scholarly journals G2/M Cell Cycle Arrest Correlates with Primate Lentiviral Vpr Interaction with the SLX4 Complex

2014 ◽  
Vol 89 (1) ◽  
pp. 230-240 ◽  
Author(s):  
Gregory Berger ◽  
Madeleine Lawrence ◽  
Stephane Hué ◽  
Stuart J. D. Neil
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Fanconi Anemia ◽  
Human Cells ◽  
Repair Pathway ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACTThe accessory genevpr, common to all primate lentiviruses, induces potent G2/M arrest in cycling cells. A recent study showed that human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) mediates this through activation of the SLX4/MUS81/EME1 exonuclease complex that forms part of the Fanconi anemia DNA repair pathway. To confirm these observations, we have examined the G2/M arrest phenotypes of a panel of simian immunodeficiency virus (SIV) Vpr proteins. We show that SIV Vpr proteins differ in their ability to promote cell cycle arrest in human cells. While this is dependent on the DCAF1/DDB1/CUL4 ubiquitin ligase complex, interaction with human DCAF1 does not predict G2/M arrest activity of SIV Vpr in human cells. In all cases, SIV Vpr-mediated cell cycle arrest in human cells correlated with interaction with human SLX4 (huSLX4) and could be abolished by small interfering RNA (siRNA) depletion of any member of the SLX4 complex. In contrast, all but one of the HIV/SIV Vpr proteins tested, including those that lacked activity in human cells, were competent for G2/M arrest in grivet cells. Correspondingly, here cell cycle arrest correlated with interaction with the grivet orthologues of the SLX4 complex, suggesting a level of host adaptation in these interactions. Phylogenetic analyses strongly suggest that G2/M arrest/SLX4 interactions are ancestral activities of primate lentiviral Vpr proteins and that the ability to dysregulate the Fanconi anemia DNA repair pathway is an essential function of Vprin vivo.IMPORTANCEThe Vpr protein of HIV-1 and related viruses is essential for the virusin vivo. The ability of Vpr to block the cell cycle at mitotic entry is well known, but the importance of this function for viral replication is unclear. Recent data have shown that HIV-1 Vpr targets the Fanconi anemia DNA repair pathway by interacting with and activating an endonuclease complex, SLX4/MUS81/EME1, that processes interstrand DNA cross-links. Here we show that the ability of a panel of SIV Vpr proteins to mediate cell cycle arrest correlates with species-specific interactions with the SLX4 complex in human and primate cells. The results of these studies suggest that the SLX4 complex is a conserved target of primate lentiviral Vpr proteins and that the ability to dysregulate members of the Fanconi anemia DNA repair pathway is essential for HIV/SIV replicationin vivo.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals XRRA1 Targets ATM/CHK1/2-Mediated DNA Repair in Colorectal Cancer

2017 ◽  
Vol 2017 ◽  
pp. 1-11 ◽  
Author(s):  
Wenjun Wang ◽  
Minzhang Guo ◽  
Xiaojun Xia ◽  
Chao Zhang ◽  
Yuan Zeng ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Human Tumor ◽  
Repair Pathway ◽  
X Ray ◽  
Cycle Arrest ◽  
Damage Response ◽  
P21 Proteins

X-ray radiation resistance associated 1 (XRRA1) has been found to regulate the response of human tumor and normal cells to X-radiation (XR). Although XRRA1 overexpression is known to be involved in cancer cell response to XR, there are no reports about whether the expression of XRRA1 in tumors can adjust radioresistance. It is widely known that cell cycle arrest could cause radioresistance. We found that blocked XRRA1 expression could lead to cell cycle G2/M arrest by the regulation of cyclin A, cyclin E, and p21 proteins in colorectal cancer (CRC) and expression of XRRA1 reduced cell cycle arrest and increased cell proliferation in CRC. However, whether regulation of the cell cycle by XRRA1 can influence radioresistance is poorly characterized. Correspondingly, DNA repair can effectively lead to radioresistance. In our study, when cancer cells were exposed to drugs and ionizing radiation, low expression of XRRA1 could increase the phosphorylation of DNA repair pathway factors CHK1, CHK2, and ATM and reduce the expression of γ-H2AX, which is believed to participate in DNA repair in the nucleus. Crucially, our results identify a novel link between XRRA1 and the ATM/CHK1/2 pathway and suggest that XRRA1 is involved in a DNA damage response that drives radio- and chemoresistance by regulating the ATM/CHK1/2 pathway.

