A new insight into viral proteins as Immunomodulatory therapeutic agents. KSHV vOX2 a homolog of human CD200 as a potent anti-inflammatory protein

Document Type: Review Article


1 Department of Physiology, Biology Division, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

2 Department of Immunology, Faculty of Medicine, Kurdistan University of Medical Sciences, Sanandaj, Iran

3 Immunology Research Centre, Bu-Ali Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran

4 Inflammation and Inflammatory Diseases Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran


The physiologic function of the immune system is defense against infectious microbes and internal tumour cells, Therefore, need to have precise modulatory mechanisms to maintain the body homeostasis. The mammalian cellular CD200 (OX2)/CD200R interaction is one of such modulatory mechanisms in which myeloid and lymphoid cells are regulated. CD200 and CD200R molecules are membrane proteins that their immunomodulatory effects are able to suppress inflammatory responses, particularly in the privilege sites such as CNS and eyes. Kaposi’s sarcoma-associated herpesvirus (KSHV), encodes a wide variety of immunoregulatory proteins which play central roles in modulating inflammatory and anti-inflammatory responses in favour of virus dissemination. One such protein is a homologue of the, encoded by open reading frame (ORF) K14 and therefore called vOX2. Based on its gene expression profile during the KSHV life cycle, it is hypothesised that vOX2 modulates host inflammatory responses. Moreover, it seems that vOX2 involves in cell adhesion and modulates innate immunity and promotes Th2 immune responses. In this review the activities of mammalian CD200 and KSHV CD200 in cell adhesion and immune system modulation are reviewed in the context of potential therapeutic agents.


1. Holmannova D, Kolackova M, Kondelkova K, Kunes P, Krejsek J, Andrys C. CD200/CD200R paired potent inhibitory molecules regulating immune and inflammatory responses; Part I: CD200/CD200R structure, activation, and function. Acta Med 2012; 55:12-17.

2.Barclay AN. Different reticular elements in rat lymphoid tissue identified by localization of Ia, Thy-1 and MRC OX 2 antigens. Immunology 1981;4:727-736.

3.Webb M, Barclay AN. Localisation of the MRC OX-2 glycoprotein on the surfaces of neurones. J Neurochem 1984; 43:1061-1067.

4. Wright GJ, Cherwinski H, Foster-Cuevas M, Brooke G, Puklavec MJ, Bigler M, et al. Characterization of the CD200 receptor family in mice and humans and their interactions with CD200. J Immunol 2003; 171:3034-3046.

5. Clark MJ, Gagnon J, Williams AF, Barclay AN. MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain. EMBO J 1985; 4:113-118.

6. Hatherley D, Lea SM, Johnson S, Barclay AN. Structures of CD200/CD200 receptor family and implications for topology, regulation, and evolution. Structure 2013; 21:820-832.

7. Chen Z, Zeng H, Gorczynski RM. Cloning and characterization of the murine homologue of the rat/human MRC OX-2 gene. Biochimica Biophys 1997; 1362:6-10.

8. Borriello F, Tizard R, Rue E, Reeves R. Characterization and localization of Mox2, the gene encoding the murine homolog of the rat MRC OX-2 membrane glycoprotein. Mamm Genome 1998; 9:114-118.

9. Gorczynski RM. CD200: CD200R-mediated regulation of immunity. ISRN Immunol 2012; 2012.

10. Gorczynski RM, Yu K, Clark D. Receptor engagement on cells expressing a ligand for the tolerance-inducing molecule OX2 induces an immunoregulatory population that inhibits alloreactivity in vitro and in vivo. J Immunol 2000; 165:4854-4860.

11. Clark DA, Dhesy-Thind S, Arredondo JL, Ellis PM, Ramsay JA. The receptor for the CD200 tolerance-signaling molecule associated with successful pregnancy is expressed by early-stage breast cancer cells in 80% of patients and by term placental trophoblasts. Am J Reprod Immunol 2015; 74:387-391.

