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

Hrecka, Kasia; Hao, Caili; Shun, Ming Chieh; Kaur, Sarabpreet; Swanson, Selene K.; Florens, Laurence; Washburn, Michael P.; Skowroński, Jacek

doi:10.1073/pnas.1605023113

Cited by 62 publications

(111 citation statements)

References 72 publications

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“…Recently, it was reported that HIV-1 recruits the CRL4 (DCAF1) E3 ligase to trigger HLTF degradation (44,45). We have confirmed the ability of HIV-1 Vpr to trigger HLTF degradation in our system (Fig.…”

Section: Discussionsupporting

confidence: 86%

“…8B, lane 2). Recent reports have indicated that Vpr can utilize CRL4 (DCAF1) E3 ligase to induce HLTF degradation (44,45). To address the role of the zinc-binding motif in HLTF degradation mediated by Vpr, we transfected the WT or mutant Vpr expression vectors and HLTF expression vector into HEK 293T cells.…”

Section: Resultsmentioning

confidence: 99%

“…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%

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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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Section: Discussionsupporting

confidence: 86%

Section: Resultsmentioning

confidence: 99%

Section: Importancementioning

confidence: 99%

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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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“…Interestingly, cluster #2 is markedly enriched for nucleic acid binding proteins, including proteins from families with known roles in DNA damage repair and nucleic acid sensing (Figure 2—figure supplement 1B and Figure 2—source data 1). Whilst this manuscript was in preparation, downregulation of a second protein in cluster #2, helicase-like transcription factor (HLTF), was also attributed to Vpr in incoming viral particles (Hrecka et al, 2016; Lahouassa et al, 2016). Remarkably, as for Vif targets APOBEC3C and PPP2R5A-E, temporal profiles of Vpr targets UNG and HLTF cluster very tightly (Figure 2—figure supplement 1C–D).…”

Section: Discussionmentioning

confidence: 99%

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

Greenwood

Matheson

Wals

et al. 2016

eLife

139

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Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function.DOI: http://dx.doi.org/10.7554/eLife.18296.001

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“…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.…”

mentioning

confidence: 99%

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

Cited by 62 publications

References 72 publications

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

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

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

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

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