scholarly journals Human Immunodeficiency Virus Type 1 (HIV-1) Vpr Enhances Expression from Unintegrated HIV-1 DNA

2003 ◽  
Vol 77 (7) ◽  
pp. 3962-3972 ◽  
Author(s):  
Betty Poon ◽  
Irvin S. Y. Chen
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Cell Cycle Arrest ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Viral Dna ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Retroviral DNA synthesized prior to integration, termed unintegrated viral DNA, is classically believed to be transcriptionally inert and to serve only as a precursor to the transcriptionally active integrated proviral DNA form. However, it has recently been found to be expressed under some circumstances during human immunodeficiency virus type 1 (HIV-1) replication and may play a significant role in HIV-1 pathogenesis. HIV-1 Vpr is a virion-associated accessory protein that is critical for HIV-1 replication in nondividing cells and induces cell cycle arrest and apoptosis. We find that Vpr, either expressed de novo or released from virions following viral entry, is essential for unintegrated viral DNA expression. HIV-1 mutants defective for integration in either the integrase catalytic domain or the cis-acting att sites can express unintegrated viral DNA at levels similar to that of wild-type HIV-1, but only in the presence of Vpr. In the absence of Vpr, the expression of unintegrated viral DNA decreases 10- to 20-fold. Vpr does not affect the efficiency of integration from integrase-defective HIV-1. Vpr-mediated enhancement of expression from integrase-defective HIV-1 requires that the viral DNA be generated in cells through infection and is mediated via a template that declines over time. Vpr activation of expression does not require exclusive nuclear localization of Vpr nor does it correlate with Vpr-mediated cell cycle arrest. These results attribute a new function to HIV-1 Vpr and implicate Vpr as a critical component in expression from unintegrated HIV-1 DNA.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2020 ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
maria Delucia ◽  
jinwoo ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background: Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods: HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein-protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results: We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions: Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether , our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

scholarly journals Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest

2019 ◽  
Author(s):  
Sara Marelli ◽  
James C Williamson ◽  
Anna V Protasio ◽  
Adi Naamati ◽  
Edward JD Greenwood ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
M Cell ◽  
Regulatory Subunits ◽  
Cycle Arrest ◽  
Identify Amino Acid ◽  
Necessary And Sufficient ◽  
Hiv 1

AbstractThe seminal description of cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. As well as APOBEC3G and its homologues, however, we have recently discovered that Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E) (Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.

scholarly journals Protein Kinase A Phosphorylation Activates Vpr-Induced Cell Cycle Arrest during Human Immunodeficiency Virus Type 1 Infection

2010 ◽  
Vol 84 (13) ◽  
pp. 6410-6424 ◽  
Author(s):  
R. Anthony Barnitz ◽  
Fengyi Wan ◽  
Vinay Tripuraneni ◽  
Diane L. Bolton ◽  
Michael J. Lenardo
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Protein Kinase ◽  
Cell Cycle Arrest ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Kinase A

ABSTRACT Infection with human immunodeficiency virus type 1 (HIV-1) causes an inexorable depletion of CD4+ T cells. The loss of these cells is particularly pronounced in the mucosal immune system during acute infection, and the data suggest that direct viral cytopathicity is a major factor. Cell cycle arrest caused by the HIV-1 accessory protein Vpr is strongly correlated with virus-induced cell death, and phosphorylation of Vpr serine 79 (S79) is required to activate G2/M cell cycle blockade. However, the kinase responsible for phosphorylating Vpr remains unknown. Our bioinformatic analyses revealed that S79 is part of a putative phosphorylation site recognized by protein kinase A (PKA). We show here that PKA interacts with Vpr and directly phosphorylates S79. Inhibition of PKA activity during HIV-1 infection abrogates Vpr cell cycle arrest. These findings provide new insight into the signaling event that activates Vpr cell cycle arrest, ultimately leading to the death of infected T cells.

