Skip to main content
PMC home page
Journal of Virology logoLink to Journal of Virology
. 1995 Jun;69(6):3308–3314. doi: 10.1128/jvi.69.6.3308-3314.1995

Requirement of N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-mediated cell fusion.

H Schaal 1, M Klein 1, P Gehrmann 1, O Adams 1, A Scheid 1
PMCID: PMC189042  PMID: 7745678

Abstract

An expression vector was designed to test the structural requirements of the gp41 N terminus for human immunodeficiency virus type 1-induced membrane fusion. Mutations in the region coding for the N terminus of gp41 were found to disrupt glycoprotein expression because of deleterious effects on the Rev-responsive element (RRE). Insertion of an additional RRE in the 3'-noncoding sequence of env made possible efficient glycoprotein expression, irrespective of the mutations introduced into the RRE in the natural location. This permitted the insertion of the unique restriction site SpeI within the N-terminal sequences of gp41, allowing convenient and efficient mutation of the gp41 N terminus by using double-stranded synthetic oligonucleotides. Mutants with deletions of 1 to 7 amino acids of the N terminus were constructed. Expression and cleavage of all mutants were confirmed by Western immunoblot analysis with anti-gp41 antibodies. The capability of mutants to induce membrane fusion was monitored following transfection of HeLa-T4+ cell lines with wild-type and mutant expression vectors by electroporation and microinjection. The efficiency of cell-fusing activity decreased drastically with deletion of 3 and 4 amino acids and was completely lost with deletion of 5 amino acids. Cotransfection of the parent and mutant expression vectors resulted in reduced cell-fusing activity. The extent of this dominant interference by mutant glycoprotein paralleled the decrease in cell-fusing activity of the mutants alone. This suggests the existence of a specific N-terminal structure required for fusing activity. However, there does not appear to be a stringent requirement for the precise length of the N terminus. This finding is supported by the length variation of this region among natural human immunodeficiency virus type 1 isolates and is in contrast to the apparent stringency in the length of analogous N-terminal structures of influenza A virus and paramyxovirus fusion glycoproteins.

Full Text

The Full Text of this article is available as a PDF (646.8 KB).

