A dissertation submitted to Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Science in Medicine Johannesburg, June 2016 / Human Leukocyte Antigen (HLA) class I (HLA-I)-restricted CD8+ cytotoxic T lymphocyte (CTL) responses are major drivers of human immunodeficiency virus type 1 (HIV-1) evolution, resulting in the selection of CTL escape mutants. This is particularly well described for HIV-1 proteins such as Gag, Pol and Nef. For the accessory proteins Vif, Vpr and Vpu though, it is still not clear to what extent HLA-I-mediated responses influence their evolution. Moreover, preliminary data from our laboratory showed that Vpu was the most targeted of the accessory proteins with regards to CD8+ CTL and natural killer (NK) cell responses in a long term non progressor/elite controller cohort. Thus, the overall aims of the study were to describe immune escape in Vif, Vpr and Vpu epitopes from HIV-1 infected patients in South Africa, and to clone, express and purify recombinant Vpu for the ultimate detection of anti-Vpu antibodies in South African patient sera. Longitudinal plasma samples were available for 25 patients from two cohorts (18 from Lung and seven from M002). Viral RNA was extracted and the vif, vpr and vpu regions were RT-PCR (Reverse transcription polymerase chain reaction) amplified and sequenced. HLA-A, B and C data for Lung cohort individuals were available, thus the HLA-A, B and C alleles were typed and assigned for the M002 cohort individuals. The Vif, Vpr and Vpu amino acid sequences were subsequently extensively analysed for HLA-driven CTL escape mutations. Lastly, three vpu sequences, Cons. C, 05ZAFV05 and 05ZAFV15 were selected for codon-optimization (humanized), each cloned into the pcDNA3.1/V5-His mammalian expression vector, and used to optimize expression of recombinant Vpu proteins in HEK 293T cells. The HEK 293T cells were harvested and the Vpu was purified using standard nickel affinity chromatography procedures. Protein expression and purification was monitored by SDS-PAGE, Western blot and dot blot analyses. Overall, longitudinal sequences were obtained from Vif, Vpr and Vpu from eight of the 18 patients in the Lung cohort and all seven patients in the M002 cohort. Of these, HLA-driven immune escape mutations were detected in four patients from the Lung cohort and six from the M002 cohort. Interestingly, for the M002 cohort, some reversions to the baseline sequences were noted over time. None of the identified escape mutations are amongst the best described and most optimal HIV-1 CD8+ CTL epitopes of HIV-1 accessory proteins in the HIV-1 databases. Eight and eleven HLA alleles contributed to immune escape in the Lung and M002 cohort Vif, Vpr and/or Vpu sequences, respectively. Overall, HLA-driven immune pressure in the Vif, Vpr and/or Vpu motifs described here may have contributed to the changes in viral loads seen in these patients over time, and likely impacted iii on disease progression, albeit in combination with escape mutations in the more highly targeted HIV-1 proteins, such as Gag, Pol and Nef. Future work looking at longitudinal sequence analysis of the entire HIV-1 proteome in these patients will likely reveal the contributing factors leading to immune escape and ultimately impact disease progression to AIDS. Expression of all three recombinant Vpu proteins in HEK 293T cells was successfully optimized however, as evidenced by SDS-PAGE, Western blot and dot blot analyses, the expression and purification protocol used in this study did not provide sufficient protein yields, nor an optimally purified protein for use in the downstream assays required. Future work will focus on optimization of Vpu expression and purification protocols to aid in the detailed elucidation of the role of Vpu and anti-Vpu immune responses in control of HIV-1 infection. / MT2016

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.

