Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1997 Dec;71(12):9732–9742. doi: 10.1128/jvi.71.12.9732-9742.1997

Human immunodeficiency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair.

E S Withers-Ward 1, J B Jowett 1, S A Stewart 1, Y M Xie 1, A Garfinkel 1, Y Shibagaki 1, S A Chow 1, N Shah 1, F Hanaoka 1, D G Sawitz 1, R W Armstrong 1, L M Souza 1, I S Chen 1
PMCID: PMC230283  PMID: 9371639

Abstract

The human immunodeficiency virus type 1 (HIV-1) vpr gene is an evolutionarily conserved gene among the primate lentiviruses HIV-1, HIV-2, and simian immunodeficiency viruses. One of the unique functions attributed to the vpr gene product is the arrest of cells in the G2 phase of the cell cycle. Here we demonstrate that Vpr interacts physically with HHR23A, one member of an evolutionarily conserved gene family involved in nucleotide excision repair. Interaction of Vpr with HHR23A was initially identified through a yeast two-hybrid screen and was confirmed by the demonstration of direct binding between bacterially expressed recombinant and transiently expressed or chemically synthesized protein products. Visualization of HHR23A and Vpr by indirect immunofluorescence and confocal microscopy indicates that the two proteins colocalize at or about the nuclear membrane. We also map the Vpr-binding domain in HHR23A to a C-terminal 45-amino-acid region of the protein previously shown to have homology to members of the ubiquitination pathway. Overexpression of HHR23A and a truncated derivative which includes the Vpr-binding domain results in a partial alleviation of the G2 arrest induced by Vpr, suggesting that the interaction between Vpr and HHR23A is critical for cell cycle arrest induced by Vpr. These results provide further support for the hypothesis that Vpr interferes with the normal function of a protein or proteins involved in the DNA repair process and, thus, in the transmission of signals that allow cells to transit from the G2 to the M phase of the cell cycle.

Full Text

The Full Text of this article is available as a PDF (465.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agostini I., Navarro J. M., Rey F., Bouhamdan M., Spire B., Vigne R., Sire J. The human immunodeficiency virus type 1 Vpr transactivator: cooperation with promoter-bound activator domains and binding to TFIIB. J Mol Biol. 1996 Sep 6;261(5):599–606. doi: 10.1006/jmbi.1996.0485. [DOI] [PubMed] [Google Scholar]
  2. Balliet J. W., Kolson D. L., Eiger G., Kim F. M., McGann K. A., Srinivasan A., Collman R. Distinct effects in primary macrophages and lymphocytes of the human immunodeficiency virus type 1 accessory genes vpr, vpu, and nef: mutational analysis of a primary HIV-1 isolate. Virology. 1994 May 1;200(2):623–631. doi: 10.1006/viro.1994.1225. [DOI] [PubMed] [Google Scholar]
  3. Bartz S. R., Rogel M. E., Emerman M. Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. J Virol. 1996 Apr;70(4):2324–2331. doi: 10.1128/jvi.70.4.2324-2331.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bouhamdan M., Benichou S., Rey F., Navarro J. M., Agostini I., Spire B., Camonis J., Slupphaug G., Vigne R., Benarous R. Human immunodeficiency virus type 1 Vpr protein binds to the uracil DNA glycosylase DNA repair enzyme. J Virol. 1996 Feb;70(2):697–704. doi: 10.1128/jvi.70.2.697-704.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bukrinsky M. I., Haggerty S., Dempsey M. P., Sharova N., Adzhubel A., Spitz L., Lewis P., Goldfarb D., Emerman M., Stevenson M. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature. 1993 Oct 14;365(6447):666–669. doi: 10.1038/365666a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bukrinsky M. I., Sharova N., McDonald T. L., Pushkarskaya T., Tarpley W. G., Stevenson M. Association of integrase, matrix, and reverse transcriptase antigens of human immunodeficiency virus type 1 with viral nucleic acids following acute infection. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6125–6129. doi: 10.1073/pnas.90.13.6125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen E. A., Dehni G., Sodroski J. G., Haseltine W. A. Human immunodeficiency virus vpr product is a virion-associated regulatory protein. J Virol. 1990 Jun;64(6):3097–3099. doi: 10.1128/jvi.64.6.3097-3099.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen E. A., Terwilliger E. F., Jalinoos Y., Proulx J., Sodroski J. G., Haseltine W. A. Identification of HIV-1 vpr product and function. J Acquir Immune Defic Syndr. 1990;3(1):11–18. [PubMed] [Google Scholar]
