Premature Activation of the SLX4 Complex by Vpr Promotes G2/M Arrest and Escape from Innate Immune Sensing

SummaryThe HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity

Editorial: Nucleic Acid-Associated Inflammation.

Editorial on the Research Topic Nucleic Acid-Associated Inflammation

Impact de la protéine Nef du VIH-1 sur le trafic intracellulaire de CD4 et le pouvoir infectieux des particules virales

Les multiples propriétés de la protéine Nef du VIH-1 sont responsables de sa capacité à accélérer la progression vers l'immunodéficience in vivo. Dans ce travail de thèse, nous avons choisi d'explorer les mécanismes qui régissent deux des fonctions les plus conservées dans les isolats primaires de nef: (i) la diminution de l'expression de surface de CD4 dans les cellules cibles du VIH et (ii) l'augmentation du pouvoir infectieux des particules virales néoformées. C'est en interférant avec les éléments de la machinerie de l'endocytose que Nef provoque la diminution de l'expression de surface de CD4, à la fois dans les lignées lymphoïdes et myéloïdes. Cependant, le trafic intracellulaire de CD4 est régit par des règles différentes dans ces cellules: dans les cellules myéloïdes, CD4 est rapidement internalisé, alors que dans les cellules lymphoïdes, CD4 est stabilisé à la surface, notamment grâce à son interaction avec la tyrosine kinase p56lck. Dans cette étude, nous montrons que Nef augmente le taux d'internalisation de CD4 uniquement dans les cellules qui expriment p56lck. Nef utilise donc des mécanismes distincts pour réduire l'expression de surface de CD4 dans les cellules lymphoïdes et myéloïdes. A ce jour il n'y a pas de consensus quant aux contributions respectives des fonctions remplies par Nef pendant la biogénèse virale ou lorsque le virus atteint les cellules cibles, à l'augmentation du pouvoir infectieux. Des protéines de fusion de Nef ont été construites, nous permettant de manipuler les quantités de Nef incorporées dans les particules virales. Cette étude nous a permis d'établir que l'incorporation de Nef dans les particules virales n'est pas suffisante pour induire une augmentation du pouvoir infectieux des particules virales ; le corolaire en est que Nef exerce probablement des fonctions pendant la biogénèse virale, qui sont déterminantes pour les propriétés infectieuses des particules virales.HIV-1 Nef accelerates progression towards immunodeficiency in vivo. During my thesis we explored two of the most conserved functions of Nef: (i) CD4 downregulation in HIV-1 target cells and (ii) viral infectivity enhancement. Interference of Nef with the endocytic machinery causes CD4 cell surface downregulation. However, CD4 trafficking is governed by different rules in the target cells of the infection: in myeloid cells CD4 is rapidly internalized from the cell surface whilst in lymphoid cells, CD4 is stabilized at the cell surface through interaction with the tyrosine kinase p56lck. In this study, we show that Nef increases CD4 internalization rate only in cells that express p56lck. Therefore Nef uses different mechanisms to downregulate CD4 from the cell surface of myeloid and lymphoid cells. To date, the relative contribution of the functions of Nef during viral biogenesis and upon arrival in the target cells to the Nef dependent increase of viral infectivity, have not been deciphered yet. We designed fusion proteins that allowed the manipulation of the amount of Nef in producer cells and incorporated into viral particles. This study allowed us to determine that the incorporation of Nef into viral particles is not sufficient to cause an increase of viral infectivity. Nef must therefore exert functions, during viral biogenesis, that are crucial for the infectivity of nascent viral particles.PARIS5-BU Méd.Cochin (751142101) / SudocSudocFranceF

Manipulation of the host cell by viral auxiliary proteins

Productive HIV infection requires completion of all the steps of the replication cycle, the success of which largely relying on the multiple interactions established by viral proteins with cellular partners. Indeed, cellular and viral fates are intertwined and this interplay may involve rerouting of cellular factors/pathways to the benefit of the viral life cycle. To gain a foothold into host cells, HIV has to take advantage of available cellular factories and overcome the numerous potential blocks opposed to its replication while ensuring cellular survival. Viral auxiliary proteins are a perfect paradigm to illustrate the complexity of the relationship between HIV and its host. Although these accessory proteins are mostly unnecessary for viral replication in permissive cells in vitro, they play a crucial role in regulating viral spread ex vivo in non-permissive cells and in vivo in hosts. Most accessory proteins are pleiotropic and instrumental in the counteraction of restriction factors and proteins involved in innate immune response. Several proteins of the "intrinsic" immune system that detect the presence of the assailant and initiate a subsequent immune response, as well as restriction factors that are directly devoted to arresting the replication cycle at precise steps have been characterized. Despite the numerous cellular mechanisms dedicated to preventing viral replication, HIV is able to efficiently replicate in humans. Indeed, as a master regulator of cellular machineries and processes, not only has HIV evolved strategies to avoid triggering of pattern recognition receptors, but HIV has also elaborated ways to counteract host restriction factors, thereby overcoming the hurdles that oppose efficient replication. This review collection is dedicated to the manipulation of host cells by HIV-1 and HIV-2, with a particular focus on viral accessory proteins