Monocytic cells survive HIV replication and consequent cytopathic effects because of their decreased sensitivity to HIV-induced apoptosis. However, the mechanism underlying this resistance to apoptosis remains poorly understood. I hypothesized that exposure to microbial products, translocated from the gut, may confer anti-apoptotic properties in human monocytic cells through interaction with their corresponding Toll-like receptors (TLRs). Using HIV-Vpr(52-96) peptide as a model apoptosis-inducing agent, I demonstrated that unlike monocyte-derived macrophages, undifferentiated primary human monocytes and pro-monocytic THP-1 cells are highly susceptible to Vpr(52-96)-induced apoptosis. Interestingly, monocytes and THP-1 cells stimulated with TLR-9 agonists, CpG and E.coli DNA, induced almost complete resistance to Vpr(52-96)-induced apoptosis albiet via a TLR-9 independent signaling pathway. Moreover, CpG and E.coli DNA selectively induced the anti- apoptotic Inhibitor of Apoptosis Protein-2 (c-IAP-2) and inhibition of the c-IAP-2 gene by either specific siRNAs or synthetic second mitochondrial activator of caspases (Smac) mimetic reversed CpG-induced resistance against Vpr(52-96)-mediated apoptosis. I demonstrated that c-IAP-2 was regulated by the c-Jun N terminal kinase (JNK) and the calcium signaling pathway in particular the calmodulin-dependent protein kinase-II (CaMK-II). Furthermore, inhibition of JNK and the calcium signaling including CaMK-II by either pharmacological inhibitors or their specific siRNAs reversed CpG-induced protection against Vpr(52-96)-mediated apoptosis. I also showed that CpG-induced JNK phosphorylation through activation of calcium signaling pathway.

Vpr is a 14 kDa accessory protein conserved amongst extant primate lentiviruses that is required for virus replication in vivo. Although many functions have been attributed to Vpr, its primary role, and the function under selective pressure in vivo, remains elusive. The minimal importance of Vpr in infection of activated CD4+ T cells in vitro suggests that its major importance lies in overcoming restriction to virus replication in quiescent CD4+ T cells and non-cycling myeloid cell populations, such as macrophages and dendritic cells. Previous studies from our laboratory demonstrated that HIV-1 replication is attenuated in the absence of Vpr in monocyte-derived dendritic cells (MDDCs) and macrophages, which is correlated with the ability of HIV-1 Vpr to overcome a post-integration transcriptional defect in these cells. In contrast to HIV-1 Vpr-mediated transcriptional enhancement of the viral LTR, here I describe a role for HIV-2 and SIVmac Vpr homologs in the suppression of innate immune sensing of primate lentiviral infection in monocyte-derived dendritic cells (MDDCs). Specifically, the Vpr proteins of HIV-2 and SIVmac, but not that of HIV-1, suppress innate immune detection and induction of type I and type III IFN at two distinct stages of the viral life cycle: prior to and during integration. We posit that HIV-2/SIVmac-lineage Vpr homologs gained this function upon the acquisition of Vpx, a Vpr paralog in the lentiviral genome, that targets the retroviral restriction factor SAMHD1 for proteasomal degradation. Mutational analysis shows that suppression of pre-integration innate immune sensing by HIV-2/SIVmac Vpr homologs is tied to their interaction with the DNA damage response protein human Uracil DNA glycosylase hUNG. Interestingly, the HIV-1 Vpr degrades hUNG, whilst the HIV-2/SIVmac Vpr homologs do not. This difference correlates with the inability of HIV-1 Vpr to suppress type I and III IFN responses in SIVmac Vpx supplemented infections of MDDCs. These results highlight how divergent lentiviruses have tailored interactions of their Vpr proteins with members of the DNA damage response to promote replication in diverse cellular contexts. This work also describes the conserved role of primate lentiviral Vpr homologs in the transcription of extrachromosomal or unintegrated viral DNA. This function is dependent on Vpr engagement with the host E3-ubiquitin ligase complex Cul4-DDB1-DCAF1 (DCAFCRL4) and ability to activate the DNA damage response. These findings give insight into the mechanisms driving transcription from an underappreciated and long-lived source of viral antigen, and further the field of non-integrating lentiviral vectors, a frequently used tool for the genetic modification of non-dividing cells. Together, both studies shed light on the way Vpr proteins from diverse primate lentiviruses converge in their manipulation of the DNA damage response to facilitate multiple stages of the virus lifecycle. / 2024-01-24T00:00:00Z

No description available.