12. Gorczynski R, Chen Z, Kai Y, Lee L, Wong S, Marsden PA. CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules. J Immunol 2004; 172:7744-7749.

13. Barclay AN, Wright GJ, Brooke G, Brown MH. CD200 and membrane protein interactions in the control of myeloid cells. Trends Immunol 2002; 23:285-290.

14. Wright GJ, Puklavec MJ, Willis AC, Hoek RM, Sedgwick JD, Brown MH, et al. Lymphoid/neuronal cell surface OX2 glycoprotein recognizes a novel receptor on macrophages implicated in the control of their function. Immunity 2000; 13:233-242.

15. Hatherley D, Cherwinski HM, Moshref M, Barclay AN. Recombinant CD200 protein does not bind activating proteins closely related to CD200 receptor.  J Immunol 2005; 175:2469-2474.

16. McCaughan GW, Clark MJ, Hurst J, Grosveld F, Barclay AN. The gene for MRC OX-2 membrane glycoprotein is localized on human chromosome 3. Immunogenetics 1987; 25:133-135.

17. Vieites JM, de la Torre R, Ortega MA, Montero T, Peco JM, Sanchez-Pozo A, et al. Characterization of human cd200 glycoprotein receptor gene located on chromosome 3q12-13. Gene 2003; 311:99-104.

18. Hoek RM, Ruuls SR, Murphy CA, Wright GJ, Goddard R, Zurawski SM, et al. Down-regulation of the macrophage lineage through interaction with OX2 (CD200). Science 2000; 290:1768-1771.

19. Broderick C, Hoek RM, Forrester JV, Liversidge J, Sedgwick JD, Dick AD. Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol 2002; 161:1669-1677.

20. Snelgrove RJ, Goulding J, Didierlaurent AM, Lyonga D, Vekaria S, Edwards L, et al. A critical function for CD200 in lung immune homeostasis and the severity of influenza infection. Nature Immunol 2008; 9:1074-1083.

21. Gorczynski RM, Cattral MS, Chen Z, Hu J, Lei J, Min WP, et al. An immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that prolongs allo- and xenograft survival. J Immunol 1999; 163:1654-1660.

22. Zhang L, Stanford M, Liu J, Barrett C, Jiang L, Barclay AN, et al. Inhibition of macrophage activation by the myxoma virus M141 protein (vCD200). J Virol 2009; 83:9602-9607.

23. Zhang S, Cherwinski H, Sedgwick JD, Phillips JH. Molecular mechanisms of CD200 inhibition of mast cell activation. J Immunol 2004; 173:6786-6793.

24. Gorczynski RM, Lee L, Boudakov I. Augmented Induction of CD4+CD25+ Treg using monoclonal antibodies to CD200R. Transplantation 2005; 79:1180-1183.

25. Platanias LC. Map kinase signaling pathways and hematologic malignancies. Blood 2003; 101:4667-4679.

26. Karin M. Mitogen activated protein kinases as targets for development of novel anti-inflammatory drugs. Ann Rheum Dis 2004; 63:ii62-ii4.

27. Saklatvala J. The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol 2004; 4:372-377.

28. Neumann H. Control of glial immune function by neurons. Glia 2001; 36:191-199.

29. Walker DG, Dalsing-Hernandez JE, Campbell NA, Lue LF. Decreased expression of CD200 and CD200 receptor in Alzheimer's disease: a potential mechanism leading to chronic inflammation. Exp Neurol 2009; 215:5-19.

30. Wang XJ, Ye M, Zhang YH, Chen SD. CD200-CD200R regulation of microglia activation in the pathogenesis of Parkinson's disease. J Neuroimmune Pharmacol 2007; 2:259-264.

31. Zhang S, Wang XJ, Tian LP, Pan J, Lu GQ, Zhang YJ, et al. CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic neurodegeneration in a rat model of Parkinson's disease. J Neuroinflamm 2011; 8:154.

