vpr gene products

Summary

Summary: Trans-acting proteins which accelerate retroviral virus replication. The vpr proteins act in trans to increase the levels of specified proteins. vpr is short for viral protein R, where R is undefined.

Top Publications

  1. Bouzar A, Guiguen F, Morin T, Villet S, Fornazero C, Garnier C, et al. Specific G2 arrest of caprine cells infected with a caprine arthritis encephalitis virus expressing vpr and vpx genes from simian immunodeficiency virus. Virology. 2003;309:41-52 pubmed
    ..The data presented here thus raise a pertinent question about the biological significance of the conservation of Vpr and Vpx functions in caprine cells despite the high phylogenic distance between primates and small ruminants...
  2. Ayyavoo V, Mahboubi A, Mahalingam S, Ramalingam R, Kudchodkar S, Williams W, et al. HIV-1 Vpr suppresses immune activation and apoptosis through regulation of nuclear factor kappa B. Nat Med. 1997;3:1117-23 pubmed
    ..The effects of Vpr could be reversed by RU486. Our finding that Vpr can regulate NF-kappa B supports the hypothesis that some aspects of viral pathogenesis are the consequence of cell dysregulation by Vpr. ..
  3. Gaynor E, Chen I. Analysis of apoptosis induced by HIV-1 Vpr and examination of the possible role of the hHR23A protein. Exp Cell Res. 2001;267:243-57 pubmed
    ..Mutation of its C-terminal UBA( 2 ) domain that is responsible for binding Vpr disrupts the apoptotic effect. This suggests that Vpr may induce apoptosis through a pathway involving hHR23A. ..
  4. Coeytaux E, Coulaud D, Le Cam E, Danos O, Kichler A. The cationic amphipathic alpha-helix of HIV-1 viral protein R (Vpr) binds to nucleic acids, permeabilizes membranes, and efficiently transfects cells. J Biol Chem. 2003;278:18110-6 pubmed
    ..Taken together, the results show that the cationic C-terminal alpha-helix of Vpr has DNA-condensing as well as membrane-destabilizing capabilities, both properties that are indispensable for efficient DNA transfection. ..
  5. Sakai K, Dimas J, Lenardo M. The Vif and Vpr accessory proteins independently cause HIV-1-induced T cell cytopathicity and cell cycle arrest. Proc Natl Acad Sci U S A. 2006;103:3369-74 pubmed
    ..We thus identify vif and vpr as necessary for T cell cytopathic effects induced by HIV-1. These findings may provide an important insight into the molecular mechanism of viral pathogenesis in AIDS. ..
  6. Waldhuber M, Bateson M, Tan J, Greenway A, McPhee D. Studies with GFP-Vpr fusion proteins: induction of apoptosis but ablation of cell-cycle arrest despite nuclear membrane or nuclear localization. Virology. 2003;313:91-104 pubmed
    ..Our findings suggest that nuclear membrane localization and nuclear import and export are strongly governed by modification of the N-terminus of Vpr. ..
  7. Hogan T, Nonnemacher M, Krebs F, Henderson A, Wigdahl B. HIV-1 Vpr binding to HIV-1 LTR C/EBP cis-acting elements and adjacent regions is sequence-specific. Biomed Pharmacother. 2003;57:41-8 pubmed
    ..These studies suggest that Vpr may regulate the interaction of members of the C/EBP transcription factor family with the viral LTR. ..
  8. Vanitharani R, Mahalingam S, Rafaeli Y, Singh S, Srinivasan A, Weiner D, et al. HIV-1 Vpr transactivates LTR-directed expression through sequences present within -278 to -176 and increases virus replication in vitro. Virology. 2001;289:334-42 pubmed
    ..The results presented here demonstrate that Vpr activates HIV-1 LTR through the host GR pathway and suggest that an intact GRE in the LTR is critical for Vpr activity. ..
  9. Eckstein D, Sherman M, Penn M, Chin P, de Noronha C, Greene W, et al. HIV-1 Vpr enhances viral burden by facilitating infection of tissue macrophages but not nondividing CD4+ T cells. J Exp Med. 2001;194:1407-19 pubmed
    ..Collectively, these data highlight the importance of tissue macrophages in local viral burden and further implicate roles for CC chemokine receptor 5, macrophages, and Vpr in the life cycle and pathogenesis of HIV-1. ..

More Information

Publications61

  1. Lai M, Zimmerman E, Planelles V, Chen J. Activation of the ATR pathway by human immunodeficiency virus type 1 Vpr involves its direct binding to chromatin in vivo. J Virol. 2005;79:15443-51 pubmed
    ..These data support a mechanism whereby HIV-1 Vpr induces ATR activation by targeting the host cell DNA and probably interfering with normal DNA replication. ..
