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. 1997 Feb 17;16(4):695–705. doi: 10.1093/emboj/16.4.695

The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells.

R Lodge 1, J P Lalonde 1, G Lemay 1, E A Cohen 1
PMCID: PMC1169671  PMID: 9049299

Abstract

Budding of retroviruses from polarized epithelial Madin-Darby canine kidney cells (MDCK) takes place specifically at the basolateral membrane surface. This sorting event is suspected to require a specific signal harbored by the viral envelope glycoprotein and it was previously shown that, as for most basolateral proteins, the intracytoplasmic domain plays a crucial role in this targeting phenomenon. It is well known that tyrosine-based motifs are a central element in basolateral targeting signals. In the present study, site-directed mutagenesis was used to generate conservative or non-conservative substitutions of each four intracytoplasmic tyrosines of the human immunodeficiency virus (HIV-1) envelope glycoprotein. This approach revealed that the membrane-proximal tyrosine is essential to ensure both the basolateral localization of envelope glycoprotein and the basolateral targeting of HIV-1 virions. Substitutions of the membrane-proximal tyrosine did not appear to affect incorporation of envelope glycoprotein into the virions, as assayed by virion infectivity and protein content, nor its capability to ensure its role in viral infection, as determined by viral multiplication kinetics. Altogether, these results indicate that the intracytoplasmic domain of the HIV-1 envelope glycoprotein harbors a unique, tyrosine-based, basolateral targeting signal. Such a tyrosine-based targeting signal may play a fundamental role in HIV transmission and pathogenesis.

