Vpx rescues HIV-1 transduction of dendritic cells from the antiviral state established by type 1 interferon

<p>Abstract</p> <p>Background</p> <p>Vpx is a virion-associated protein encoded by SIV_SM, a lentivirus endemic to the West African sooty mangabey (<it>Cercocebus atys</it>). HIV-2 and SIV_MAC, zoonoses resulting from SIV_SMtransmission to humans or Asian rhesus macaques (<it>Macaca mulatta</it>), also encode Vpx. In myeloid cells, Vpx promotes reverse transcription and transduction by these viruses. This activity correlates with Vpx binding to DCAF1 (VPRBP) and association with the DDB1/RBX1/CUL4A E3 ubiquitin ligase complex. When delivered experimentally to myeloid cells using VSV G-pseudotyped virus-like particles (VLPs), Vpx promotes reverse transcription of retroviruses that do not normally encode Vpx.</p> <p>Results</p> <p>Here we show that Vpx has the extraordinary ability to completely rescue HIV-1 transduction of human monocyte-derived dendritic cells (MDDCs) from the potent antiviral state established by prior treatment with exogenous type 1 interferon (IFN). The magnitude of rescue was up to 1,000-fold, depending on the blood donor, and was also observed after induction of endogenous IFN and IFN-stimulated genes (ISGs) by LPS, poly(I:C), or poly(dA:dT). The effect was relatively specific in that Vpx-associated suppression of soluble IFN-β production, of mRNA levels for ISGs, or of cell surface markers for MDDC differentiation, was not detected. Vpx did not rescue HIV-2 or SIV_MACtransduction from the antiviral state, even in the presence of SIV_MACor HIV-2 VLPs bearing additional Vpx, or in the presence of HIV-1 VLPs bearing all accessory genes. In contrast to the effect of Vpx on transduction of untreated MDDCs, HIV-1 rescue from the antiviral state was not dependent upon Vpx interaction with DCAF1 or on the presence of DCAF1 within the MDDC target cells. Additionally, although Vpx increased the level of HIV-1 reverse transcripts in MDDCs to the same extent whether or not MDDCs were treated with IFN or LPS, Vpx rescued a block specific to the antiviral state that occurred after HIV-1 cDNA penetrated the nucleus.</p> <p>Conclusion</p> <p>Vpx provides a tool for the characterization of a potent, new HIV-1 restriction activity, which acts in the nucleus of type 1 IFN-treated dendritic cells.</p

Polarization of Migrating Monocytic Cells Is Independent of PI 3-Kinase Activity

BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A)-adrenoceptor (alpha(2A)AR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of alpha(2A)AR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity

Cyclosporine A-Sensitive, Cyclophilin B-Dependent Endoplasmic Reticulum-Associated Degradation

Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells

Lv4, an activity that restricts nuclear entry of SIVMAC/SIVSM in human blood cells

SIVSM is a lentivirus endemic to the West African sooty mangabey (Cercocebus atys). HIV-2 and SIVMAC are zoonoses that resulted from SIVSM transmission to humans and Asian rhesus macaques (Macaca mulatto), respectively. Human leukemia cell lines, human peripheral blood mononuclear cells and CD4+ T cells, were 4 to 50-fold less permissive for SIVMAC and SIVSM than for HIV-1. In contrast, SIVMAC transduction of human adherent cell lines was equivalent to that of HIV-1. Consistent with adaptation to human cells, HIV-2 was not restricted as potently as was SIVMAC. SIVMAC transduction of human blood cells was rescued up to the level of HIV-1 by As2O3, a compound that increases the infectivity of viruses in the context of TRIM5-mediated restriction. Nonetheless, efficient knockdown of TRIM5 or cyclophilin A, a cytoplasmic factor that sometimes regulates TRIM5 restriction activity, did not rescue SIVMAC tranduction of these cells. Substitution of HIV-1 CA with the CA from SIVMAC rendered HIV-1 poorly infectious for Jurkat T cells. The block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Heterokaryons resulting from fusion of permissive with restrictive cells exhibited the restrictive phenotype, indicating that SIV transduction of human blood cells is inefficient due to a dominant-acting restriction factor. These results demonstrate that the nucleus of human blood cells possesses a TRIM5-like restriction factor specific for the SIVMAC/SIVSM capsid and that, by extension, cross-species transmission of SIVSM to human cells necessitated adaptation of HIV-2 to this restriction factor