32. Lyons A, Downer EJ, Costello DA, Murphy N, Lynch MA. Dok2 mediates the CD200Fc attenuation of Abeta-induced changes in glia. J Neuroinflamm 2012; 9:107.

33. Yue X, Qiao D, Wang A, Tan X, Li Y, Liu C, et al. CD200 attenuates methamphetamine-induced microglial activation and dopamine depletion. J Huazhong Univ Sci Technolog Med Sci 2012; 32:415-421.

34. Dick AD, Broderick C, Forrester JV, Wright GJ. Distribution of OX2 antigen and OX2 receptor within retina. Invest Ophthalmol Vis Sci 2001; 42:170-176.

35.Szekeres-Bartho J. Immunological relationship between the mother and the fetus. Int Rev Immunol 2002; 21:471-495.

36. Wright GJ, Jones M, Puklavec MJ, Brown MH, Barclay AN. The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans. Immunology 2001; 102:173-179.

37. Bukovsky A, Presl J, Zidovsky J, Mancal P. The localization of Thy-1.1, MRC OX 2 and Ia antigens in the rat ovary and fallopian tube. Immunology 1983; 48:587-596.

38. Clark DA, Ding JW, Yu G, Levy GA, Gorczynski RM. Fgl2 prothrombinase expression in mouse trophoblast and decidua triggers abortion but may be countered by OX-2. Mol Hum Reprod 2001; 7:185-194.

39. Clark DA, Yu G, Levy GA, Gorczynski RM. Procoagulants in fetus rejection: the role of the OX-2 (CD200) tolerance signal. Semin Immunol 2001; 13:255-263.

40. Clark DA, Arredondo JL, Dhesy-Thind S. The CD200 tolerance-signaling molecule and its receptor, CD200R1, are expressed in human placental villus trophoblast and in peri-implant decidua by 5 weeks' gestation. J Reprod Immunol 2015; 112:20-23.

41. Gorczynski RM, Chen Z, Zeng H, Fu XM. A role for persisting antigen, antigen presentation, and ICAM-1 in increased renal graft survival after oral or portal vein donor-specific immunization. Transplantation 1998; 66:339-349.

42. Ragheb R, Abrahams S, Beecroft R, Hu J, Ni J, Ramakrishna V, et al. Preparation and functional properties of monoclonal antibodies to human, mouse and rat OX-2. Immun Lett 1999; 68:311-315.

43. Gorczynski RM, Bransom J, Cattral M, Huang X, Lei J, Xiaorong L, et al. Synergy in induction of increased renal allograft survival after portal vein infusion of dendritic cells transduced to express TGFbeta and IL-10, along with administration of CHO cells expressing the regulatory molecule OX-2. Clin Immunol 2000; 95:182-189.

44. Nicholls SM, Copland DA, Vitova A, Kuffova L, Forrester JV, Dick AD. Local targeting of the CD200-CD200R axis does not promote corneal graft survival. Exp Eye Res 2015; 130:1-18.

45. Gorczynski R, Chen Z, Shivagnahnam S, Taseva A, Wong K, Yu K, et al. CD200Fc (Gly) 6TGF {beta} suppresses transplant rejection and MLCs in vitro.  J Immunol 2010; 184:49.15.

46. Gorczynski RM, Chen Z, Shivagnahnam S, Taseva A, Wong K, Yu K, et al. Potent immunosuppression by a bivalent molecule binding to CD200R and TGF-betaR. Transplantation 2010; 90:150-159.

47. Gorczynski RM, Chen Z, Hu J, Kai Y, Lei J. Evidence of a role for CD200 in regulation of immune rejection of leukaemic tumour cells in C57BL/6 mice. Clin Exp Immunol 2001; 126:220-229.

48. Gorczynski RM, Chen Z, Diao J, Khatri I, Wong K, Yu K, et al. Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice. Breast Cancer Res Treat 2010; 123:405-415.