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Selected References

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  1. Aiken C., Konner J., Landau N. R., Lenburg M. E., Trono D. Nef induces CD4 endocytosis: requirement for a critical dileucine motif in the membrane-proximal CD4 cytoplasmic domain. Cell. 1994 Mar 11;76(5):853–864. doi: 10.1016/0092-8674(94)90360-3. [DOI] [PubMed] [Google Scholar]
  2. Aroeti B., Kosen P. A., Kuntz I. D., Cohen F. E., Mostov K. E. Mutational and secondary structural analysis of the basolateral sorting signal of the polymeric immunoglobulin receptor. J Cell Biol. 1993 Dec;123(5):1149–1160. doi: 10.1083/jcb.123.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bugelski P. J., Maleeff B. E., Klinkner A. M., Ventre J., Hart T. K. Ultrastructural evidence of an interaction between Env and Gag proteins during assembly of HIV type 1. AIDS Res Hum Retroviruses. 1995 Jan;11(1):55–64. doi: 10.1089/aid.1995.11.55. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Collawn J. F., Stangel M., Kuhn L. A., Esekogwu V., Jing S. Q., Trowbridge I. S., Tainer J. A. Transferrin receptor internalization sequence YXRF implicates a tight turn as the structural recognition motif for endocytosis. Cell. 1990 Nov 30;63(5):1061–1072. doi: 10.1016/0092-8674(90)90509-d. [DOI] [PubMed] [Google Scholar]
  6. Compans R. W., Srinivas R. V. Protein sorting in polarized epithelial cells. Curr Top Microbiol Immunol. 1991;170:141–181. doi: 10.1007/978-3-642-76389-2_5. [DOI] [PubMed] [Google Scholar]
  7. Dargemont C., Le Bivic A., Rothenberger S., Iacopetta B., Kühn L. C. The internalization signal and the phosphorylation site of transferrin receptor are distinct from the main basolateral sorting information. EMBO J. 1993 Apr;12(4):1713–1721. doi: 10.1002/j.1460-2075.1993.tb05816.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deng W. P., Nickoloff J. A. Site-directed mutagenesis of virtually any plasmid by eliminating a unique site. Anal Biochem. 1992 Jan;200(1):81–88. doi: 10.1016/0003-2697(92)90280-k. [DOI] [PubMed] [Google Scholar]
  9. Dorfman T., Mammano F., Haseltine W. A., Göttlinger H. G. Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. J Virol. 1994 Mar;68(3):1689–1696. doi: 10.1128/jvi.68.3.1689-1696.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dubay J. W., Roberts S. J., Hahn B. H., Hunter E. Truncation of the human immunodeficiency virus type 1 transmembrane glycoprotein cytoplasmic domain blocks virus infectivity. J Virol. 1992 Nov;66(11):6616–6625. doi: 10.1128/jvi.66.11.6616-6625.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fais S., Capobianchi M. R., Abbate I., Castilletti C., Gentile M., Cordiali Fei P., Ameglio F., Dianzani F. Unidirectional budding of HIV-1 at the site of cell-to-cell contact is associated with co-polarization of intercellular adhesion molecules and HIV-1 viral matrix protein. AIDS. 1995 Apr;9(4):329–335. [PubMed] [Google Scholar]
  12. Fantini J., Baghdiguian S., Yahi N., Chermann J. C. Selected human immunodeficiency virus replicates preferentially through the basolateral surface of differentiated human colon epithelial cells. Virology. 1991 Dec;185(2):904–907. doi: 10.1016/0042-6822(91)90570-2. [DOI] [PubMed] [Google Scholar]
  13. Fantini J., Yahi N., Baghdiguian S., Chermann J. C. Human colon epithelial cells productively infected with human immunodeficiency virus show impaired differentiation and altered secretion. J Virol. 1992 Jan;66(1):580–585. doi: 10.1128/jvi.66.1.580-585.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fath K. R., Mamajiwalla S. N., Burgess D. R. The cytoskeleton in development of epithelial cell polarity. J Cell Sci Suppl. 1993;17:65–73. doi: 10.1242/jcs.1993.supplement_17.10. [DOI] [PubMed] [Google Scholar]
  15. Fisher A. G., Collalti E., Ratner L., Gallo R. C., Wong-Staal F. A molecular clone of HTLV-III with biological activity. Nature. 1985 Jul 18;316(6025):262–265. doi: 10.1038/316262a0. [DOI] [PubMed] [Google Scholar]
  16. Freed E. O., Martin M. A. Virion incorporation of envelope glycoproteins with long but not short cytoplasmic tails is blocked by specific, single amino acid substitutions in the human immunodeficiency virus type 1 matrix. J Virol. 1995 Mar;69(3):1984–1989. doi: 10.1128/jvi.69.3.1984-1989.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gabuzda D. H., Lawrence K., Langhoff E., Terwilliger E., Dorfman T., Haseltine W. A., Sodroski J. Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes. J Virol. 1992 Nov;66(11):6489–6495. doi: 10.1128/jvi.66.11.6489-6495.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gabuzda D. H., Lever A., Terwilliger E., Sodroski J. Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins. J Virol. 1992 Jun;66(6):3306–3315. doi: 10.1128/jvi.66.6.3306-3315.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gao F., Morrison S. G., Robertson D. L., Thornton C. L., Craig S., Karlsson G., Sodroski J., Morgado M., Galvao-Castro B., von Briesen H. Molecular cloning and analysis of functional envelope genes from human immunodeficiency virus type 1 sequence subtypes A through G. The WHO and NIAID Networks for HIV Isolation and Characterization. J Virol. 1996 Mar;70(3):1651–1667. doi: 10.1128/jvi.70.3.1651-1667.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Geffen I., Fuhrer C., Leitinger B., Weiss M., Huggel K., Griffiths G., Spiess M. Related signals for endocytosis and basolateral sorting of the asialoglycoprotein receptor. J Biol Chem. 1993 Oct 5;268(28):20772–20777. [PubMed] [Google Scholar]