  2. Kootstra N, Schuitemaker H. Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages. Virology. 1999;253:170-80 pubmed
    ..In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection. ..
  3. Kino T, Gragerov A, Kopp J, Stauber R, Pavlakis G, Chrousos G. The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor. J Exp Med. 1999;189:51-62 pubmed
    ..The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS. ..
  4. Zhu Y, Roshal M, Li F, Blackett J, Planelles V. Upregulation of survivin by HIV-1 Vpr. Apoptosis. 2003;8:71-9 pubmed
    ..Blocking Vpr-induced G(2)/M arrest leads to elimination of the survivin promoter transactivation by Vpr. Our results suggest that Survivin may be actively involved in regulating cell viability during HIV-1 infection. ..
  5. Rey F, Bouhamdan M, Navarro J, Agostini I, Willetts K, Bouyac M, et al. A role for human immunodeficiency virus type 1 Vpr during infection of peripheral blood mononuclear cells. J Gen Virol. 1998;79 ( Pt 5):1083-7 pubmed
    ..Our data indicate that Vpr is involved in the virus life-cycle during infection of dividing PBMC, presumably as it is during infection of MDMs. ..
  6. Tan L, Ehrlich E, Yu X. DDB1 and Cul4A are required for human immunodeficiency virus type 1 Vpr-induced G2 arrest. J Virol. 2007;81:10822-30 pubmed
    ..These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest. ..
  7. Le Rouzic E, Mousnier A, Rustum C, Stutz F, Hallberg E, Dargemont C, et al. Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1. J Biol Chem. 2002;277:45091-8 pubmed
    ..This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA. ..
  8. Thotala D, Schafer E, Tungaturthi P, Majumder B, Janket M, Wagner M, et al. Structure-functional analysis of human immunodeficiency virus type 1 (HIV-1) Vpr: role of leucine residues on Vpr-mediated transactivation and virus replication. Virology. 2004;328:89-100 pubmed
    ..These results suggest a contrasting role for the leucine residues on HIV-1 LTR-directed transactivation dependent upon their location in Vpr. ..
  9. He J, Choe S, Walker R, Di Marzio P, Morgan D, Landau N. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol. 1995;69:6705-11 pubmed
    ..In vivo, Vpr might, by preventing p34cdc2 activation, delay or prevent apoptosis of infected cells. This would increase the amount of virus each infected cell produced. ..
  10. Janket M, Manickam P, Majumder B, Thotala D, Wagner M, Schafer E, et al. Differential regulation of host cellular genes by HIV-1 viral protein R (Vpr): cDNA microarray analysis using isogenic virus. Biochem Biophys Res Commun. 2004;314:1126-32 pubmed
    ..These results for the first time reveal the involvement of several potential cellular genes, which may be useful, both for understanding Vpr functions and for the development of therapeutics targeting the Vpr molecule. ..
  11. Iijima S, Nitahara Kasahara Y, Kimata K, Zhong Zhuang W, Kamata M, Isogai M, et al. Nuclear localization of Vpr is crucial for the efficient replication of HIV-1 in primary CD4+ T cells. Virology. 2004;327:249-61 pubmed
    ..Our results suggest that the nuclear import of Vpr might be a good target in efforts to block the early stages of replication of HIV-1. ..
  12. Mirani M, Elenkov I, Volpi S, Hiroi N, Chrousos G, Kino T. HIV-1 protein Vpr suppresses IL-12 production from human monocytes by enhancing glucocorticoid action: potential implications of Vpr coactivator activity for the innate and cellular immunity deficits observed in HIV-1 infection. J Immunol. 2002;169:6361-8 pubmed
    ..Through this effect, Vpr may contribute to the suppression of innate and cellular immunities of HIV-1-infected individuals and AIDS patients. ..
  13. Rücker E, Grivel J, Munch J, Kirchhoff F, Margolis L. Vpr and Vpu are important for efficient human immunodeficiency virus type 1 replication and CD4+ T-cell depletion in human lymphoid tissue ex vivo. J Virol. 2004;78:12689-93 pubmed
    ..Our results demonstrate that nef, vpr, and vpu are all essential for efficient viral spread in HLT, suggesting an important role in AIDS pathogenesis. ..