Human Immunodeficiency Virus (HIV) is the causal agent of acquired immunodeficiency syndrome (AIDS), a disease characterized by the depletion of CD4+ T cells which impairs immune response. Analysis of HIV-1 infected patients has identified two distinctive phenotypes that differ in length of time towards the development of AIDS, Rapid Progressor (RP) and Long-Term Non-Progressor (LTNP) patients. The cause of the differences between these two groups is a process that is still under investigation. Hints about a possible cause have been attributed to the discovery of mutations in the viral protein R (Vpr) that have been associated with these phenotypes: mutations producing the isoforms R36W to RP patients and R77Q to LTNP patients. It has been suggested that these mutations play an important role in the depletion of CD4+ T cells, however more studies are needed to clarify and support this idea. This study examines the effect of the two isoforms in the infection of human primary CD4+ T cells and a model cell line, using the viral strain HIV-1 JR-CSF and derived strains to which the aforementioned mutations have been induced. Our results show that after infection, isoform R77Q has the capacity to significantly induce more apoptosis (identified by Annexin V staining) than Vpr wild type (WT) and the viral strain expressing the isoform R36W. Moreover, R36W significantly induces more cell death by necrosis than R77Q. Notably, the differences found in the way these isoforms of Vpr induce necrosis and apoptosis support the idea of the correlation between strains harboring these mutations and the phenotypes of RP and LTNP patients.

Si la découverte récente de la souche virale JFH1, capable de réaliser un cycle viral complet en culture cellulaire, a permis de mieux caractériser le déroulement de l’infection du virus de l’hépatite C VHC, de nombreux aspects de la biologie du virus demeurent méconnus. Parmi ceux-ci, les mécanismes gouvernant l’encapsidation de l’ARN génomique viral au sein des particules restent à élucider. Cette thèse décrit la mise point d’un système de mobilisation hétérologue permettant l’analyse de l’interaction de la protéine de capside Core avec l’ARN viral. Ce système nous a permis d’identifier les régions de la protéine impliquées dans la liaison au génome viral, tout en autorisant son transfert dans des cellules naïves. Cette approche de mobilisation dépendante de la Core est complétée d’une étude de recrutement hétérologue basée sur la reconnaissance de l’ARN du phage MS2 avec la protéine de manteau Coat. Cette stratégie novatrice permet le recrutement efficace des ARNs marqués tout en autorisant leur localisation cellulaire. / The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. To study packaging in vivo, we developed a novel heterologous system to evaluate the interactions of HCV Core capside with viral RNA. This system allowed us to pinpoint Core domains involved in RNA binding, and package and transfer HCV RNA into a lentiviral vector. Aside from this Core dependent recruitment, we engineered retoviral vectors to trans-package MS2-tagged RNAs using MS2 Coat protein interaction. This system allowed us to efficiently recruit MS2-tagged replicons into naive cells and offers the opportunity to visualize HCV RNAs in Huh7.5 cells.

Thesis (PhD)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Background: Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is an accessory protein that interacts with a number of host cellular and other viral proteins. Vpr exerts several functions such as induction of apoptosis, induction of cell cycle G2 arrest, modulation of gene expression, and suppression of immune activation. The functionality of subtype C Vpr, especially South African strains, has not been studied. The aim of this study was to describe the diversity of South African HIV-1 subtype C vpr genes and to investigate selected functions of these Vpr proteins. Methodology: The HIV-1 vpr region of 58 strains was amplified, sequenced, and subtyped using phylogenetic analysis. Fragments containing natural mutations were cloned in mammalian expression vectors. A consensus subtype C vpr gene was constructed and site-directed mutagenesis was used to induce mutations in postions in which no natural mutations have been described. The functionality of all constructs was compared with the wild-type subtype B Vpr, by transfecting