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HIV-1 Vpr Triggers Natural Killer Cell–Mediated Lysis of Infected Cells through Activation of the ATR-Mediated DNA Damage Response  [PDF]
Jeffrey Ward,Zachary Davis,Jason DeHart,Erik Zimmerman,Alberto Bosque,Enrico Brunetta,Domenico Mavilio,Vicente Planelles equal contributor,Edward Barker equal contributor
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000613
Abstract: Natural killer (NK) cells are stimulated by ligands on virus-infected cells. We have recently demonstrated that NK cells respond to human immunodeficiency virus type-1 (HIV-1)-infected autologous T-cells, in part, through the recognition of ligands for the NK cell activating receptor NKG2D on the surface of the infected cells. Uninfected primary CD4pos T-cell blasts express little, if any, NKG2D ligands. In the present study we determined the mechanism through which ligands for NKG2D are induced on HIV-1-infected cells. Our studies reveal that expression of vpr is necessary and sufficient to elicit the expression of NKG2D ligands in the context of HIV-1 infection. Vpr specifically induces surface expression of the unique-long 16 binding proteins (ULBP)-1 and ULBP-2, but not ULBP-3, MHC class I-related chain molecules (MIC)-A or MIC-B. In these studies we also demonstrated that Vpr increases the level of ULBP-1 and ULBP-2 mRNA in primary CD4pos T-cell blasts. The presence of ULBP-1 and ULBP-2 on HIV-1 infected cells is dependent on the ability of Vpr to associate with a protein complex know as Cullin 4a (Cul4a)/damaged DNA binding protein 1 (DDB1) and Cul4a-associated factor-1(DCAF-1) E3 ubiquitin ligase (Cul4aDCAF-1). ULBP-1 and -2 expression by Vpr is also dependent on activation of the DNA damage sensor, ataxia telangiectasia and rad-3-related kinase (ATR). When T-cell blasts are infected with a vpr-deficient HIV-1, NK cells are impaired in killing the infected cells. Thus, HIV-1 Vpr actively triggers the expression of the ligands to the NK cell activation receptor.
Anti-Tumor Effects of Mfn2 in Gastric Cancer  [PDF]
Ge-Er Zhang,Hai-Long Jin,Xian-Ke Lin,Chao Chen,Xiao-Sun Liu,Qing Zhang,Ji-Ren Yu
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms140713005
Abstract: Mitofusin-2 (Mfn2) is a mitochondrial outer membrane protein involved in mitochondrial fusion. Its mutation can cause Charcot-Marie-Tooth disease. Recent studies of Mfn2 in cancer research have not included gastric cancer. We confirmed that Mfn2 expression was lower in tumor tissue than in normal gastric mucosal tissue and that it was negatively correlated with tumor size, indicating an anti-tumor role for Mfn2. In vitro experiments showed that Mfn2 overexpression suppressed gastric cancer cell proliferation and colony formation, weakened the invasion and migratory ability of cancer cells by downregulating MMP-2 and MMP-9, halted the cell cycle and induced apoptosis. Western blotting indicated the likely involvement of P21 and PI3K/Akt signaling. Therefore, Mfn2 is a potential anti-tumor gene and a potential therapeutic target for treating gastric cancer. The progress of gastric cancer may be delayed by controlling Mfn2 expression.
Direct Vpr-Vpr Interaction in Cells monitored by two Photon Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging
Jo?lle V Fritz, Pascal Didier, Jean-Pierre Clamme, Emmanuel Schaub, Delphine Muriaux, Charlotte Cabanne, Nelly Morellet, Serge Bouaziz, Jean-Luc Darlix, Yves Mély, Hugues de Rocquigny
Retrovirology , 2008, DOI: 10.1186/1742-4690-5-87
Abstract: Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization.We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.As for any replication competent retrovirus, the human immunodeficiency virus type 1 (HIV-1) encodes the precursors to the major structural proteins, enzymes and envelope glycoproteins of the viral particle. In addition, HIV-1 codes for essential regulatory factors, notably Tat, Rev and Vpr. Over the past decade, Vpr has been the subject of many studies because it was suspected to play a direct role in the physiopathology of the viral infection. In fact, Vpr was found to interact with the C-terminus of Gag, causing its virion incorporation [1-4], and with cellular proteins in infected cells. Due to these interactions Vpr promotes the transactivation of HIV-1 long terminal repeat (LTR) and can cause a G2/M arrest and apoptosis of cells, but the relationship between these two roles of Vpr is still a matter of debate (reviewed in [5-7]). Also Vpr appears to contribute to the nuclear import of the pre-integration complex (PIC) and thus of the viral DNA [8,9]. This last function is supported by the nuclear envelope (NE) localization of Vpr, which is medi
Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr functions and virus replication in macrophages
Guillaume Jacquot, Erwann Le Rouzic, Annie David, Julie Mazzolini, Jér?me Bouchet, Serge Bouaziz, Florence Niedergang, Gianfranco Pancino, Serge Benichou
Retrovirology , 2007, DOI: 10.1186/1742-4690-4-84