  14. Yoshizuka N, Yoshizuka Chadani Y, Krishnan V, Zeichner S. Human immunodeficiency virus type 1 Vpr-dependent cell cycle arrest through a mitogen-activated protein kinase signal transduction pathway. J Virol. 2005;79:11366-81 pubmed
    ..This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication. ..
  15. Ardon O, Zimmerman E, Andersen J, Dehart J, Blackett J, Planelles V. Induction of G2 arrest and binding to cyclophilin A are independent phenotypes of human immunodeficiency virus type 1 Vpr. J Virol. 2006;80:3694-700 pubmed
    ..The interaction between Vpr and CypA is intriguing, and further studies should examine its potential effects on other functions of Vpr. ..
  16. Le Rouzic E, Belaïdouni N, Estrabaud E, Morel M, Rain J, Transy C, et al. HIV1 Vpr arrests the cell cycle by recruiting DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle. 2007;6:182-8 pubmed
    ..This in turn suggests that both proteins have evolved to preserve interaction with the same Cul4 ubiquitin ligase while diverging in the recognition of host substrates targeted for proteasomal degradation. ..
  17. Caly L, Saksena N, Piller S, Jans D. Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor. Retrovirology. 2008;5:67 pubmed publisher
    ..Importantly, an F72L point mutation in the LTNP was identified for the first time as being uniquely responsible for decreased Vpr nuclear import. ..
  18. Muthumani K, Zhang D, Hwang D, Kudchodkar S, Dayes N, Desai B, et al. Adenovirus encoding HIV-1 Vpr activates caspase 9 and induces apoptotic cell death in both p53 positive and negative human tumor cell lines. Oncogene. 2002;21:4613-25 pubmed
    ..Such a vector may give insight into approaches designed to limit the growth of pathogenic human cells. ..
  19. Watanabe N, Yamaguchi T, Akimoto Y, Rattner J, Hirano H, Nakauchi H. Induction of M-phase arrest and apoptosis after HIV-1 Vpr expression through uncoupling of nuclear and centrosomal cycle in HeLa cells. Exp Cell Res. 2000;258:261-9 pubmed
    ..Thus, the expression of Vpr appears to be responsible for abnormal centrosome duplication, which in turn contributes in part to the rapid cell death following HIV-1 infection. ..
  20. Agostini I, Popov S, Hao T, Li J, Dubrovsky L, Chaika O, et al. Phosphorylation of Vpr regulates HIV type 1 nuclear import and macrophage infection. AIDS Res Hum Retroviruses. 2002;18:283-8 pubmed
    ..The replication defect was mapped by polymerase chain reaction analysis to the step of nuclear import. These results suggest that phosphorylation of Vpr regulates its activity in the nuclear import of the HIV-1 preintegration complex. ..
  21. Yasuda J, Miyao T, Kamata M, Aida Y, Iwakura Y. T cell apoptosis causes peripheral T cell depletion in mice transgenic for the HIV-1 vpr gene. Virology. 2001;285:181-92 pubmed
    ..Involvement of Bcl-x, Bax, and Caspase-1, but not of the Fas-Fas ligand system, was suggested in the apoptotic processes. These observations suggest that Vpr is involved in the pathogenesis of T cell depletion in HIV-infected people. ..
  22. Patel C, Mukhtar M, Pomerantz R. Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells. J Virol. 2000;74:9717-26 pubmed
  23. Kichler A, Pages J, Leborgne C, Druillennec S, Lenoir C, Coulaud D, et al. Efficient DNA transfection mediated by the C-terminal domain of human immunodeficiency virus type 1 viral protein R. J Virol. 2000;74:5424-31 pubmed
    ..Vpr(52-96)-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection. ..
  24. Decrion A, Dichamp I, Varin A, Herbein G. HIV and inflammation. Curr HIV Res. 2005;3:243-59 pubmed
    ..Understanding the role of inflammation in HIV infection could lead to new therapeutic strategies that could ultimately enhance immune restoration and limit the formation of viral reservoirs in HIV-infected patients. ..
  25. Kino T, Gragerov A, Valentin A, Tsopanomihalou M, Ilyina Gragerova G, Erwin Cohen R, et al. Vpr protein of human immunodeficiency virus type 1 binds to 14-3-3 proteins and facilitates complex formation with Cdc25C: implications for cell cycle arrest. J Virol. 2005;79:2780-7 pubmed
    ..These results indicate that Vpr promotes cell cycle arrest at the G(2)/M phase by facilitating association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status. ..
  26. Andersen J, Planelles V. The role of Vpr in HIV-1 pathogenesis. Curr HIV Res. 2005;3:43-51 pubmed
    ..This review presents the latest findings about the pathogenic events triggered by Vpr, the cellular pathways involved, and the molecular and cellular consequences of the action of Vpr in the context of HIV-1 infection. ..