49. Stumpfova M, Ratner D, Desciak EB, Eliezri YD, Owens DM. The immunosuppressive surface ligand CD200 augments the metastatic capacity of squamous cell carcinoma. Cancer Res 2010; 70:2962-2972.

50. Vela-Ojeda J, Garcia-Ruiz Esparza MA, Padilla-Gonzalez Y, Perez-Retiguin F, Reyes-Maldonado E, Maillet D, et al. [CD200 protein, bad prognostic in patients with multiple myeloma]. Rev Med Inst Mex Seguro Soc 2015; 53:438-443.

51. Zhang XL, Shen AL, Guo R, Wang Y, Qiu HR, Qiao C, et al. [Expression characteristics of CD200 in acute myeloid leukemia and its clinical significance]. Zhongguo shi Yan Xue Ye Xue Za Zhi 2014; 22:1531-1534.

52. Holmannova D, Kolackova M, Kondelkova K, Kunes P, Krejsek J, Ctirad A. CD200/CD200R paired potent inhibitory molecules regulating immune and inflammatory responses; Part II: CD200/CD200R potential clinical applications. Acta Med 2012; 55:59-65.

53. Lauzon-Joset JF, Langlois A, Lai LJ, Santerre K, Lee-Gosselin A, Bosse Y, et al. Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma. Am J Respir Cell  Mol Biol 2015; 53:276-284.

54. Liu Y, Bando Y, Vargas-Lowy D, Elyaman W, Khoury SJ, Huang T, et al. CD200R1 agonist attenuates mechanisms of chronic disease in a murine model of multiple sclerosis. J Neurosci 2010; 30:2025-2038.

55. Simelyte E, Criado G, Essex D, Uger RA, Feldmann M, Williams RO. CD200-Fc, a novel antiarthritic biologic agent that targets proinflammatory cytokine expression in the joints of mice with collagen-induced arthritis. Arthritis Rheum 2008; 58:1038-1043.

56. Simelyte E, Alzabin S, Boudakov I, Williams R. CD200R1 regulates the severity of arthritis but has minimal impact on the adaptive immune response. Clin Exp Immunol 2010; 162:163-168.

57. Cary Chisholm, Lopez L. Cutaneous Infections Caused by Herpesviridae: A Review. Arch Pathol Lab Med 2011; 135:1357-1362.

58. Plancoulaine S, Gessain A. Epidemiological aspects of human herpesvirus 8 infection and of Kaposi's sarcoma. Med Mal Infect 2005; 35:314-321.

59. Dukers NH, Rezza G. Human herpesvirus 8 epidemiology: what we do and do not know. AIDS 2003; 17:1717-1730.

60. Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles DM, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi's sarcoma. Science 1994; 266:1865-1869.

61. Soulier J, Grollet L, Oksenhendler E, Cacoub P, Cazals-Hatem D, Babinet P, et al. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in multicentric Castleman's disease. Blood 1995; 86:1276-1280.

62. Cesarman E, Chang Y, Moore PS, Said JW, Knowles DM. Kaposi's sarcoma-associated herpesvirus-like DNA sequences in AIDS-related body-cavity-based lymphomas. N Engl J Med 1995; 332:1186-1191.

63. Gessain A. Human herpesvirus 8 (HHV-8): clinical and epidemiological aspects and clonality of associated tumors. Bull Acad Natl Med 2008; 192:1189-11204.

64. Friedman-Kien AE, Laubenstein L, Marmor M, Hymes K, Green J, Ragaz A, et al. Kaposi's sarcoma and Pneumocystis pneumonia among homosexual men-New York City and California. MMWR 1981; 30:305-308.

65. Jacobson LP, Jenkins FJ, Springer G, Munoz A, Shah KV, Phair J, et al. Interaction of human immunodeficiency virus type 1 and human herpesvirus type 8 infections on the incidence of Kaposi's sarcoma. J Infect Dis 2000; 181:1940-1949.