Polarization of migrating monocytic cells is independent of PI 3-Kinase activity

Background: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. Methodology/Principal Findings: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the a2A-adrenoceptor (a2AAR) display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homlogue UK 14’304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microcopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET) of a2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinse activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes – in contrast to neutrophils – rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. Conclusions/Significance: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the attractant cue. Polarized monocytes, which display typical amoeboid like motility, can rapidly develop a new leading edge facing the highest chemoattractant concentration at any site of the plasma membrane, including the uropod. The process appears to be independent of PI 3-kinase activity

Cyclosporine A-sensitive, cyclophilin B-dependent endoplasmic reticulum-associated degradation

Peptidyl-prolyl cis/trans isomerases (PPIs) catalyze cis/trans isomerization of peptide bonds preceding proline residues. The involvement of PPI family members in protein refolding has been established in test tube experiments. Surprisingly, however, no data is available on the involvement of endoplasmic reticulum (ER)-resident members of the PPI family in protein folding, quality control or disposal in the living cell. Here we report that the immunosuppressive drug cyclosporine A (CsA) selectively inhibits the degradation of a subset of misfolded proteins generated in the ER. We identify cyclophilin B (CyPB) as the ER-resident target of CsA that catalytically enhances disposal from the ER of ERAD-LS substrates containing cis proline residues. Our manuscript presents the first evidence for enzymatic involvement of a PPI in protein quality control in the ER of living cells

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to \u3e 100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1 degrees sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor

Comprehensive Identification of Host Modulators of HIV-1 Replication using Multiple Orthologous RNAi Reagents

SummaryRNAi screens have implicated hundreds of host proteins as HIV-1 dependency factors (HDFs). While informative, these early studies overlap poorly due to false positives and false negatives. To ameliorate these issues, we combined information from the existing HDF screens together with new screens performed with multiple orthologous RNAi reagents (MORR). In addition to being traditionally validated, the MORR screens and the historical HDF screens were quantitatively integrated by the adaptation of an established analysis program, RIGER, for the collective interpretation of each gene’s phenotypic significance. False positives were addressed by the removal of poorly expressed candidates through gene expression filtering, as well as with GESS, which identifies off-target effects. This workflow produced a quantitatively integrated network of genes that modulate HIV-1 replication. We further investigated the roles of GOLGI49, SEC13, and COG in HIV-1 replication. Collectively, the MORR-RIGER method minimized the caveats of RNAi screening and improved our understanding of HIV-1–host cell interactions

CEACAM1 regulates TIM-3-mediated tolerance and exhaustion

T-cell immunoglobulin domain and mucin domain-3 (TIM-3, also known as HAVCR2) is an activation-induced inhibitory molecule involved in tolerance and shown to induce T-cell exhaustion in chronic viral infection and cancers[superscript 1, 2, 3, 4, 5]. Under some conditions, TIM-3 expression has also been shown to be stimulatory. Considering that TIM-3, like cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death 1 (PD-1), is being targeted for cancer immunotherapy, it is important to identify the circumstances under which TIM-3 can inhibit and activate T-cell responses. Here we show that TIM-3 is co-expressed and forms a heterodimer with carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1), another well-known molecule expressed on activated T cells and involved in T-cell inhibition[superscript 6, 7, 8, 9, 10]. Biochemical, biophysical and X-ray crystallography studies show that the membrane-distal immunoglobulin-variable (IgV)-like amino-terminal domain of each is crucial to these interactions. The presence of CEACAM1 endows TIM-3 with inhibitory function. CEACAM1 facilitates the maturation and cell surface expression of TIM-3 by forming a heterodimeric interaction in cis through the highly related membrane-distal N-terminal domains of each molecule. CEACAM1 and TIM-3 also bind in trans through their N-terminal domains. Both cis and trans interactions between CEACAM1 and TIM-3 determine the tolerance-inducing function of TIM-3. In a mouse adoptive transfer colitis model, CEACAM1-deficient T cells are hyper-inflammatory with reduced cell surface expression of TIM-3 and regulatory cytokines, and this is restored by T-cell-specific CEACAM1 expression. During chronic viral infection and in a tumour environment, CEACAM1 and TIM-3 mark exhausted T cells. Co-blockade of CEACAM1 and TIM-3 leads to enhancement of anti-tumour immune responses with improved elimination of tumours in mouse colorectal cancer models. Thus, CEACAM1 serves as a heterophilic ligand for TIM-3 that is required for its ability to mediate T-cell inhibition, and this interaction has a crucial role in regulating autoimmunity and anti-tumour immunity.American Association for Cancer Research. Pancreatic Cancer Action Networ