human 293T cell line to investigate subcellular localization, induction of apoptosis and cell cycle G2 arrest. The modulation of genes expressed in the induction of apoptosis using TaqMan Low density arrays (TLDA) was also investigated. Results: Phylogenetic analysis characterized 54 strains as HIV-1 subtype C and 4 strains as HIV-1 subtype B. The overall amino acid sequence of Vpr was conserved including motifs FPRPWL and TYGDTW, but the C-terminal was more variable. The following mutations were constructed using site-directed mutagenesis: P14I, W18C, Y47N, Q65H and Q88S. Subtype B and all natural mutants of subtype C Vpr localized to the nucleus but the W18C mutation disturbed the nuclear localization of Vpr. The cell cycle G2 arrest activity of all the mutants, as well as consensus-C, was lower than that of subtype B Vpr. All the natural mutants of subtype C Vpr induced cell cycle G2 arrest in 54.0-66.3% of the cells, while subtype B Vpr induced cell cycle G2 arrest in 71.5% of the cells. Subtype B and the natural mutant Vpr proteins induced apoptosis in a similar manner, ranging from 95.3-98.6% of transfected cells. However, an artificially designed Vpr protein containing the consensus sequences of subtype C Vpr indicated a reduced ability to induce apoptosis. While consensus-C Vpr induced apoptosis in only 82.0% of the transfected cells, the artificial mutants of Vpr induced apoptosis in 88.4 to 96.2% of the cells. The induction of apoptosis associated gene expression was similar for all constructs, indicated that apoptosis was efficiently induced through the intrinsic pathway by the mutants. Conclusion: This study indicated that both HIV-1 subtype B and C Vpr display a similar ability for nuclear localization and apoptosis induction. The induction of cell cycle G2 arrest by HIV-1 subtype B Vpr may be more robust than many subtype C Vpr proteins. The natural mutations studied in the isolates did not disturb the functions of subtype C Vpr and in some cases even potentiated the protein to induce apoptosis. Naturally occurring mutations in HIV-1 Vpr cannot be regarded as defective, since enhanced functionality would be more indicative of an adaptive role. The increased potency of the mutated Vpr proteins suggests that Vpr may increase the pathogenicity of HIV-1 by adapting apoptotic enhancing mutations. / AFRIKAANSE OPSOMMING: Agtergrond: Die virus protein R (Vpr) van Menslike Immuungebrek Virus tipe 1 (MIV-1) is ‘n bykomstige protein wat met ‘n aantal sellulêre proteine van die gasheer en ander virus proteine in wisselwerking tree. Vpr het 'n invloed op verskeie funksies onder andere die induksie van apoptose, die induksie van selsiklus G2 staking, modulering van geen uitdrukking en onderdrukking van immuun aktivering. Die funksionaliteit van subtipe C Vpr, en veral die van Suid-Afrikaanse stamme, is nie beskryf nie. Die doelwit van die studie was om die diversiteit van Suid Afrikaanse MIV-1 subtipe C vpr gene te beskryf en ook om selektiewe funksies van die Vpr proteine te ondersoek Metodiek: Die MIV-1 vpr streek van 58 stamme is vermeerder, die DNA volgordes is bepaal en die stamme is gesubtipeer deur filogenetiese analise. Fragmente met natuurlike mutasies is in ekspressie vektore gekloon. ‘n Konsensus subtipe C Vpr geen is ontwerp en mutasies in posisies waar geen natuurlike mutasies beskryf is nie, is ontwerp deur mutagenese. Die funksionaliteit van die konstrukte is met die wilde tipe subtype B vergelyk deur 293T sellyn te transfekteer en te ondersoek vir subsellulêre lokalisering, induksie van apoptose, en G2 selsiklus stilstand. Die modulering van geen uitdrukking in die induksie van apoptose is deur TLDA ondersoek. Resultate: Filogenetiese analise het 54 stamme as HIV-1 subtipe C geklassifiseer en 4 stamme as subtype B. Die Vpr aminosuur volgordes was konstant insluitend die FPRPWL en TYGDTW motiewe, maar die C-terminaal was meer variëerbaar. Deur mutagenese is die volgende mutasies ontwerp: P14I, W18C, Y47N, Q65H and Q88S. Subtipe B en al die natuurlike mutante van subtipe C het in die selkern gelokaliseer, maar die W18C mutasie het die lokalisasie versteur. Die G2 selsiklus stilstand van alle mutante en konsensus C was laer as die van subtype B. Al die natuurlike subtipe C mutante het G2 selsiklus tot stilstand gebring in 54.0-66.3% van die selle, terwyl subtype B selsiklus tot stilstand gebring het in 71.5% van die