66. Davison AJ. Comparative analysis of the genomes. In: Arvin A, Campadelli-Fiume G, Mocarski E, Moore PS, Roizman B, Whitley R, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007.

67. Goedert J, Jenkins F, Hoffman L. Overview of Herpesviruses.  Infectious Causes of Cancer. Infectous Disease: Humana Press; 2000. p. 33-49.

68. Areste C, Blackbourn DJ. Modulation of the immune system by Kaposi's sarcoma-associated herpesvirus. Trends Microbiol 2009; 17:119-129.

69. Lee HR, Lee S, Chaudhary PM, Gill P, Jung JU. Immune evasion by Kaposi's sarcoma-associated herpesvirus. Future Microbiol 2010; 5:1349-1365.

70. Dialyna IA, Graham D, Rezaee R, Blue CE, Stavrianeas NG, Neisters HG, et al. Anti-HHV-8/KSHV antibodies in infected individuals inhibit infection in vitro. AIDS 2004; 18:1263-1270.

71. Glauser DL, Gillet L, Stevenson PG. Virion endocytosis is a major target for murid herpesvirus-4 neutralization. J Gen Virol 2012; 93:1316-1327.

72. Micheletti F, Monini P, Fortini C, Rimessi P, Bazzaro M, Andreoni M, et al. Identification of cytotoxic T lymphocyte epitopes of human herpesvirus 8. Immunology 2002; 106:395-403.

73. Sirianni MC, Vincenzi L, Topino S, Giovannetti A, Mazzetta F, Libi F, et al. NK cell activity controls human herpesvirus 8 latent infection and is restored upon highly active antiretroviral therapy in AIDS patients with regressing Kaposi's sarcoma. Eur J Immunol 2002; 32:2711-2720.

74. Spiller OB, Robinson M, O'Donnell E, Milligan S, Morgan BP, Davison AJ, et al. Complement regulation by Kaposi's sarcoma-associated herpesvirus ORF4 protein. J Virol 2003; 77:592-599.

75. Moore PS, Boshoff C, Weiss RA, Chang Y. Molecular mimicry of human cytokine and cytokine response pathway genes by KSHV. Science 1996; 274:1739-1744.

76. Cunningham C, Barnard S, Blackbourn DJ, Davison AJ. Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors. J Gen Virol 2003; 84:1471-1483.

77. Rezaee SAR, Cunningham C, Davison AJ, Blackbourn DJ. Kaposi's sarcoma-associated herpesvirus immune modulation: an overview. J Gen Virol 2006; 87:1781-1804.

78. Ishido S, Wang C, Lee BS, Cohen GB, Jung JU. Downregulation of major histocompatibility complex class I molecules by Kaposi's sarcoma-associated herpesvirus K3 and K5 proteins. J Virol 2000; 74:5300-5309.

79. Gompels UA, Nicholas J, Lawrence G, Jones M, Thomson BJ, Martin ME, et al. The DNA sequence of human herpesvirus-6: structure, coding content, and genome evolution. Virology 1995; 209:29-51.

80. Nicholas J. Determination and analysis of the complete nucleotide sequence of human herpesvirus. J Virol 1996; 70:5975-5989.

81. Voigt S, Sandford GR, Hayward GS, Burns WH. The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht. J Gen Virol 2005; 86:263-274.

82. Searles RP, Bergquam EP, Axthelm MK, Wong SW. Sequence and genomic analysis of a Rhesus macaque rhadinovirus with similarity to Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8. J Virol 1999; 73:3040-3053.

83. Russo JJ, Bohenzky RA, Chien MC, Chen J, Yan M, Maddalena D, et al. Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8). Proc Natl Acad Sci U S A 1996; 93:14862-14867.

84. Lee HJ, Essani K, Smith GL. The genome sequence of Yaba-like disease virus, a yatapoxvirus. Virology 2001; 281:170-192.

85. Willer DO, McFadden G, Evans DH. The complete genome sequence of shope (rabbit) fibroma virus. Virology 1999; 264:319-343.

