Characterisation of Lentiviral Vpr Function and Mechanism

Genetic conflict between viruses and their hosts has driven an ‘arms race’, forcing the evolution of both immune defencesdefenses and multiple viral strategies to counteract and evade them. In the case of HIV many of these processes are well characterised – the virus carries with it a set of accessory proteins which target specific host restriction factors. Of these accessory proteins Vpr is the least well understood, with no described role that adequately explains its conservation across all known primate lentiviruses. Unpublished data from our lab indicate that Vpr is able to rescue infection in macrophages from addition of cGAMP, a second messenger protein produced by the cytosolic DNA sensor cGAS which activates antiviral immune signalling pathways. This study first sought to test the hypothesis that Vpr has evolved to counteract cGAS/STING mediated cytosolic DNA sensing using a co-transfection assay to test Vpr proteins from all groups of primate lentiviruses. Initial observations appeared to demonstrate specific degradation of innate immune signalling proteins. It was subsequently shown that HIV-1 M Vpr antagonises expression from all tested co-transfected plasmids. This phenotype was demonstrated to be species specific, and to correlate with both the history of zoonotic transmission and localisation of Vpr to the nuclear rim. Additionally, it was shown that Vpr antagonises NFB signalling activated by TNF, independent of an effect on expression from transfected plasmids, but with the same dependence on nuclear localisation, putatively by the same mechanism. Next, this study characterised an observation that the Vpr from the lentivirus infecting a mona monkey (SIVmon) stimulates NFB signalling. It was hypothesised that the SIVmon Vpr might have molecular binding partners in common with the HIV-1 M Vpr and conditions were optimised for proteomics studies to determine these binding partners. This study provides insights into the role of Vpr in antagonising innate immune sensing. Additionally, overexpression assays have been used widely in the literature describing Vpr. The data presented here indicate that observations using these assays, apparently demonstrating specific degradation of host cellular proteins, should be interpreted cautiously

Role of viral protein R in infection of human dendritic cells by primate lentiviruses

Viral protein R (Vpr) is an evolutionarily conserved but poorly understood protein encoded by all primate lentiviruses, including the lineages that gave rise to both human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2), the causative agents of AIDS in humans. In this work, I sought to define the contribution of primate lentiviral Vpr to viral replication and evasion from cell-intrinsic antiviral defenses. I found that HIV-1 infection of human dendritic cells (MDDCs) is substantially attenuated upon infection with Vpr-deficient (HIV-1/ΔVpr) virus compared to wild-type (WT) infection. This replication defect to HIV-1/ΔVpr is evident in a single round of infection, results in reduced levels of viral transcription, and is relieved upon complementation by virion-associated Vpr. The block to transcription is alleviated through Vpr-engagement with the Cul4A/DCAF/DDB1 (DCAFCRL4) ubiquitin ligase complex and a yet-to-be identified host factor, hypothesized to induce the DNA damage response (DDR) in infected cells. MDDCs are critical immune cells that are poised to detect invading viruses through a variety of cell-intrinsic antiviral sensors, resulting in the production of type I interferon (IFN) and restriction of virus replication. Surprisingly, infection of MDDCs with Vpr-deficient lentiviruses (HIV-2 or SIVmac) resulted in production of type I IFN indicating that this pathway is targeted by Vpr. I determined that signaling cascades that induce NF-κB-dependent type I IFN production are triggered in response to lentiviral integration, an obligatory process in lentivirus life cycle that results in host DNA lesions and subsequent repair by cellular DNA repair machinery. I also demonstrated that mutations in SIVmac Vpr that ablate the ability to initiate DDR are unable to counteract the antiviral type I IFN response. Together, our work suggests the existence of a novel host factor that detects lentiviral integration in MDDCs to trigger an innate immune response that blocks virus dissemination. I hypothesize that Vpr by overcoming this cell intrinsic block to integration would be a critical viral adaptation to facilitate cross-species transmission that resulted in the HIV pandemic.2018-11-01T00:00:00

Vpr Mediates Immune Evasion and HIV-1 Spread.

The molecular mechanisms by which human immunodeficiency virus (HIV-1) evades immunity to cause persistent infection remain incompletely characterized. Viral protein R (Vpr) is conserved in all primate lentiviruses, including HIV-1. Previous studies have demonstrated that Vpr is required for maximal infection of T lymphocytes in vivo. However, Vpr does not enhance HIV-1 infection of T lymphocytes under standard in vitro infection conditions, and the mechanism of Vpr function is poorly understood. Our work demonstrates that Vpr prevents the induction of a type I interferon-stimulated antiviral response in macrophages that targets Env and Env-containing virions for lysosomal degradation. By preventing this response, Vpr promotes Env-dependent virological synapse formation and enables efficient spread of HIV-1 from macrophages to activated T lymphocytes. This mode of spread requires direct cell-to-cell contact and is highly resistant to neutralizing antibodies. These studies provide a mechanistic explanation for the evolutionary conservation and function of Vpr.PHDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/113293/1/davrcol_1.pd

Étude de l'interactome et identification de nouvelles cibles de la protéine virale Vpr du VIH-1