  9. Connor R. I., Chen B. K., Choe S., Landau N. R. Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes. Virology. 1995 Feb 1;206(2):935–944. doi: 10.1006/viro.1995.1016. [DOI] [PubMed] [Google Scholar]
  10. Di Marzio P., Choe S., Ebright M., Knoblauch R., Landau N. R. Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J Virol. 1995 Dec;69(12):7909–7916. doi: 10.1128/jvi.69.12.7909-7916.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feilotter H. E., Hannon G. J., Ruddell C. J., Beach D. Construction of an improved host strain for two hybrid screening. Nucleic Acids Res. 1994 Apr 25;22(8):1502–1503. doi: 10.1093/nar/22.8.1502. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  13. Fletcher T. M., 3rd, Brichacek B., Sharova N., Newman M. A., Stivahtis G., Sharp P. M., Emerman M., Hahn B. H., Stevenson M. Nuclear import and cell cycle arrest functions of the HIV-1 Vpr protein are encoded by two separate genes in HIV-2/SIV(SM). EMBO J. 1996 Nov 15;15(22):6155–6165. [PMC free article] [PubMed] [Google Scholar]
  14. Freed E. O., Englund G., Martin M. A. Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infection. J Virol. 1995 Jun;69(6):3949–3954. doi: 10.1128/jvi.69.6.3949-3954.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gibbs J. S., Lackner A. A., Lang S. M., Simon M. A., Sehgal P. K., Daniel M. D., Desrosiers R. C. Progression to AIDS in the absence of a gene for vpr or vpx. J Virol. 1995 Apr;69(4):2378–2383. doi: 10.1128/jvi.69.4.2378-2383.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  17. Guzder S. N., Bailly V., Sung P., Prakash L., Prakash S. Yeast DNA repair protein RAD23 promotes complex formation between transcription factor TFIIH and DNA damage recognition factor RAD14. J Biol Chem. 1995 Apr 14;270(15):8385–8388. doi: 10.1074/jbc.270.15.8385. [DOI] [PubMed] [Google Scholar]
  18. Guzder S. N., Habraken Y., Sung P., Prakash L., Prakash S. Reconstitution of yeast nucleotide excision repair with purified Rad proteins, replication protein A, and transcription factor TFIIH. J Biol Chem. 1995 Jun 2;270(22):12973–12976. doi: 10.1074/jbc.270.22.12973. [DOI] [PubMed] [Google Scholar]
  19. Hannon G. J., Demetrick D., Beach D. Isolation of the Rb-related p130 through its interaction with CDK2 and cyclins. Genes Dev. 1993 Dec;7(12A):2378–2391. doi: 10.1101/gad.7.12a.2378. [DOI] [PubMed] [Google Scholar]
  20. He J., Choe S., Walker R., Di Marzio P., Morgan D. O., Landau N. R. 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 Nov;69(11):6705–6711. doi: 10.1128/jvi.69.11.6705-6711.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heinzinger N. K., Bukrinsky M. I., Haggerty S. A., Ragland A. M., Kewalramani V., Lee M. A., Gendelman H. E., Ratner L., Stevenson M., Emerman M. The Vpr protein of human immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing host cells. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7311–7315. doi: 10.1073/pnas.91.15.7311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hoch J., Lang S. M., Weeger M., Stahl-Hennig C., Coulibaly C., Dittmer U., Hunsmann G., Fuchs D., Müller J., Sopper S. vpr deletion mutant of simian immunodeficiency virus induces AIDS in rhesus monkeys. J Virol. 1995 Aug;69(8):4807–4813. doi: 10.1128/jvi.69.8.4807-4813.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hofmann K., Bucher P. The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway. Trends Biochem Sci. 1996 May;21(5):172–173. [PubMed] [Google Scholar]