Abstract: In order to define the functional role of Vpr localization at the NE, we have characterized a set of single-point Vpr mutants, and selected two new mutants with substitutions within the first α-helix of the protein, Vpr-L23F and Vpr-K27M, that failed to associate with hCG1, but were still able to interact with other known relevant host partners of Vpr. In mammalian cells, these mutants failed to localize at the NE resulting in a diffuse nucleocytoplasmic distribution both in HeLa cells and in primary human monocyte-derived macrophages. Other mutants with substitutions in the first α-helix (Vpr-A30L and Vpr-F34I) were similarly distributed between the nucleus and cytoplasm, demonstrating that this helix contains the determinants required for localization of Vpr at the NE. All these mutations also impaired the Vpr-mediated G2-arrest of the cell cycle and the subsequent cell death induction, indicating a functional link between these activities and the Vpr accumulation at the NE. However, this localization is not sufficient, since mutations within the C-terminal basic region of Vpr (Vpr-R80A and Vpr-R90K), disrupted the G2-arrest and apoptotic activities without altering NE localization. Finally, the replication of the Vpr-L23F and Vpr-K27M hCG1-binding deficient mutant viruses was also affected in primary macrophages from some but not all donors.These results indicate that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis; they also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can improve HIV-1 replication in macrophages.In contrast to oncoretroviruses that replicate only in dividing cells and require nuclear envelope (NE) disassembly during mitosis to integrate their genetic material into the host cell genome, HIV-1 and other lentiviruses have the ability to productively infect non-dividing cells, such as terminally-differentiated macrophages [1
Visualizing Vpr-Induced G2 Arrest and Apoptosis  [PDF]
Tomoyuki Murakami, Yoko Aida
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0086840
Abstract: Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the dynamics of the morphological changes that occur during Vpr-induced G2 arrest and apoptosis.
HIV-1 Vpr—a still “enigmatic multitasker”  [PDF]
Carolin A. Guenzel,Cécile Hérate
Frontiers in Microbiology , 2014, DOI: 10.3389/fmicb.2014.00127
Abstract: Like other HIV-1 auxiliary proteins, Vpr is conserved within all the human (HIV-1, HIV-2) and simian (SIV) immunodeficiency viruses. However, Vpr and homologous HIV-2, and SIV Vpx are the only viral auxiliary proteins specifically incorporated into virus particles through direct interaction with the Gag precursor, indicating that this presence in the core of the mature virions is mainly required for optimal establishment of the early steps of the virus life cycle in the newly infected cell. In spite of its small size, a plethora of effects and functions have been attributed to Vpr, including induction of cell cycle arrest and apoptosis, modulation of the fidelity of reverse transcription, nuclear import of viral DNA in macrophages and other non-dividing cells, and transcriptional modulation of viral and host cell genes. Even if some more recent studies identified a few cellular targets that HIV-1 Vpr may utilize in order to perform its different tasks, the real role and functions of Vpr during the course of natural infection are still enigmatic. In this review, we will summarize the main reported functions of HIV-1 Vpr and their significance in the context of the viral life cycle.
Human immunodeficiency virus type 1 Vpr: oligomerization is an essential feature for its incorporation into virus particles
Narasimhan J Venkatachari, Leah A Walker, Oznur Tastan, Thien Le, Timothy M Dempsey, Yaming Li, Naveena Yanamala, Alagarsamy Srinivasan, Judith Klein-Seetharaman, Ronald C Montelaro, Velpandi Ayyavoo
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-119
Abstract: HIV-1 vpr gene encodes a protein of 96 amino acids with a predicted molecular weight of 14 kDa, which is conserved in both HIV and SIV [1]. Vpr is packaged into assembling virions by binding to the p6 domain of viral p55Gag precursor protein. The presence of a functional Vpr is necessary for the efficient translocation of the pre-integration complex (PIC) into the nucleus and subsequent infection of primary monocytes/macrophages and other non-dividing cells [2-4]. Analysis of HIV-1 accessory genes (including vpr) in long-term non-progressors and asymptomatic patients suggests that defects in accessory genes are related to non-progressive status [5,6]. In this regard, the presence of defective or mutated vpr quasispecies has been shown to be associated with long-term non-progressive mothers [6-8]. Though vpr is selected against in tissue culture, selection for an intact Vpr occurs in vivo [9,10]. This finding suggests that vpr is required for optimal virus production and pathogenesis in vivo [11]. These observations clearly indicate the importance of Vpr in viral pathogenesis and disease progression.HIV-1 Vpr is known to oligomerize both in vitro and in vivo [12,13]. This has been demonstrated by using cells in which Vpr was expressed either in the context of transfection of plasmid DNAs or through virus infection. Similar observations have also been reported with the purified Vpr protein generated using the prokaryotic expression system. Vpr has been shown to exist as dimers, trimers, tetramers and higher order multimers [13]. In general, protein oligomerization is thought to be an advantageous feature for the stability of the protein, interaction/binding with other proteins, allosteric control and the establishment of higher-order complexity [14]. HIV-1 Vpr, a non-structural protein, is incorporated into the virus particles and possesses several characteristic features that are known to play important roles in HIV-1 replication and disease progression. Vpr interact