selle. Subtipe B en die natuurlike Vpr mutante het apoptose op ‘n soortgelyke wyse geinduseer, wat wissel tussen 95.3-98.6% van getransfekteerde selle. Die protein met die kunsmatig ontwerpte konsensus C volgorde het egter ‘n verlaagde vermoë gehad om apoptose te induseer. Die konsensus subtipe C het apoptose in 82.0% van getransfekteerde selle geinduseer en die kunsmatige mutante in 88.4 – 96.2% van die selle. Die induksie van die apoptose verwante geen ekspressie deur die mutante was soortgelyk as die van konsensus C en subtipe B Vpr wat ’n aangeduiding is dat apoptose effektief veroorsaak is deur die intrinsieke roete. Gevolgtrekking: Hierdie studie het aangetoon dat kern lokalisering en apoptose op ‘n soortgelyke wyse by beide MIV-1 subtipe B en C Vpr plaasvind. Die induksie van selsiklus G2 stilstand deur MIV-1 subtipe B Vpr is egter meer robuust as baie van die subtipe C Vpr proteïene. Natuurlike mutasies in MIV-1 Vpr kan nie as gebrekkig beskou word nie, aangesien beter funksionaliteit 'n aanduiding is vandie aanpasbare rol. Die verhoogde krag van die gemuteerde Vpr proteïen dui daarop dat Vpr die patogenisiteit van MIV-1 kan verbeter deur die aanpassing van mutasies.

Le VIH-1 est l'agent infectieux qui cause le SIDA. De nombreuses thérapies existent pour combattre le SIDA mais aucune ne permet son éradication et des résistances apparaissent. Le développement de nouvelles thérapies est donc nécessaire. Les anticorps simple-domaine (sdAb) de lamas présentent les propriétés idéales pour le développement de molécules neutralisantes. Des lamas ont donc été immunisés avec Vpr et les formes native, ou induite par un miniCD4, du trimère de gp140 (partie extracellulaire de l'enveloppe (Env)). Des banques de sdAbs ont ensuite été construites et des sélections par phage display et par double hybride ont été réalisées. Trois sdAbs se liant au site de liaison du co- récepteur de l'Env et un sdAb se liant au site de liaison du CD4 ont ainsi été sélectionnés. Ces sites sont conservés mais difficile d'accès pour des immunoglobulines conventionnelles. Ces sdAbs ont ensuite été caractérisés par ELISA, SPR et cytométrie de flux pour leur capacité de liaison à différentes Env, et en « single round assay » pour leur capacité de neutralisation d'un large spectre (LS) de pseudovirus. Des protéines multidomaines (plusieurs sdAbs reliés par un linker) ont ensuite été construites et testées pour leur neutralisation. Plusieurs de ces molécules, neutralisant un LS de virus, pourraient être utilisées dans des microbicides. La stabilité caractéristique des sdAbs, même en absence de formation de pont disulfure, par exemple dans un environnement réducteur tel que le cytoplasme, est primordiale dans le développement d'anticorps intracellulaires (intrabodies). / HIV-1 is the infectious agent of AIDS. Numerous therapies exist to fight AIDS, but they are not able to eradicate it, and resistances appear. So, new therapy development is necessary. Single-domain antibodies (sdAb) of llamas have ideal properties to develop neutralizing molecules. So, llamas have been immunized with Vpr and with free or miniCD4 induced trimeric gp140 (extracellular part of the envelope (Env)). SdAb libraries have been built and selections were done by phage display and yeast two hybrid. Three sdAbs targeting the co-receptor binding site of the Env and one sdAb targeting the CD4 binding site have been selected. These sites are conserved but inaccessible by conventional immunoglobulins. These sdAbs have been characterized by ELISA, SPR and FACS for their ability to bind different Env and by single-round assay for their neutralization ability. Multimeric proteins (linked sdAbs) have been built and tested for their neutralization ability. Several of these molecules are able to neutralize a broad spectrum of pseudoviruses. They can be used in microbicides. The characteristic stability of these sdAbs, even without disulfide bound formation, ie into reducing environment, as the cytoplasm, is primordial for intracellular antibody (intrabody) development. One sdAb anti-Vpr has been selected using the Sos Recruitment System (SRS), an yeast two-hybrid system allowing detection of cytoplasmic protein-protein interactions. This sdAb is able to alter the localization of its antigen into eukaryotic cells. It is a proof of concept ot the use of SRS in the selection of intracellularly functional sdAbs.