  24. Jowett J. B., Planelles V., Poon B., Shah N. P., Chen M. L., Chen I. S. The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle. J Virol. 1995 Oct;69(10):6304–6313. doi: 10.1128/jvi.69.10.6304-6313.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kondo E., Mammano F., Cohen E. A., Göttlinger H. G. The p6gag domain of human immunodeficiency virus type 1 is sufficient for the incorporation of Vpr into heterologous viral particles. J Virol. 1995 May;69(5):2759–2764. doi: 10.1128/jvi.69.5.2759-2764.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol. 1975 Jul;66(1):188–193. doi: 10.1083/jcb.66.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lang S. M., Weeger M., Stahl-Hennig C., Coulibaly C., Hunsmann G., Müller J., Müller-Hermelink H., Fuchs D., Wachter H., Daniel M. M. Importance of vpr for infection of rhesus monkeys with simian immunodeficiency virus. J Virol. 1993 Feb;67(2):902–912. doi: 10.1128/jvi.67.2.902-912.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lavallée C., Yao X. J., Ladha A., Göttlinger H., Haseltine W. A., Cohen E. A. Requirement of the Pr55gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles. J Virol. 1994 Mar;68(3):1926–1934. doi: 10.1128/jvi.68.3.1926-1934.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lee M. G., Nurse P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature. 1987 May 7;327(6117):31–35. doi: 10.1038/327031a0. [DOI] [PubMed] [Google Scholar]
  30. Levy D. N., Fernandes L. S., Williams W. V., Weiner D. B. Induction of cell differentiation by human immunodeficiency virus 1 vpr. Cell. 1993 Feb 26;72(4):541–550. doi: 10.1016/0092-8674(93)90073-y. [DOI] [PubMed] [Google Scholar]
  31. Levy D. N., Refaeli Y., MacGregor R. R., Weiner D. B. Serum Vpr regulates productive infection and latency of human immunodeficiency virus type 1. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10873–10877. doi: 10.1073/pnas.91.23.10873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Li L., Lu X., Peterson C., Legerski R. XPC interacts with both HHR23B and HHR23A in vivo. Mutat Res. 1997 May 1;383(3):197–203. doi: 10.1016/s0921-8777(97)00002-5. [DOI] [PubMed] [Google Scholar]
  33. Lu Y. L., Bennett R. P., Wills J. W., Gorelick R., Ratner L. A leucine triplet repeat sequence (LXX)4 in p6gag is important for Vpr incorporation into human immunodeficiency virus type 1 particles. J Virol. 1995 Nov;69(11):6873–6879. doi: 10.1128/jvi.69.11.6873-6879.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Lu Y. L., Spearman P., Ratner L. Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions. J Virol. 1993 Nov;67(11):6542–6550. doi: 10.1128/jvi.67.11.6542-6550.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Macreadie I. G., Castelli L. A., Hewish D. R., Kirkpatrick A., Ward A. C., Azad A. A. A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2770–2774. doi: 10.1073/pnas.92.7.2770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Masutani C., Sugasawa K., Yanagisawa J., Sonoyama T., Ui M., Enomoto T., Takio K., Tanaka K., van der Spek P. J., Bootsma D. Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J. 1994 Apr 15;13(8):1831–1843. doi: 10.1002/j.1460-2075.1994.tb06452.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mueller J. P., Smerdon M. J. Rad23 is required for transcription-coupled repair and efficient overrall repair in Saccharomyces cerevisiae. Mol Cell Biol. 1996 May;16(5):2361–2368. doi: 10.1128/mcb.16.5.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. O'Connor P. M., Ferris D. K., Pagano M., Draetta G., Pines J., Hunter T., Longo D. L., Kohn K. W. G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently. J Biol Chem. 1993 Apr 15;268(11):8298–8308. [PubMed] [Google Scholar]
  39. O'Connor P. M., Ferris D. K., White G. A., Pines J., Hunter T., Longo D. L., Kohn K. W. Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard. Cell Growth Differ. 1992 Jan;3(1):43–52. [PubMed] [Google Scholar]