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Akrigg A., Wilkinson G. W., Angliss S., Greenaway P. J. HIV-1 indicator cell lines. AIDS. 1991 Feb;5(2):153–158. doi: 10.1097/00002030-199102000-00004. [DOI] [PubMed] [Google Scholar]
  3. Ansorge W. Improved system for capillary microinjection into living cells. Exp Cell Res. 1982 Jul;140(1):31–37. doi: 10.1016/0014-4827(82)90152-5. [DOI] [PubMed] [Google Scholar]
  4. Brasseur R., Lorge P., Goormaghtigh E., Ruysschaert J. M., Espion D., Burny A. The mode of insertion of the paramyxovirus F1 N-terminus into lipid matrix, an initial step in host cell/virus fusion. Virus Genes. 1988 Jul;1(4):325–332. doi: 10.1007/BF00257096. [DOI] [PubMed] [Google Scholar]
  5. Capecchi M. R. High efficiency transformation by direct microinjection of DNA into cultured mammalian cells. Cell. 1980 Nov;22(2 Pt 2):479–488. doi: 10.1016/0092-8674(80)90358-x. [DOI] [PubMed] [Google Scholar]
  6. Ceriotti A., Colman A. Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes. J Cell Biol. 1990 Aug;111(2):409–420. doi: 10.1083/jcb.111.2.409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choppin P. W., Richardson C. D., Merz D. C., Hall W. W., Scheid A. The functions and inhibition of the membrane glycoproteins of paramyxoviruses and myxoviruses and the role of the measles virus M protein in subacute sclerosing panencephalitis. J Infect Dis. 1981 Mar;143(3):352–363. doi: 10.1093/infdis/143.3.352. [DOI] [PubMed] [Google Scholar]
  8. Choppin P. W., Scheid A. The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Rev Infect Dis. 1980 Jan-Feb;2(1):40–61. doi: 10.1093/clinids/2.1.40. [DOI] [PubMed] [Google Scholar]
  9. Cochrane A. W., Chen C. H., Rosen C. A. Specific interaction of the human immunodeficiency virus Rev protein with a structured region in the env mRNA. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1198–1202. doi: 10.1073/pnas.87.3.1198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cook K. S., Fisk G. J., Hauber J., Usman N., Daly T. J., Rusche J. R. Characterization of HIV-1 REV protein: binding stoichiometry and minimal RNA substrate. Nucleic Acids Res. 1991 Apr 11;19(7):1577–1583. doi: 10.1093/nar/19.7.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crise B., Ruusala A., Zagouras P., Shaw A., Rose J. K. Oligomerization of glycolipid-anchored and soluble forms of the vesicular stomatitis virus glycoprotein. J Virol. 1989 Dec;63(12):5328–5333. doi: 10.1128/jvi.63.12.5328-5333.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dayton A. I., Terwilliger E. F., Potz J., Kowalski M., Sodroski J. G., Haseltine W. A. Cis-acting sequences responsive to the rev gene product of the human immunodeficiency virus. J Acquir Immune Defic Syndr. 1988;1(5):441–452. [PubMed] [Google Scholar]
  13. Dayton E. T., Konings D. A., Powell D. M., Shapiro B. A., Butini L., Maizel J. V., Dayton A. I. Extensive sequence-specific information throughout the CAR/RRE, the target sequence of the human immunodeficiency virus type 1 Rev protein. J Virol. 1992 Feb;66(2):1139–1151. doi: 10.1128/jvi.66.2.1139-1151.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dayton E. T., Powell D. M., Dayton A. I. Functional analysis of CAR, the target sequence for the Rev protein of HIV-1. Science. 1989 Dec 22;246(4937):1625–1629. doi: 10.1126/science.2688093. [DOI] [PubMed] [Google Scholar]
  15. Dewar R. L., Vasudevachari M. B., Natarajan V., Salzman N. P. Biosynthesis and processing of human immunodeficiency virus type 1 envelope glycoproteins: effects of monensin on glycosylation and transport. J Virol. 1989 Jun;63(6):2452–2456. doi: 10.1128/jvi.63.6.2452-2456.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Earl P. L., Doms R. W., Moss B. Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. Proc Natl Acad Sci U S A. 1990 Jan;87(2):648–652. doi: 10.1073/pnas.87.2.648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  18. Emerman M., Vazeux R., Peden K. The rev gene product of the human immunodeficiency virus affects envelope-specific RNA localization. Cell. 1989 Jun 30;57(7):1155–1165. doi: 10.1016/0092-8674(89)90053-6. [DOI] [PubMed] [Google Scholar]
  19. Feinberg M. B., Jarrett R. F., Aldovini A., Gallo R. C., Wong-Staal F. HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA. Cell. 1986 Sep 12;46(6):807–817. doi: 10.1016/0092-8674(86)90062-0. [DOI] [PubMed] [Google Scholar]
  20. Felber B. K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G. N. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1495–1499. doi: 10.1073/pnas.86.5.1495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Felser J. M., Klimkait T., Silver J. A syncytia assay for human immunodeficiency virus type I (HIV-I) envelope protein and its use in studying HIV-I mutations. Virology. 1989 Jun;170(2):566–570. doi: 10.1016/0042-6822(89)90448-0. [DOI] [PubMed] [Google Scholar]
  22. Freed E. O., Delwart E. L., Buchschacher G. L., Jr, Panganiban A. T. A mutation in the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 dominantly interferes with fusion and infectivity. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):70–74. doi: 10.1073/pnas.89.1.70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Freed E. O., Myers D. J., Risser R. Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4650–4654. doi: 10.1073/pnas.87.12.4650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gallaher W. R. Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus. Cell. 1987 Jul 31;50(3):327–328. doi: 10.1016/0092-8674(87)90485-5. [DOI] [PubMed] [Google Scholar]