  21. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Helseth E., Kowalski M., Gabuzda D., Olshevsky U., Haseltine W., Sodroski J. Rapid complementation assays measuring replicative potential of human immunodeficiency virus type 1 envelope glycoprotein mutants. J Virol. 1990 May;64(5):2416–2420. doi: 10.1128/jvi.64.5.2416-2420.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hopkins C. R. Selective membrane protein trafficking: vectorial flow and filter. Trends Biochem Sci. 1992 Jan;17(1):27–32. doi: 10.1016/0968-0004(92)90423-7. [DOI] [PubMed] [Google Scholar]
  24. Hunziker W., Harter C., Matter K., Mellman I. Basolateral sorting in MDCK cells requires a distinct cytoplasmic domain determinant. Cell. 1991 Sep 6;66(5):907–920. doi: 10.1016/0092-8674(91)90437-4. [DOI] [PubMed] [Google Scholar]
  25. Kilpatrick D. R., Srinivas R. V., Compans R. W. Expression of the spleen focus-forming virus envelope gene in a polarized epithelial cell line. Virology. 1988 Jun;164(2):547–550. doi: 10.1016/0042-6822(88)90571-5. [DOI] [PubMed] [Google Scholar]
  26. LaBranche C. C., Sauter M. M., Haggarty B. S., Vance P. J., Romano J., Hart T. K., Bugelski P. J., Hoxie J. A. Biological, molecular, and structural analysis of a cytopathic variant from a molecularly cloned simian immunodeficiency virus. J Virol. 1994 Sep;68(9):5509–5522. doi: 10.1128/jvi.68.9.5509-5522.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lee M. H., Sano K., Morales F. E., Imagawa D. T. Sensitive reverse transcriptase assay to detect and quantitate human immunodeficiency virus. J Clin Microbiol. 1987 Sep;25(9):1717–1721. doi: 10.1128/jcm.25.9.1717-1721.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lodge R., Göttlinger H., Gabuzda D., Cohen E. A., Lemay G. The intracytoplasmic domain of gp41 mediates polarized budding of human immunodeficiency virus type 1 in MDCK cells. J Virol. 1994 Aug;68(8):4857–4861. doi: 10.1128/jvi.68.8.4857-4861.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matter K., Hunziker W., Mellman I. Basolateral sorting of LDL receptor in MDCK cells: the cytoplasmic domain contains two tyrosine-dependent targeting determinants. Cell. 1992 Nov 27;71(5):741–753. doi: 10.1016/0092-8674(92)90551-m. [DOI] [PubMed] [Google Scholar]
  31. Matter K., Mellman I. Mechanisms of cell polarity: sorting and transport in epithelial cells. Curr Opin Cell Biol. 1994 Aug;6(4):545–554. doi: 10.1016/0955-0674(94)90075-2. [DOI] [PubMed] [Google Scholar]
  32. Mellman I., Yamamoto E., Whitney J. A., Kim M., Hunziker W., Matter K. Molecular sorting in polarized and non-polarized cells: common problems, common solutions. J Cell Sci Suppl. 1993;17:1–7. doi: 10.1242/jcs.1993.supplement_17.1. [DOI] [PubMed] [Google Scholar]
  33. Mouland A. J., Bevan S., White J. H., Hendy G. N. Human chromogranin A gene. Molecular cloning, structural analysis, and neuroendocrine cell-specific expression. J Biol Chem. 1994 Mar 4;269(9):6918–6926. [PubMed] [Google Scholar]
  34. Ohno H., Stewart J., Fournier M. C., Bosshart H., Rhee I., Miyatake S., Saito T., Gallusser A., Kirchhausen T., Bonifacino J. S. Interaction of tyrosine-based sorting signals with clathrin-associated proteins. Science. 1995 Sep 29;269(5232):1872–1875. doi: 10.1126/science.7569928. [DOI] [PubMed] [Google Scholar]
  35. Owens R. J., Dubay J. W., Hunter E., Compans R. W. Human immunodeficiency virus envelope protein determines the site of virus release in polarized epithelial cells. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3987–3991. doi: 10.1073/pnas.88.9.3987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pearce-Pratt R., Malamud D., Phillips D. M. Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus. J Virol. 1994 May;68(5):2898–2905. doi: 10.1128/jvi.68.5.2898-2905.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Phillips D. M., Bourinbaiar A. S. Mechanism of HIV spread from lymphocytes to epithelia. Virology. 1992 Jan;186(1):261–273. doi: 10.1016/0042-6822(92)90080-9. [DOI] [PubMed] [Google Scholar]
  38. Phillips D. M., Zacharopoulos V. R., Tan X., Pearce-Pratt R. Mechanisms of sexual transmission of HIV: does HIV infect intact epithelia? Trends Microbiol. 1994 Nov;2(11):454–458. doi: 10.1016/0966-842x(94)90804-4. [DOI] [PubMed] [Google Scholar]
