating ligand upregulation by targeting A3G for degradation through Vif. Additionally, we propose that Vpr counteracts uracilation of HIV DNA by recruiting the host uracil DNA glycosylase UNG2. We also explore the ability of Vpr to suppress the antiviral response to HIV, and identify novel restrictions to HIV infection in primary macrophages counteracted by Vpr. Vpr enhances the spread of HIV-1 in macrophages by increasing virus production per infected cell. Surprisingly, Vpr also increases the intracellular level and virion incorporation of Env by preventing Env lysosomal degradation. Importantly, these novel Vpr activities are dependent on the DCAF1-DDB1-Cul4 E3 ubiquitin ligase complex. We also identify novel cell culture conditions that increase the requirement for Vpr to infect macrophages with HIV. Mechanisms of action of eight of the nine HIV-1 genes have been determined. However, vpr is unique because its function has not yet been characterized. This dissertation describes a systematic study of the mechanism through which Vpr enhances HIV replication in primary macrophages. Mechanistic insight into the function of Vpr in HIV-infected macrophages may lead to novel strategies to cure HIV.PHDImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/120706/1/mmashiba_1.pd

Fracture Callus Under Anti-resorptive Agent Treatment Evaluated by pQCT

Abstract Effects of estrogen, raloxifene, and alendronate on fracture healing were evaluated by a peripheral quantitative computed tomography (pQCT) in an osteoporotic fracture rat model. Three-month-old ovariectomized (OVX; except sham-operated controls) Sprague-Dawley rats were pretreated with vehicle (sham and OVX controls), 0.1mg kg day −1 estrogen (α-ethynyl estradiol), 1mg kg day −1 raloxifene, or 0.01mg kg day −1 alendronate for weeks before fracture induction. At this point, the pre-fracture groups were killed while transverse osteotomy was performed at the midshaft of both femora in the remaining animals and kept for weeks with drug treatment, and then killed weeks after fracture induction. Excised femora and fracture calluses were analyzed by high-resolution pQCT. At weeks after fracture, the alendronate and OVX groups showed larger calluses at a larger cross-sectional moment of inertia (CSMI) than that of other groups. At weeks after fracture, the calluses in OVX rats were significantly smaller than those observed at weeks, whereas the calluses treated with alendronate did not change in size; therefore, calluses in OVX rats without drug treatment remodeled towards the original geometry in the femoral midshaft faster than drug-treated rats, and on the contrary, the fracture calluses in rats treated with alendronate were the slowest. In conclusion, OVX-induced higher bone turnover and resulted in the fastest remodeling of fracture callus, which was, however, delayed under alendronate treatment. Estrogen and raloxifene treatment showed intermediate callus remodeling between OVX and sham

The Adenoviral E1B 55-Kilodalton Protein Controls Expression of Immune Response Genes but Not p53-Dependent Transcription▿

The human adenovirus type 5 (Ad5) E1B 55-kDa protein modulates several cellular processes, including activation of the tumor suppressor p53. Binding of the E1B protein to the activation domain of p53 inhibits p53-dependent transcription. This activity has been correlated with the transforming activity of the E1B protein, but its contribution to viral replication is not well understood. To address this issue, we used microarray hybridization methods to examine cellular gene expression in normal human fibroblasts (HFFs) infected by Ad5, the E1B 55-kDa-protein-null mutant Hr6, or a mutant carrying substitutions that impair repression of p53-dependent transcription. Comparison of the changes in cellular gene expression observed in these and our previous experiments (D. L. Miller et al., Genome Biol. 8:R58, 2007) by significance analysis of microarrays indicated excellent reproducibility. Furthermore, we again observed that Ad5 infection led to efficient reversal of the p53-dependent transcriptional program. As this same response was also induced in cells infected by the two mutants, we conclude that the E1B 55-kDa protein is not necessary to block activation of p53 in Ad5-infected cells. However, groups of cellular genes that were altered in expression specifically in the absence of the E1B protein were identified by consensus k-means clustering of the hybridization data. Statistical analysis of the enrichment of genes associated with specific functions in these clusters established that the E1B 55-kDa protein is necessary for repression of genes encoding proteins that mediate antiviral and immune defenses

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

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

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

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