  27. Bell C, Connell B, Capovilla A, Venter W, Stevens W, Papathanasopoulos M. Molecular characterization of the HIV type 1 subtype C accessory genes vif, vpr, and vpu. AIDS Res Hum Retroviruses. 2007;23:322-30 pubmed
    ..These results indicate that antiviral drugs that target the conserved functional domains within Vif, Vpr or Vpu could be active against all circulating subtypes, including HIV-1 subtype C. ..
  28. McDonald D, Vodicka M, Lucero G, Svitkina T, Borisy G, Emerman M, et al. Visualization of the intracellular behavior of HIV in living cells. J Cell Biol. 2002;159:441-52 pubmed
    ..Based on these observations, we propose that HIV uses dynein and the microtubule network to facilitate the delivery of the viral genome to the nucleus of the cell during early postentry steps of the HIV life cycle. ..
  29. Elder R, Yu M, Chen M, Zhu X, Yanagida M, Zhao Y. HIV-1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe) through a pathway involving regulatory and catalytic subunits of PP2A and acting on both Wee1 and Cdc25. Virology. 2001;287:359-70 pubmed
    ..These two genes may be part of the uncharacterized pathway for Vpr-induced G2 arrest in which Vpr upregulates PP2A to activate Wee1 and inhibit Cdc25. ..
  30. Piller S, Caly L, Jans D. Nuclear import of the pre-integration complex (PIC): the Achilles heel of HIV?. Curr Drug Targets. 2003;4:409-29 pubmed
  31. Mansky L, Preveral S, Le Rouzic E, Bernard L, Selig L, Depienne C, et al. Interaction of human immunodeficiency virus type 1 Vpr with the HHR23A DNA repair protein does not correlate with multiple biological functions of Vpr. Virology. 2001;282:176-85 pubmed
    ..Further analysis is needed to determine the functional role(s) of the Vpr-HHR23A association during the HIV-1 life cycle. ..
  32. Stark L, Hay R. Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) interacts with Lys-tRNA synthetase: implications for priming of HIV-1 reverse transcription. J Virol. 1998;72:3037-44 pubmed
    ..Since tRNA(Lys) is the primer for reverse transcription of the HIV-1 genome, this suggests that the interaction between Vpr and LysRS may influence the initiation of HIV-1 reverse transcription. ..
  33. Altfeld M, Livingston B, Reshamwala N, Nguyen P, Addo M, Shea A, et al. Identification of novel HLA-A2-restricted human immunodeficiency virus type 1-specific cytotoxic T-lymphocyte epitopes predicted by the HLA-A2 supertype peptide-binding motif. J Virol. 2001;75:1301-11 pubmed
  34. Sherman M, de Noronha C, Eckstein L, Hataye J, Mundt P, Williams S, et al. Nuclear export of Vpr is required for efficient replication of human immunodeficiency virus type 1 in tissue macrophages. J Virol. 2003;77:7582-9 pubmed
  35. Le Rouzic E, Benichou S. The Vpr protein from HIV-1: distinct roles along the viral life cycle. Retrovirology. 2005;2:11 pubmed
    ..In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle. ..
  36. Patel C, Mukhtar M, Harley S, Kulkosky J, Pomerantz R. Lentiviral expression of HIV-1 Vpr induces apoptosis in human neurons. J Neurovirol. 2002;8:86-99 pubmed
    ..Thus, Vpr delivered intracellularly, as well as extracellularly, is involved in the induction of significant neuronal apoptosis and may be one of the molecular mechanisms in HIV-1-induced encephalopathy. ..
  37. Muthumani K, Hwang D, Choo A, Mayilvahanan S, Dayes N, Thieu K, et al. HIV-1 Vpr inhibits the maturation and activation of macrophages and dendritic cells in vitro. Int Immunol. 2005;17:103-16 pubmed
    ..These studies have importance for understanding the interaction of HIV with the host immune system. ..
  38. Stewart S, Poon B, Jowett J, Chen I. Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest. J Virol. 1997;71:5579-92 pubmed
    ..These studies indicate that Vpr is capable of inducing apoptosis, and we propose that both the initial arrest of cells and subsequent apoptosis may contribute to CD4 cell depletion in HIV-1 disease. ..
  39. Fouchier R, Meyer B, Simon J, Fischer U, Albright A, Gonzalez Scarano F, et al. Interaction of the human immunodeficiency virus type 1 Vpr protein with the nuclear pore complex. J Virol. 1998;72:6004-13 pubmed
    ..These findings not only demonstrate that Vpr harbors a bona fide NLS but also raise the possibility that one (or more) of Vpr's functions may take place at the NPC. ..