scholarly journals Functional Complementation of Human Centromere Protein A (CENP-A) by Cse4p from Saccharomyces cerevisiae

2004 ◽  
Vol 24 (15) ◽  
pp. 6620-6630 ◽  
Author(s):  
Gerhard Wieland ◽  
Sandra Orthaus ◽  
Sabine Ohndorf ◽  
Stephan Diekmann ◽  
Peter Hemmerich
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Budding Yeast ◽  
Protein A ◽  
Human Cells ◽  
Cycle Arrest ◽  
Centromere Protein ◽  
Centromere Protein A

ABSTRACT We have employed a novel in vivo approach to study the structure and function of the eukaryotic kinetochore multiprotein complex. RNA interference (RNAi) was used to block the synthesis of centromere protein A (CENP-A) and Clip-170 in human cells. By coexpression, homologous kinetochore proteins from Saccharomyces cerevisiae were then tested for the ability to complement the RNAi-induced phenotypes. Cse4p, the budding yeast CENP-A homolog, was specifically incorporated into kinetochore nucleosomes and was able to complement RNAi-induced cell cycle arrest in CENP-A-depleted human cells. Thus, Cse4p can structurally and functionally substitute for CENP-A, strongly suggesting that the basic features of centromeric chromatin are conserved between yeast and mammals. Bik1p, the budding yeast homolog of human CLIP-170, also specifically localized to kinetochores during mitosis, but Bik1p did not rescue CLIP-170 depletion-induced cell cycle arrest. Generally, the newly developed in vivo complementation assay provides a powerful new tool for studying the function and evolutionary conservation of multiprotein complexes from yeast to humans.

scholarly journals Functional and Structural Insights into a Vif/PPP2R5 Complex Elucidated Using Patient HIV-1 Isolates and Computational Modeling

2020 ◽  
Vol 94 (21) ◽  
Author(s):  
Daniel J. Salamango ◽  
Jennifer L. McCann ◽  
Özlem Demir ◽  
Jordan T. Becker ◽  
Jiayi Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Protein Docking ◽  
Targeted Mutagenesis ◽  
Imaging Studies ◽  
M Cell ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Degradation Activity ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vif recruits a cellular ubiquitin ligase complex to degrade antiviral APOBEC3 enzymes (APOBEC3C-H) and PP2A phosphatase regulators (PPP2R5A to PPP2R5E). While APOBEC3 antagonism is the canonical function of HIV-1 Vif, this viral accessory protein is also known to trigger G2/M cell cycle arrest. Vif initiates G2/M arrest by degrading multiple PPP2R5 family members, an activity prevalent among diverse HIV-1 and simian immunodeficiency virus (SIV) isolates. Here, computational protein-protein docking was used to delineate a Vif/CBF-β/PPP2R5 complex in which Vif is predicted to bind the same PPP2R5 surface as physiologic phosphatase targets. This model was tested using targeted mutagenesis of amino acid residues within or adjacent to the putative interface to show loss or retention, respectively, of Vif-induced PPP2R5 degradation activity. Additionally, expression of a peptide that mimics cellular targets of PPP2R5s robustly inhibited Vif-mediated degradation of PPP2R5A but not APOBEC3G. Moreover, live-cell imaging studies examining Vif-mediated degradation of PPP2R5A and APOBEC3G within the same cell revealed that PPP2R5A degradation kinetics are comparable to those of APOBEC3G with a half-life of roughly 6 h postinfection, demonstrating that Vif can concurrently mediate the degradation of distinct cellular substrates. Finally, experiments with a panel of patient-derived Vif isolates indicated that PPP2R5A degradation activity is common in patient-derived isolates. Taken together, these results support a model in which PPP2R5 degradation and global changes in the cellular phosphoproteome are likely to be advantageous for viral pathogenesis. IMPORTANCE A critical function of HIV-1 Vif is to counteract the family of APOBEC3 innate immune proteins. It is also widely accepted that Vif induces G2/M cell cycle arrest in several different cell types. Recently, it has been shown that Vif degrades multiple PPP2R5 phosphoregulators to induce the G2/M arrest phenotype. Here, computational approaches are used to test a structural model of the Vif/PPP2R5 complex. In addition, imaging studies are used to show that Vif degrades these PPP2R5 substrates in roughly the same time frame as APOBEC3 degradation and that this activity is prevalent in patient-derived Vif isolates. These studies are important by further defining PPP2R5 proteins as a bona fide substrate of HIV-1 Vif.