86. Cameron C, Hota-Mitchell S, Chen L, Barrett J, Cao JX, Macaulay C, et al. The complete DNA sequence of myxoma virus. Virology 1999; 264:298-318.

87. McGeoch DJ, Davison AJ. The descent of human herpesvirus 8. Semin Cancer Biol 1999; 9:201-209.

88. Foster-Cuevas M, Wright GJ, Puklavec MJ, Brown MH, Barclay AN. Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor. J Virol 2004; 78:7667-2676.

89. Chung YH, Means RE, Choi JK, Lee BS, Jung JU. Kaposi's sarcoma-associated herpesvirus OX2 glycoprotein activates myeloid-lineage cells to induce inflammatory cytokine production. J Virol 2002; 76:4688-4698.

90. Amini AA, Solovyova AS, Sadeghian H, Blackbourn DJ, Rezaee SA. Structural properties of a viral orthologue of cellular CD200 protein: KSHV vOX2. Virology 2015; 474:94-104.

91. Barczyk M, Carracedo S, Gullberg D. Integrins. Cell Tissue Res 2010; 339:269-280.

92. Weis SM, Cheresh DA. Tumor angiogenesis: molecular pathways and therapeutic targets. Nat Med 2011; 17:1359-1370.

93. Sutherland M, Gordon A, Shnyder SD, Patterson LH, Sheldrake HM. RGD-binding integrins in prostate cancer: expression patterns and therapeutic prospects against bone metastasis. Cancers 2012; 4:1106-1145.

94. Akula SM, Pramod NP, Wang FZ, Chandran B. Integrin alpha3beta1 (CD 49c/29) is a cellular receptor for Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) entry into the target cells. Cell 2002; 108:407-419.

95.Rezaee SR, Gracie JA, McInnes IB, Blackbourn DJ. Inhibition of neutrophil function by the Kaposi's sarcoma-associated herpesvirus vOX2 protein. Aids 2005;19:1907-1910.

96.Shiratori I, Yamaguchi M, Suzukawa M, Yamamoto K, Lanier LL, Saito T, et al. Down-regulation of basophil function by human CD200 and human herpesvirus-8 CD200. J Immunol 2005;175:4441-4449.

97.Misstear K, Chanas SA, Rezaee SA, Colman R, Quinn LL, Long HM, et al. Suppression of antigen-specific T cell responses by the Kaposi's sarcoma-associated herpesvirus viral OX2 protein and its cellular orthologue, CD200. J Virol 2012;86:6246-6257.

98.Salata C, Curtarello M, Calistri A, Sartori E, Sette P, de Bernard M, et al. vOX2 glycoprotein of human herpesvirus 8 modulates human primary macrophages activity. J Cell Physiol 2009;219:698-6706.

99.Alcami A, Koszinowski UH. Viral mechanisms of immune evasion. Immunol Today 2000;21:447-455.

100.Jenner RG, Alba MM, Boshoff C, Kellam P. Kaposi's sarcoma-associated herpesvirus latent and lytic gene expression as revealed by DNA arrays. J Virol 2001;75:891-902.

101.Tullman MJ. Overview of the epidemiology, diagnosis, and disease progression associated with multiple sclerosis. Am J Manag Care 2013;19:S15-20.

102.Tsokos GC. Systemic lupus erythematosus. N Engl J Med 2011;365:2110-2121.

103. Danchenko N, Satia JA, Anthony MS. Epidemiology of systemic lupus erythematosus: a comparison of worldwide disease burden. Lupus 2006; 15:308-318.

104. Stucker F, Ackermann D. Immunosuppressive drugs - how they work, their side effects and interactions. Ther Umsch 1993; 68:679-686.

105. Naesens M, Kuypers DR, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol 2009; 4:481-508.

106. Focosi D, Maggi F, Pistello M, Boggi U, Scatena F. Immunosuppressive monoclonal antibodies: current and next generation. Clin Microbiol Infect 2011; 17:1759-1768.