An Unusual Splice Defect in the Mitofusin 2 Gene (MFN2) Is Associated with Degenerative Axonopathy in Tyrolean Grey Cattle  [PDF]
Cord Dr?gemüller,Ursula Reichart,Torsten Seuberlich,Anna Oevermann,Martin Baumgartner,Kathrin Kühni Boghenbor,Michael H. Stoffel,Claudia Syring,Mireille Meylan,Simone Müller,Mathias Müller,Birgit Gredler,Johann S?lkner,Tosso Leeb
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018931
Abstract: Tyrolean Grey cattle represent a local breed with a population size of ~5000 registered cows. In 2003, a previously unknown neurological disorder was recognized in Tyrolean Grey cattle. The clinical signs of the disorder are similar to those of bovine progressive degenerative myeloencephalopathy (weaver syndrome) in Brown Swiss cattle but occur much earlier in life. The neuropathological investigation of an affected calf showed axonal degeneration in the central nervous system (CNS) and femoral nerve. The pedigrees of the affected calves suggested a monogenic autosomal recessive inheritance. We localized the responsible mutation to a 1.9 Mb interval on chromosome 16 by genome-wide association and haplotype mapping. The MFN2 gene located in this interval encodes mitofusin 2, a mitochondrial membrane protein. A heritable human axonal neuropathy, Charcot-Marie-Tooth disease-2A2 (CMT2A2), is caused by MFN2 mutations. Therefore, we considered MFN2 a positional and functional candidate gene and performed mutation analysis in affected and control Tyrolean Grey cattle. We did not find any non-synonymous variants. However, we identified a perfectly associated silent SNP in the coding region of exon 20 of the MFN2 gene. This SNP is located within a putative exonic splice enhancer (ESE) and the variant allele leads to partial retention of the entire intron 19 and a premature stop codon in the aberrant MFN2 transcript. Thus we have identified a highly unusual splicing defect, where an exonic single base exchange leads to the retention of the preceding intron. This splicing defect represents a potential explanation for the observed degenerative axonopathy. Marker assisted selection can now be used to eliminate degenerative axonopathy from Tyrolean Grey cattle.
Preliminary Association Study of SNPs in MFN2 Gene Showing Marbling-Associated Expression Changes
Bin Tong,Youji Muramatsu,Takuji Yamamoto,Hideki Tanomura,Takeshi Ohta,Hiroyuki Kose,Toshie Sugiyama,Takahisa Yamada
Journal of Animal and Veterinary Advances , 2012, DOI: 10.3923/javaa.2012.2796.2800
Abstract: Researchers have previously showed that the mitofusin 2 (MFN2) gene involved in energy balance through mitochondrial fusion and expressed in slow-twitch oxidative fiber that is observed in high-marbled muscle, possesses expression differences in musculus longissimus muscle between low-marbled Holstein and high-marbled Japanese Black steer groups. In the present study, researchers found that a marker (IDVGA-49) close to the MFN2 was polymorphic between low-marbled Holstein and high-marbled Japanese Black steer groups and exhibited significantly different allelic distribution between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. Further, researchers detected Single Nucleotide Polymorphism (SNP) in the MFN2 gene between low-marbled Holstein and high-marbled Japanese Black steer groups. The allelic distributions of the 6 SNPs in the MFN2 were indistinguishable between Japanese Black sires with extremely high predicted breeding value for marbling and with extremely low one. The findings suggest that an unidentified true causal mutation which is in linkage disequilibrium with the IDVGA-49 marker but not the 6 SNPs may be related to changes in MFN2 gene expression and/or marbling. The IDVGA-49 marker may be useful for effective marker-assisted selection to increase the levels of marbling.
Characterization of the Molecular Determinants of Primary HIV-1 Vpr Proteins: Impact of the Q65R and R77Q Substitutions on Vpr Functions  [PDF]
Guillaume Jacquot,Erwann Le Rouzic,Priscilla Maidou-Peindara,Marion Maizy,Jean-Jacques Lefrère,Vincent Daneluzzi,Carlos M. R. Monteiro-Filho,Duanping Hong,Vicente Planelles,Laurence Morand-Joubert,Serge Benichou
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0007514
Abstract: Although HIV-1 Vpr displays several functions in vitro, limited information exists concerning their relevance during infection. Here, we characterized Vpr variants isolated from a rapid and a long-term non-progressor (LTNP). Interestingly, vpr alleles isolated from longitudinal samples of the LTNP revealed a dominant sequence that subsequently led to diversity similar to that observed in the progressor patient. Most of primary Vpr proteins accumulated at the nuclear envelope and interacted with host-cell partners of Vpr. They displayed cytostatic and proapoptotic activities, although a LTNP allele, harboring the Q65R substitution, failed to bind the DCAF1 subunit of the Cul4a/DDB1 E3 ligase and was inactive. This Q65R substitution correlated with impairment of Vpr docking at the nuclear envelope, raising the possibility of a functional link between this property and the Vpr cytostatic activity. In contradiction with published results, the R77Q substitution, found in LTNP alleles, did not influence Vpr proapoptotic activity.
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