  40. Sherman M, de Noronha C, Pearce D, Greene W. Human immunodeficiency virus type 1 Vpr contains two leucine-rich helices that mediate glucocorticoid receptor coactivation independently of its effects on G(2) cell cycle arrest. J Virol. 2000;74:8159-65 pubmed
  41. Zimmerman E, Chen J, Andersen J, Ardon O, Dehart J, Blackett J, et al. Human immunodeficiency virus type 1 Vpr-mediated G2 arrest requires Rad17 and Hus1 and induces nuclear BRCA1 and gamma-H2AX focus formation. Mol Cell Biol. 2004;24:9286-94 pubmed
    ..These findings should contribute to a greater understanding of how HIV-1 manipulates the CD4(+)-lymphocyte cell cycle and apoptosis induction in the progressive CD4(+)-lymphocyte depletion characteristic of HIV-1 pathogenesis. ..
  42. Jacotot E, Ferri K, El Hamel C, Brenner C, Druillennec S, Hoebeke J, et al. Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2. J Exp Med. 2001;193:509-19 pubmed
    ..Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT. ..
  43. Lai M, Chen J. The role of Vpr in HIV-1 disease progression is independent of its G2 arrest induction function. Cell Cycle. 2006;5:2275-80 pubmed
    ..We propose that the G(2) arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr. ..
  44. Gallay P, Stitt V, Mundy C, Oettinger M, Trono D. Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import. J Virol. 1996;70:1027-32 pubmed
    ..In addition, our data suggest that Vpr governs HIV-1 nuclear import through a distinct pathway. ..
  45. Jacotot E, Ravagnan L, Loeffler M, Ferri K, Vieira H, Zamzami N, et al. The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. J Exp Med. 2000;191:33-46 pubmed
    ..Hence, Vpr induces apoptosis via a direct effect on the mitochondrial PTPC. ..
  46. Zhao R, Bukrinsky M, Elder R. HIV-1 viral protein R (Vpr) & host cellular responses. Indian J Med Res. 2005;121:270-86 pubmed
    ..Strategies with potential application for future antiviral therapies directed at suppressing Vpr activities are described. ..
  47. Dehart J, Andersen J, Zimmerman E, Ardon O, An D, Blackett J, et al. The ataxia telangiectasia-mutated and Rad3-related protein is dispensable for retroviral integration. J Virol. 2005;79:1389-96 pubmed
    ..We conclude that cells in which ATR has been depleted are competent for retroviral integration. We also conclude that the presence of Vpr as a virion-bound protein does not enhance integration of a lentivirus vector in dividing cells. ..
  48. Singh S, Tungaturthi P, Cartas M, Tomkowicz B, Rizvi T, Khan S, et al. Virion-associated HIV-1 Vpr: variable amount in virus particles derived from cells upon virus infection or proviral DNA transfection. Virology. 2001;283:78-83 pubmed
    ..The differences in virus particles with respect to Vpr as revealed by these studies should be taken into account in assessing the effect of Vpr. ..
  49. Ramanathan M, Curley E, Su M, Chambers J, Weiner D. Carboxyl terminus of hVIP/mov34 is critical for HIV-1-Vpr interaction and glucocorticoid-mediated signaling. J Biol Chem. 2002;277:47854-60 pubmed
    ..These results support the view that hVIP is a member of the complex array of nucleocytoplasmic shuttling proteins that are regulated by HIV infection and glucocorticoids. ..
  50. Bukrinsky M, Zhao Y. Heat-shock proteins reverse the G2 arrest caused by HIV-1 viral protein R. DNA Cell Biol. 2004;23:223-5 pubmed
    ..This effect appears to be mediated by an interaction between heat shock proteins and Vpr. These results illustrate another example of antagonistic interactions between the viral and cellular proteins. ..
  51. Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson S, Florens L, et al. Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle. Proc Natl Acad Sci U S A. 2007;104:11778-83 pubmed publisher
  52. Somasundaran M, Sharkey M, Brichacek B, Luzuriaga K, Emerman M, Sullivan J, et al. Evidence for a cytopathogenicity determinant in HIV-1 Vpr. Proc Natl Acad Sci U S A. 2002;99:9503-8 pubmed
    ..This study suggests the presence of a determinant of in vivo cytopathogenicity within HIV-1 Vpr and further indicates that viral replication can be uncoupled from cytopathicity in vitro and in vivo. ..