Le virus de l’immunodéficience humaine de type 1 (VIH-1) est l’agent étiologique du SIDA, un rétrovirus complexe encodant les protéines accessoires : Nef, Vif, Vpr et Vpu. La fonction principale de ces protéines est de moduler l’environnement cellulaire afin de promouvoir la réplication virale. Les travaux présentés dans cette thèse portent sur la protéine virale Vpr, une protéine bien connue pour son activité d’arrêt du cycle cellulaire en phase G2/M dans les cellules en division et pour l’avantage réplicatif qu’elle confère au virus durant l’infection de cellules myéloïdes. Les évènements sous-jacents à ces deux activités restent pour l’heure mal compris. Le but des travaux regroupés dans cet ouvrage est d’identifier de nouveaux facteurs cellulaires pouvant éventuellement expliquer les activités de Vpr précédemment décrites. Pour ce faire, nous avons utilisé une approche d’identification des partenaires de proximité par biotinylation, appelée BioID. L’avantage du BioID est de permettre un marquage in cellulo des protéines à proximité de la protéine d’intérêt. La mise en place et la caractérisation de cette approche font l’objet de la première section de cette thèse. En utilisant cette approche, nous avons défini un réseau de 352 partenaires cellulaires de la protéine Vpr. Parmi ces partenaires de Vpr, plusieurs sont organisés sous forme de complexes ou réseaux protéiques incluant notamment le complexe promoteur de l’anaphase/cyclosome (APC/C) et les centrosomes. Étant donné que le complexe APC/C est l’un des principaux régulateurs du cycle cellulaire, nous avons décidé d’analyser sa relation avec Vpr. Nous avons découvert que Vpr formait un complexe non seulement avec APC1, une sous-unité essentielle du complexe APC/C, mais aussi avec les coactivateurs (CDH1 et CDC20) de ce complexe. Nous avons par la suite démontré que Vpr induisait la dégradation d’APC1 et que celle-ci pouvait être prévenue par une double-mutation N28S-G41N de Vpr. Cette dégradation d’APC1 ne semblerait pas être reliée aux activités précédemment décrites de Vpr. Ces travaux font l’objet de la seconde section de cette thèse. Enfin, dans une troisième section, des travaux effectués en collaboration et analysant la relation entre les centrosomes et Vpr sont présentés. Cette thèse identifie 200 nouveaux partenaires de Vpr, ouvrant la porte à l’exploration de nouvelles cibles et activités de Vpr. Elle décrit également une nouvelle cible de Vpr : le complexe APC/C. Globalement nos résultats contribuent à une meilleure compréhension de la façon dont le VIH-1 manipule l’environnement cellulaire de l’hôte à travers la protéine virale Vpr.Human immunodeficiency virus (HIV-1) is the AIDS causal agent. This complex retrovirus encodes several accessory proteins; namely Nef, Vif, Vpr and Vpu; whose functions are to manipulate the cellular host environment in order to favor HIV-1 viral replication. This thesis focused on Vpr whose main activities are to induce a cell cycle arrest in the G2/M phase in dividing cells and to provide a replicative advantage to HIV-1 during infection of myeloid cells such as macrophages. The cellular mechanisms underlying these two activities are up to now misunderstood. The main goal of the work presented in this thesis is to identify new cellular factors that could potentially explain the previously described Vpr activities. To do so, we used the proximity labelling approach called BioID. The main strength of BioID is to tag in cellulo partners of the protein of interest. The development as well as optimization of the BioID approach is presented in the thesis first section. Using BioID, we defined a network containing 352 cellular partners in close proximity with the viral protein Vpr. Amongst these cellular partners, several were organized into protein complexes or networks such as the anaphase promoting complex/cyclosome (APC/C) or the centrosome. Given that APC/C is a cell cycle master-regulator, we analyzed the interplay governing Vpr and APC/C interactions. We first demonstrated that Vpr could form a complex containing the scaffolding subunit APC1. APC/C coactivators, namely CDH1 and CDC20, could also be found in association with Vpr. We next showed that Vpr was inducing APC1 degradation and that Vpr residues N28 and G41 were essential to this activity. Surprisingly, the APC1-Vpr interplay does not relate to previously described Vpr activities. This work is presented in the second section of this thesis. Lastly, in the third section, a work done in collaboration analyzed the interplay between Vpr and the centrosomes. In this thesis we identified 200 new potential partners of Vpr, opening the doors to discover novel Vpr targets and activities. This thesis also defined APC/C as new Vpr target. Taken together our results allow a better understanding on how HIV-1 modulates the cellular environment by using the viral accessory protein Vpr

Characterisation of lentiviral accessory protein Vpx modulation of innate immune signalling

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京都大学ウイルス研究所年報, Volume.54, 2011

CHRONOLOGICAL TABLE / 沿革Organization and Staff / Organization and StaffStaff Changes of The Institute / 異動教官Science Lecture Meeting / ウイルス研究所学術講演会Seminars / 年間セミナーResearch Activities / 研究活動Department of Viral Oncology / がんウイルス研究部門Department of Genetics and Molecular Biology / 遺伝子動態調節研究部門Department of Biological Responses / 生体応答学研究部門Department of Cell Biology / 細胞生物学研究部門Center for Human Retrovirus Research / 附属ヒトレトロウイルス研究施設Experimental Research Center for Infectious Diseases / 附属感染症モデル研究センターCenter for Emerging Virus Research / 附属新興ウイルス感染症研究センター外部資金獲得状況構成