  39. Prill V., Lehmann L., von Figura K., Peters C. The cytoplasmic tail of lysosomal acid phosphatase contains overlapping but distinct signals for basolateral sorting and rapid internalization in polarized MDCK cells. EMBO J. 1993 May;12(5):2181–2193. doi: 10.1002/j.1460-2075.1993.tb05866.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Roth M. G., Srinivas R. V., Compans R. W. Basolateral maturation of retroviruses in polarized epithelial cells. J Virol. 1983 Mar;45(3):1065–1073. doi: 10.1128/jvi.45.3.1065-1073.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sargiacomo M., Lisanti M., Graeve L., Le Bivic A., Rodriguez-Boulan E. Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells. J Membr Biol. 1989 Mar;107(3):277–286. doi: 10.1007/BF01871942. [DOI] [PubMed] [Google Scholar]
  42. Shimizu H., Hasebe F., Tsuchie H., Morikawa S., Ushijima H., Kitamura T. Analysis of a human immunodeficiency virus type 1 isolate carrying a truncated transmembrane glycoprotein. Virology. 1992 Aug;189(2):534–546. doi: 10.1016/0042-6822(92)90577-c. [DOI] [PubMed] [Google Scholar]
  43. Simons K., Wandinger-Ness A. Polarized sorting in epithelia. Cell. 1990 Jul 27;62(2):207–210. doi: 10.1016/0092-8674(90)90357-k. [DOI] [PubMed] [Google Scholar]
  44. Subbramanian R. A., Cohen E. A. Molecular biology of the human immunodeficiency virus accessory proteins. J Virol. 1994 Nov;68(11):6831–6835. doi: 10.1128/jvi.68.11.6831-6835.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tashiro M., Yamakawa M., Tobita K., Seto J. T., Klenk H. D., Rott R. Altered budding site of a pantropic mutant of Sendai virus, F1-R, in polarized epithelial cells. J Virol. 1990 Oct;64(10):4672–4677. doi: 10.1128/jvi.64.10.4672-4677.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Terwilliger E. F., Godin B., Sodroski J. G., Haseltine W. A. Construction and use of a replication-competent human immunodeficiency virus (HIV-1) that expresses the chloramphenicol acetyltransferase enzyme. Proc Natl Acad Sci U S A. 1989 May;86(10):3857–3861. doi: 10.1073/pnas.86.10.3857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Thomas D. C., Roth M. G. The basolateral targeting signal in the cytoplasmic domain of glycoprotein G from vesicular stomatitis virus resembles a variety of intracellular targeting motifs related by primary sequence but having diverse targeting activities. J Biol Chem. 1994 Jun 3;269(22):15732–15739. [PubMed] [Google Scholar]
  48. Trono D. HIV accessory proteins: leading roles for the supporting cast. Cell. 1995 Jul 28;82(2):189–192. doi: 10.1016/0092-8674(95)90306-2. [DOI] [PubMed] [Google Scholar]
  49. Tucker S. P., Compans R. W. Virus infection of polarized epithelial cells. Adv Virus Res. 1993;42:187–247. doi: 10.1016/S0065-3527(08)60086-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Upender M., Raj L., Weir M. Megaprimer method for in vitro mutagenesis using parallel templates. Biotechniques. 1995 Jan;18(1):29-30, 32. [PubMed] [Google Scholar]
  51. Wandinger-Ness A., Bennett M. K., Antony C., Simons K. Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells. J Cell Biol. 1990 Sep;111(3):987–1000. doi: 10.1083/jcb.111.3.987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Welker R., Kottler H., Kalbitzer H. R., Kräusslich H. G. Human immunodeficiency virus type 1 Nef protein is incorporated into virus particles and specifically cleaved by the viral proteinase. Virology. 1996 May 1;219(1):228–236. doi: 10.1006/viro.1996.0240. [DOI] [PubMed] [Google Scholar]
  53. Willems L., Gatot J. S., Mammerickx M., Portetelle D., Burny A., Kerkhofs P., Kettmann R. The YXXL signalling motifs of the bovine leukemia virus transmembrane protein are required for in vivo infection and maintenance of high viral loads. J Virol. 1995 Jul;69(7):4137–4141. doi: 10.1128/jvi.69.7.4137-4141.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Willey R. L., Maldarelli F., Martin M. A., Strebel K. Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes. J Virol. 1992 Jan;66(1):226–234. doi: 10.1128/jvi.66.1.226-234.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yao X. J., Göttlinger H., Haseltine W. A., Cohen E. A. Envelope glycoprotein and CD4 independence of vpu-facilitated human immunodeficiency virus type 1 capsid export. J Virol. 1992 Aug;66(8):5119–5126. doi: 10.1128/jvi.66.8.5119-5126.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yao X. J., Subbramanian R. A., Rougeau N., Boisvert F., Bergeron D., Cohen E. A. Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J Virol. 1995 Nov;69(11):7032–7044. doi: 10.1128/jvi.69.11.7032-7044.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. van Meer G. Transport and sorting of membrane lipids. Curr Opin Cell Biol. 1993 Aug;5(4):661–673. doi: 10.1016/0955-0674(93)90137-f. [DOI] [PubMed] [Google Scholar]

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