Activation of STING in the pancreatic tumor microenvironment: a novel therapeutic opportunity

International audiencePancreatic ductal adenocarcinoma (PDAC) is a cancer of poor prognosis that presents with a dense desmoplastic stroma that contributes to therapeutic failure. PDAC patients are mostly unresponsive to immunotherapy. However, hopes to elicit response to immunotherapy have emerged with novel strategies targeting the Stimulator of Interferon Genes (STING) protein, which is a major regulator of tumor-associated inflammation. Combination of STING agonists with conventional immunotherapy approaches has proven to potentiate therapeutic benefits in several cancers. However, recent data underscore that the output of STING activation varies depending on the cellular and tissue context. This suggests that tumor heterogeneity, and in particular the heterogeneity of the tumor microenvironment (TME), is a key factor determining whether STING activation would bear benefits for patients. In this review, we discuss the potential benefits of STING activation in PDAC. To this aim, we describe the major components of the PDAC TME, and the expected consequences of STING activation

Animal Models for the Study of Nucleic Acid Immunity: Novel Tools and New Perspectives

International audienceUnresolved inflammation fosters and supports a wide range of human pathologies. There is growing evidence for a role played by cytosolic nucleic acids in initiating and supporting pathological chronic inflammation. In particular, the cGAS-STING pathway has emerged as central to the mounting of nucleic acid-dependent type I interferon responses, leading to the identification of small-molecule modulators of STING that have raised clinical interest. However, several new challenges have emerged, representing potential obstacles to efficient clinical translation. Indeed, the current literature underscores that nucleic acid-induced inflammatory responses are subjected to several layers of regulation, further suggesting complex coordination at the cell-type, tissue or organism level. Untangling the underlying processes is paramount to the identification of specific therapeutic strategies targeting deleterious inflammation. Herein, we present an overview of human pathologies presenting with deregulated interferon levels and with accumulation of cytosolic nucleic acids. We focus on the central role of the STING adaptor protein in these pathologies and discuss how in vivo models have forged our current understanding of nucleic acid immunity. We present our opinion on the advantages and limitations of zebrafish and mice models to highlight their complementarity for the study of inflammatory human pathologies and the development of therapeutics. Finally, we discuss high-throughput screening strategies that generate multi-parametric datasets that allow integrative analysis of heterogeneous information (imaging and omics approaches). These approaches are likely to structure the future of screening strategies for the treatment of human pathologies

Phosphorylation of SAMHD1 by Cyclin A2/CDK1 Regulates Its Restriction Activity toward HIV-1

SAMHD1 restricts HIV-1 replication in myeloid and quiescent CD4+ T cells. Here, we show that SAMHD1 restriction activity is regulated by phosphorylation. SAMHD1 interacts with cyclin A2/cdk1 only in cycling cells. Cyclin A2/CDK1 phosphorylates SAMHD1 at the Threonine 592 residue both in vitro and in vivo. Phosphorylation of SAMHD1 Thr592 correlates with loss of its ability to restrict HIV-1. Indeed, while PMA treatment of proliferating THP1 cells results in reduced Thr592 phosphorylation, activation of resting peripheral blood mononuclear cells (PBMCs) and purified quiescent CD4+ T cells results in increased phosphorylation of SAMHD1 Thr592. Interestingly, we found that treatment of cells by type 1 interferon reduced Thr592 phosphorylation, reinforcing the link between the phosphorylation of SAMHD1 and its antiviral activity. Unlike wild-type SAMHD1, a phosphorylation-defective mutant was able to restrict HIV-1 replication in both PMA-treated and untreated cells. Our results uncover the phosphorylation of SAMHD1 at Thr592 by cyclin A2/CDK1 as a key regulatory mechanism of its antiviral activity