  40. Paxton W., Connor R. I., Landau N. R. Incorporation of Vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J Virol. 1993 Dec;67(12):7229–7237. doi: 10.1128/jvi.67.12.7229-7237.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Planelles V., Jowett J. B., Li Q. X., Xie Y., Hahn B., Chen I. S. Vpr-induced cell cycle arrest is conserved among primate lentiviruses. J Virol. 1996 Apr;70(4):2516–2524. doi: 10.1128/jvi.70.4.2516-2524.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Poon B., Jowett J. B., Stewart S. A., Armstrong R. W., Rishton G. M., Chen I. S. Human immunodeficiency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA alkylating agent, nitrogen mustard. J Virol. 1997 May;71(5):3961–3971. doi: 10.1128/jvi.71.5.3961-3971.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Re F., Braaten D., Franke E. K., Luban J. Human immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the activation of p34cdc2-cyclin B. J Virol. 1995 Nov;69(11):6859–6864. doi: 10.1128/jvi.69.11.6859-6864.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Refaeli Y., Levy D. N., Weiner D. B. The glucocorticoid receptor type II complex is a target of the HIV-1 vpr gene product. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3621–3625. doi: 10.1073/pnas.92.8.3621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rogel M. E., Wu L. I., Emerman M. The human immunodeficiency virus type 1 vpr gene prevents cell proliferation during chronic infection. J Virol. 1995 Feb;69(2):882–888. doi: 10.1128/jvi.69.2.882-888.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sugasawa K., Masutani C., Uchida A., Maekawa T., van der Spek P. J., Bootsma D., Hoeijmakers J. H., Hanaoka F. HHR23B, a human Rad23 homolog, stimulates XPC protein in nucleotide excision repair in vitro. Mol Cell Biol. 1996 Sep;16(9):4852–4861. doi: 10.1128/mcb.16.9.4852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tristem M., Marshall C., Karpas A., Hill F. Evolution of the primate lentiviruses: evidence from vpx and vpr. EMBO J. 1992 Sep;11(9):3405–3412. doi: 10.1002/j.1460-2075.1992.tb05419.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang L., Mukherjee S., Jia F., Narayan O., Zhao L. J. Interaction of virion protein Vpr of human immunodeficiency virus type 1 with cellular transcription factor Sp1 and trans-activation of viral long terminal repeat. J Biol Chem. 1995 Oct 27;270(43):25564–25569. doi: 10.1074/jbc.270.43.25564. [DOI] [PubMed] [Google Scholar]
  49. Yu X., Matsuda Z., Yu Q. C., Lee T. H., Essex M. Vpx of simian immunodeficiency virus is localized primarily outside the virus core in mature virions. J Virol. 1993 Jul;67(7):4386–4390. doi: 10.1128/jvi.67.7.4386-4390.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yuan X., Matsuda Z., Matsuda M., Essex M., Lee T. H. Human immunodeficiency virus vpr gene encodes a virion-associated protein. AIDS Res Hum Retroviruses. 1990 Nov;6(11):1265–1271. doi: 10.1089/aid.1990.6.1265. [DOI] [PubMed] [Google Scholar]
  51. Zhao L. J., Mukherjee S., Narayan O. Biochemical mechanism of HIV-I Vpr function. Specific interaction with a cellular protein. J Biol Chem. 1994 Jun 3;269(22):15577–15582. [PubMed] [Google Scholar]
  52. Zhao Y., Cao J., O'Gorman M. R., Yu M., Yogev R. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J Virol. 1996 Sep;70(9):5821–5826. doi: 10.1128/jvi.70.9.5821-5826.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. van der Spek P. J., Eker A., Rademakers S., Visser C., Sugasawa K., Masutani C., Hanaoka F., Bootsma D., Hoeijmakers J. H. XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes. Nucleic Acids Res. 1996 Jul 1;24(13):2551–2559. doi: 10.1093/nar/24.13.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. van der Spek P. J., Visser C. E., Hanaoka F., Smit B., Hagemeijer A., Bootsma D., Hoeijmakers J. H. Cloning, comparative mapping, and RNA expression of the mouse homologues of the Saccharomyces cerevisiae nucleotide excision repair gene RAD23. Genomics. 1996 Jan 1;31(1):20–27. doi: 10.1006/geno.1996.0004. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES