Restricted Access to Myeloid Cells Explained

The lentiviral accessory protein, Vpx, is known to counteract a restriction factor that is specific to myeloid cells, such as macrophages and dendritic cells. This review summarizes the findings in two seminal studies that identify SAMHD1 as the cellular protein that is responsible for myeloid cell restriction, and establish the existence of other types of restriction in these cells

An HIV-encoded antisense long non-coding RNA epigenetically regulates viral transcription

pre-printThe abundance of long non-coding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus (HIV) encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this HIV-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5'LTR and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating HIV gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter

Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation

pre-printDespite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1 infected T cells is altered relative to uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28 responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host-cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation

Method for obtaining an enriched population of sirna-expressing cells

ReportProblems with transience of siRNA-mediated knock-down and transfection efficiency have limited the scope of RNAi-based experiments. The invention provides a tool for employing RNAi more efficiently and effectively by integrating RNAi expression with methods of cell enrichment

Differential effects of Vpr on single-cycle and spreading HIV-1 infections in CD4 + T-cells and dendritic cells

Journal ArticleThe Vpr protein of human immunodeficiency virus type 1 (HIV-1) contributes to viral replication in non-dividing cells, specifically those of the myeloid lineage. However, the effects of Vpr in enhancing HIV-1 infection in dendritic cells have not been extensively investigated. Here, we evaluated the role of Vpr during infection of highly permissive peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells and compared it to that of monocyte-derived dendritic cells (MDDCs), which are less susceptible to HIV-1 infection. Infections of dividing PBMCs and non-dividing MDDCs were carried out with singlecycle and replication-competent HIV-1 encoding intact Vpr or Vpr-defective mutants. In contrast to previous findings, we observed that single-cycle HIV-1 infection of both PBMCs and MDDCs was significantly enhanced in the presence of Vpr when the viral stocks were carefully characterized and titrated. HIV-1 DNA quantification revealed that Vpr only enhanced the reverse transcription and nuclear import processes in single-cycle HIV-1 infected MDDCs, but not in CD4+ T-cells. However, a significant enhancement in HIV-1 gag mRNA expression was observed in both CD4+ T-cells and MDDCs in the presence of Vpr. Furthermore, Vpr complementation into HIV-1 virions did not affect single-cycle viral infection of MDDCs, suggesting that newly synthesized Vpr plays a significant role to facilitate single-cycle HIV-1 infection. Over the course of a spreading infection, Vpr significantly enhanced replication-competent HIV-1 infection in MDDCs, while it modestly promoted viral infection in activated PBMCs. Quantification of viral DNA in replication-competent HIV-1 infected PBMCs and MDDCs revealed similar levels of reverse transcription products, but increased nuclear import in the presence of Vpr independent of the cell types. Taken together, our results suggest that Vpr has differential effects on single-cycle and spreading HIV-1 infections, which are dependent on the permissiveness of the target cell

Host factors influencing susceptibility to HIV infection and AIDS progression

Transmission of HIV first results in an acute infection, followed by an apparently asymptomatic period that averages ten years. In the absence of antiretroviral treatment, most patients progress into a generalized immune dysfunction that culminates in death. The length of the asymptomatic period varies, and in rare cases infected individuals never progress to AIDS. Other individuals whose behavioral traits put them at high-risk of HIV transmission, surprisingly appear resistant and never succumb to infection. These unique cases highlight the fact that susceptibility to HIV infection and progression to disease are complex traits modulated by environmental and genetic factors. Recent evidence has indicated that natural variations in host genes can influence the outcome of HIV infection and its transmission. In this review we summarize the available literature on the roles of cellular factors and their genetic variation in modulating HIV infection and disease progression

Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP

Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy

<p>Abstract</p> <p>Background</p> <p>Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their <it>in vivo </it>role as long-lived HIV-1 reservoirs.</p> <p>Results</p> <p>Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat – a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.</p> <p>Conclusion</p> <p>Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.</p

HIV-1 Accessory Proteins Impart a Modest Interferon Response and Upregulate Cell Cycle-Related Genes in Macrophages

HIV-1 infection of myeloid cells is associated with the induction of an IFN response. How HIV-1 manipulates and subverts the IFN response is of key interest for the design of therapeutics to improve immune function and mitigate immune dysregulation in people living with HIV. HIV-1 accessory genes function to improve viral fitness by altering host pathways in ways that enable transmission to occur without interference from the immune response. We previously described changes in transcriptomes from HIV-1 infected and from IFN-stimulated macrophages and noted that transcription of IFN-regulated genes and genes related to cell cycle processes were upregulated during HIV-1 infection. In the present study, we sought to define the roles of individual viral accessory genes in upregulation of IFN-regulated and cell cycle-related genes using RNA sequencing. We observed that Vif induces a set of genes involved in mitotic processes and that these genes are potently downregulated upon stimulation with type-I and -II IFNs. Vpr also upregulated cell cycle-related genes and was largely responsible for inducing an attenuated IFN response. We note that the induced IFN response most closely resembled a type-III IFN response. Vpu and Nef-regulated smaller sets of genes whose transcriptomic signatures upon infection related to cytokine and chemokine processes. This work provides more insight regarding processes that are manipulated by HIV-1 accessory proteins at the transcriptional level.This research was partially funded by the National Institutes of Health, grants AI143567-02 (V.P. and M.C.) and AI122377-05 (V.P.) and by startup funds from the Department of Pathology, University of Utah School of Medicine (T.M.H.).S

Downmodulation of CCR7 by HIV-1 Vpu Results in Impaired Migration and Chemotactic Signaling within CD4+ T Cells

SummaryThe chemokine receptor CCR7 plays a crucial role in the homing of central memory and naive T cells to peripheral lymphoid organs. Here, we show that the HIV-1 accessory protein Vpu downregulates CCR7 on the surface of CD4+ T cells. Vpu and CCR7 were found to specifically interact and colocalize within the trans-Golgi network, where CCR7 is retained. Downmodulation of CCR7 did not involve degradation or endocytosis and was strictly dependent on Vpu expression. Stimulation of HIV-1-infected primary CD4+ T cells with the CCR7 ligand CCL19 resulted in reduced mobilization of Ca2+, reduced phosphorylation of Erk1/2, and impaired migration toward CCL19. Specific amino acid residues within the transmembrane domain of Vpu that were previously shown to be critical for BST-2 downmodulation (A14, A18, and W22) were also necessary for CCR7 downregulation. These results suggest that BST-2 and CCR7 may be downregulated via similar mechanisms