scholarly journals Phosphorylation of Human Immunodeficiency Virus Type 1 Vpr Regulates Cell Cycle Arrest

2000 ◽  
Vol 74 (14) ◽  
pp. 6520-6527 ◽  
Author(s):  
Yi Zhou ◽  
Lee Ratner
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Cell Cycle Arrest ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Cell Cycle Progression ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vpr regulates nuclear transport of the viral preintegration complex, G2 cell cycle arrest, and transcriptional transactivation. We asked whether phosphorylation could affect Vpr activity. Vpr was found to be phosphorylated on serine residues in transiently transfected and infected cells. Residues 79, 94, and 96 were all found to be phosphorylated, as assessed by alanine mutations. Mutation of Ser-79 to Ala abrogated effects of Vpr on cell cycle progression, whereas mutation of Ser-94 and Ser-96 had no effect. Simultaneous mutation of all three Vpr serine residues attenuated HIV-1 replication in macrophages, whereas single and double Ser mutations had no effect.

scholarly journals Genetic Studies with the Fission YeastSchizosaccharomyces pombe Suggest Involvement of Wee1, Ppa2, and Rad24 in Induction of Cell Cycle Arrest by Human Immunodeficiency Virus Type 1 Vpr

2000 ◽  
Vol 74 (6) ◽  
pp. 2636-2646 ◽  
Author(s):  
Michiaki Masuda ◽  
Yukiko Nagai ◽  
Norihito Oshima ◽  
Koichi Tanaka ◽  
Hiroshi Murakami ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Cell Proliferation ◽  
Cell Cycle Arrest ◽  
Wild Type ◽  
Genetic Studies ◽  
Cycle Arrest ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Accessory protein Vpr of human immunodeficiency virus type 1 (HIV-1) arrests cell cycling at G2/M phase in human and simian cells. Recently, it has been shown that Vpr also causes cell cycle arrest in the fission yeast Schizosaccharomyces pombe, which shares the cell cycle regulatory mechanisms with higher eukaryotes including humans. In this study, in order to identify host cellular factors involved in Vpr-induced cell cycle arrest, the ability of Vpr to cause elongated cellular morphology (cdcphenotype) typical of G2/M cell cycle arrest in wild-type and various mutant strains of S. pombe was examined. Our results indicated that Vpr caused the cdc phenotype in wild-type S. pombe as well as in strains carrying mutations, such as the cdc2-3w, Δcdc25,rad1-1, Δchk1, Δmik1, and Δppa1 strains. However, other mutants, such as thecdc2-1w, Δwee1, Δppa2, and Δrad24 strains, failed to show a distinct cdcphenotype in response to Vpr expression. Results of these genetic studies suggested that Wee1, Ppa2, and Rad24 might be required for induction of cell cycle arrest by HIV-1 Vpr. Cell proliferation was inhibited by Vpr expression in all of the strains examined including the ones that did not show the cdc phenotype. The results supported the previously suggested possibility that Vpr affects the cell cycle and cell proliferation through different pathways.

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