HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

Lahouassa, Hichem; Blondot, Marie-Lise; Chauveau, Lise; Chougui, Ghina; Morel, Marie Hélène; Leduc, Marjorie; Guilhot, François; Ramirez, Bertha Cécilia; Schwartz, Olivier; Margottin-Goguet, Florence

doi:10.1073/pnas.1600485113

Cited by 93 publications

(103 citation statements)

References 95 publications

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“…Previous work in this field suggests that Vpr likely regulates a complex network of host interactions that may vary depending on the cell type infected. We find that, unlike what has been previously observed in activated CD4 ϩ T cells and monocyte-derived macrophages (20,(22)(23)(24)(25)29), infection of MDDCs with ΔVpr viruses was significantly attenuated compared to WT HIV-1 infections (Fig. 1), similar to the findings reported previously by de Silva et al (41).…”

Section: Discussionsupporting

confidence: 89%

“…It is well established that Vpr-mediated G 2 /M cell cycle arrest is mediated through its association with the Cul4A/DCAF/DDB1 E3 (CRL4 DCAF1 ) ubiquitin ligase complex (15)(16)(17). In addition, HIV-1 Vpr has been shown to recruit and degrade a number of DNA damage response (DDR) proteins, including the SLX4-SLX1/MUS81-EME1 structure-specific endonuclease complex (SLX4com), uracil DNA glycosylase 2 (UNG2), and helicase-like transcription factor (HLTF) (18)(19)(20)(21), via the CRL4 DCAF1 complex, resulting in G 2 /M cell cycle arrest, although the role that this process plays during HIV-1 infection still remains unclear.…”

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confidence: 99%

“…Although a number of previous studies have examined the requirement of Vpr for HIV-1 replication in various cell types, including primary CD4 ϩ T cells and monocytederived macrophages (MDMs), differences in virus replication have not been consistently observed (18,20,(22)(23)(24)(25)(26). Vpr expression is dispensable for infection in activated CD4 ϩ T cells in vitro (22)(23)(24)(25)27), presumably due to the well-characterized cytostatic and cytopathic functions of Vpr in cycling cells (12).…”

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confidence: 99%

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Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

Miller

Akiyama

Agosto

et al. 2017

J Virol

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Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4 ϩ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication.IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G 2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

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Section: Discussionsupporting

confidence: 89%

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confidence: 99%

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confidence: 99%

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Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

Miller

Akiyama

Agosto

et al. 2017

J Virol

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“…To this end, we performed a proteomic screen, which led to the identification of HLTF DNA helicase as a specific target of the CRL4 DCAF1-H1.Vpr E3 ubiquitin ligase. Of note, HLTF was also identified as HIV-1 Vpr target by Lahouassa et al (49).…”

Section: Discussionmentioning

confidence: 88%

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

Hrecka

Hao

Shun

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

103

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HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4DCAF1 E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4DCAF1 E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4DCAF1 E3. Thus, separate functions of HIV-1 Vpr usurp CRL4DCAF1 E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host.

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“…The major phenotype ascribed to Vpr is the induction of G 2 /M cell cycle arrest in dividing cells, and this activity has been previously reported to be a feature of several SIV Vpr proteins (41)(42)(43). Recently, Vpr has been identified as inducing the degradation of host helicase transcription factor (HLTF) (44,45) and removing viral DNA by recruiting SLX4-MUS81 complexes (46,47).…”

Section: Importancementioning

confidence: 99%

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly

Wang

Guo

et al. 2017

J Virol

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The lentiviral accessory proteins Vpx and Vpr are known to utilize CRL4 (DCAF1) E3 ligase to induce the degradation of the host restriction factor SAMHD1 or host helicase transcription factor (HLTF), respectively. Selective disruption of viral CRL4 (DCAF1) E3 ligase could be a promising antiviral strategy. Recently, we have determined that posttranslational modification (neddylation) of Cullin-4 is required for the activation of Vpx-CRL4 (DCAF1) E3 ligase. However, the mechanism of Vpx/Vpr-CRL4 (DCAF1) E3 ligase assembly is still poorly understood. Here, we report that zinc coordination is an important regulator of Vpx-CRL4 E3 ligase assembly. Residues in a conserved zinc-binding motif of Vpx were essential for the recruitment of the CRL4 (DCAF1) E3 complex and Vpx-induced SAMHD1 degradation. Importantly, altering the intracellular zinc concentration by treatment with the zinc chelator N,N,N=-tetrakis-(2=-pyridylmethyl)ethylenediamine (TPEN) potently blocked Vpx-mediated SAMHD1 degradation and inhibited wild-type SIVmac (simian immunodeficiency virus of macaques) infection of myeloid cells, even in the presence of Vpx. TPEN selectively inhibited Vpx and DCAF1 binding but not the Vpx-SAMHD1 interaction or Vpx virion packaging. Moreover, we have shown that zinc coordination is also important for the assembly of the HIV-1 Vpr-CRL4 E3 ligase. In particular, Vpr zinc-binding motif mutation or TPEN treatment efficiently inhibited Vpr-CRL4 (DCAF1) E3 ligase assembly and Vpr-mediated HLTF degradation or Vpr-induced G 2 cell cycle arrest. Collectively, our study sheds light on a conserved strategy by the viral proteins Vpx and Vpr to recruit host CRL4 (DCAF1) E3 ligase, which represents a target for novel antihuman immunodeficiency virus (HIV) drug development.IMPORTANCE The Vpr and its paralog Vpx are accessory proteins encoded by different human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) lentiviruses. To facilitate viral replication, Vpx has evolved to induce SAMHD1 degradation and Vpr to mediate HLTF degradation. Both Vpx and Vpr perform their functions by recruiting CRL4 (DCAF1) E3 ligase. In this study, we demonstrate that the assembly of the Vpx-or Vpr-CRL4 E3 ligase requires a highly conserved zinc-binding motif. This motif is specifically required for the DCAF1 interaction but not for the interaction of Vpx or Vpr with its substrate. Selective disruption of Vpx-or Vpr-CRL4 E3 ligase function was achieved by zinc sequestration using N,N,N=-tetrakis-(2=-pyridylmethyl) ethylenediamine (TPEN). At the same time, zinc sequestration had no effect on zinc-

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HIV-1 Vpr degrades the HLTF DNA translocase in T cells and macrophages

Cited by 93 publications

References 95 publications

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins

Inhibition of Vpx-Mediated SAMHD1 and Vpr-Mediated Host Helicase Transcription Factor Degradation by Selective Disruption of Viral CRL4 (DCAF1) E3 Ubiquitin Ligase Assembly

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