La phagocytose, fonction fondamentale des macrophages, est un processus qui se décompose en deux étapes bien distinctes : les étapes précoces d’internalisation menant à la formation du phagosome et les étapes tardives de maturation du phagosome. Le virus de l’immunodéficience humaine de type I (VIH-1) infecte les macrophages, ce qui perturbe leurs fonctions. L’effet de l’infection virale dans ces cellules est peu caractérisé en comparaison des lymphocytes T. Des travaux antérieurs ont montré d’une part que l’étape précoce d’internalisation de larges particules et bactéries était bloquée de moitié dans les macrophages primaires humains infectés par le VIH-1 via Nef, la protéine de virulence majeure du virus et d’autres part, que la réponse cytokinique était atténuée chez les patients infectés. Ainsi, nous avons étudié l’effet du VIH-1 sur les étapes tardives de la phagocytose : la maturation du phagosome et l’activation des macrophages qui en résulte. Nous avons montré que le VIH-1 altère les étapes tardives de la phagocytose en inhibant la maturation du phagosome, définie par le recrutement de marqueurs tardifs de la voie d’endocytose, d’hydrolases et la production d’espèces réactives oxygénées. Malgré une pré-activation basale, les macrophages infectés par le VIH-1 sont incapables de répondre efficacement à une stimulation induite par phagocytose, ce qui conduit à une modulation de la réponse transcriptionnelle et cytokinique. La dynamique des microtubules et la migration centripète des phagosomes sont profondément affectées par le virus. De façon inattendue, la protéine virale Vpr est impliquée dans ces perturbations, alors que Nef ne joue pas de rôle notable. Nos résultats indiquent que les composants intracellulaires de la machinerie de tri endosomal sont détournés par le compartiment viral dans les macrophages infectés. Par cette étude, nous avons donc identifié la protéine Vpr comme nouveau modulateur de la dynamique des microtubules et du trafic intracellulaire, entraînant ainsi une altération profonde de la maturation du phagosome et de la clairance bactérienne dans les macrophages infectés. Ce travail contribue à mieux comprendre l’établissement d’infections opportunistes chez les patients infectés. / Phagocytosis, a crucial function of macrophages, is composed of two well defined steps : the early step of internalization leading to phagosome formation and the late step of phagosome maturation. The immunodeficiency virus type I (HIV-1) infects macrophages, which disturbs theirs functions. The effects of HIV-1 infection are poorly characterized in this cell type compared to T lymphocytes. Previous results have already shown that the early step of internalization of large particles and bacteria are half blocked by Nef in HIV-1 infected primary macrophages and that the cytokine response is attenuated in infected patients. Thus, we have studied the effect of HIV-1 infection on the late step of phagocytosis : phagosome maturation and the resulting macrophage activation. We shown that HIV-1 impairs late phagocytic events affecting the phagosome maturation, as defined by late endocytic markers and hydrolases recruitment, and reactives oxygens species production. HIV-1 infected macrophages exhibited a basal preactivation but appeared unable to respond efficiently to phagocytic triggers leading to cytokine and transcriptional modifications. Centripetal migration of phagosomes and microtubule dynamics were deeply altered upon viral infection. Surprisingly, the Vpr viral protein was implicated in these pertubations, while Nef was not. Our results revealed that elements of the endosomal sorting machinery were hijacked to the virus-containing compartments in HIV-infected macrophages. With this study, we identify Vpr as a modulator of the microtubule dynamics and intracellular trafficking, leading to alterations in phagosome maturation and bacterial clearance in HIV-1 infected macrophages. This work contribute to better understanding of the establishment of opportunistic infections in HIV-infected patients.