  25. Gething M. J., White J. M., Waterfield M. D. Purification of the fusion protein of Sendai virus: analysis of the NH2-terminal sequence generated during precursor activation. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2737–2740. doi: 10.1073/pnas.75.6.2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Gonzalez-Scarano F., Waxham M. N., Ross A. M., Hoxie J. A. Sequence similarities between human immunodeficiency virus gp41 and paramyxovirus fusion proteins. AIDS Res Hum Retroviruses. 1987 Fall;3(3):245–252. doi: 10.1089/aid.1987.3.245. [DOI] [PubMed] [Google Scholar]
  27. Gorman C. M., Howard B. H., Reeves R. Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate. Nucleic Acids Res. 1983 Nov 11;11(21):7631–7648. doi: 10.1093/nar/11.21.7631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hadzopoulou-Cladaras M., Felber B. K., Cladaras C., Athanassopoulos A., Tse A., Pavlakis G. N. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J Virol. 1989 Mar;63(3):1265–1274. doi: 10.1128/jvi.63.3.1265-1274.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hammarskjöld M. L., Heimer J., Hammarskjöld B., Sangwan I., Albert L., Rekosh D. Regulation of human immunodeficiency virus env expression by the rev gene product. J Virol. 1989 May;63(5):1959–1966. doi: 10.1128/jvi.63.5.1959-1966.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Heaphy S., Dingwall C., Ernberg I., Gait M. J., Green S. M., Karn J., Lowe A. D., Singh M., Skinner M. A. HIV-1 regulator of virion expression (Rev) protein binds to an RNA stem-loop structure located within the Rev response element region. Cell. 1990 Feb 23;60(4):685–693. doi: 10.1016/0092-8674(90)90671-z. [DOI] [PubMed] [Google Scholar]
  31. Horth M., Lambrecht B., Khim M. C., Bex F., Thiriart C., Ruysschaert J. M., Burny A., Brasseur R. Theoretical and functional analysis of the SIV fusion peptide. EMBO J. 1991 Oct;10(10):2747–2755. doi: 10.1002/j.1460-2075.1991.tb07823.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Horvath C. M., Lamb R. A. Studies on the fusion peptide of a paramyxovirus fusion glycoprotein: roles of conserved residues in cell fusion. J Virol. 1992 Apr;66(4):2443–2455. doi: 10.1128/jvi.66.4.2443-2455.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Huang X. J., Hope T. J., Bond B. L., McDonald D., Grahl K., Parslow T. G. Minimal Rev-response element for type 1 human immunodeficiency virus. J Virol. 1991 Apr;65(4):2131–2134. doi: 10.1128/jvi.65.4.2131-2134.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kjems J., Sharp P. A. The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J Virol. 1993 Aug;67(8):4769–4776. doi: 10.1128/jvi.67.8.4769-4776.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kowalski M., Bergeron L., Dorfman T., Haseltine W., Sodroski J. Attenuation of human immunodeficiency virus type 1 cytopathic effect by a mutation affecting the transmembrane envelope glycoprotein. J Virol. 1991 Jan;65(1):281–291. doi: 10.1128/jvi.65.1.281-291.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kowalski M., Potz J., Basiripour L., Dorfman T., Goh W. C., Terwilliger E., Dayton A., Rosen C., Haseltine W., Sodroski J. Functional regions of the envelope glycoprotein of human immunodeficiency virus type 1. Science. 1987 Sep 11;237(4820):1351–1355. doi: 10.1126/science.3629244. [DOI] [PubMed] [Google Scholar]
  37. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  38. Maddon P. J., Dalgleish A. G., McDougal J. S., Clapham P. R., Weiss R. A., Axel R. The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell. 1986 Nov 7;47(3):333–348. doi: 10.1016/0092-8674(86)90590-8. [DOI] [PubMed] [Google Scholar]
  39. Malim M. H., Cullen B. R. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993 Oct;13(10):6180–6189. doi: 10.1128/mcb.13.10.6180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Malim M. H., Hauber J., Le S. Y., Maizel J. V., Cullen B. R. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989 Mar 16;338(6212):254–257. doi: 10.1038/338254a0. [DOI] [PubMed] [Google Scholar]
  41. Malim M. H., Tiley L. S., McCarn D. F., Rusche J. R., Hauber J., Cullen B. R. HIV-1 structural gene expression requires binding of the Rev trans-activator to its RNA target sequence. Cell. 1990 Feb 23;60(4):675–683. doi: 10.1016/0092-8674(90)90670-a. [DOI] [PubMed] [Google Scholar]
  42. McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosek J. C., Reyes G. R., Weissman I. L. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell. 1988 Apr 8;53(1):55–67. doi: 10.1016/0092-8674(88)90487-4. [DOI] [PubMed] [Google Scholar]
  43. Olsen H. S., Nelbock P., Cochrane A. W., Rosen C. A. Secondary structure is the major determinant for interaction of HIV rev protein with RNA. Science. 1990 Feb 16;247(4944):845–848. doi: 10.1126/science.2406903. [DOI] [PubMed] [Google Scholar]
  44. Owens R. J., Tanner C. C., Mulligan M. J., Srinivas R. V., Compans R. W. Oligopeptide inhibitors of HIV-induced syncytium formation. AIDS Res Hum Retroviruses. 1990 Nov;6(11):1289–1296. doi: 10.1089/aid.1990.6.1289. [DOI] [PubMed] [Google Scholar]
  45. Pinter A., Honnen W. J., Tilley S. A., Bona C., Zaghouani H., Gorny M. K., Zolla-Pazner S. Oligomeric structure of gp41, the transmembrane protein of human immunodeficiency virus type 1. J Virol. 1989 Jun;63(6):2674–2679. doi: 10.1128/jvi.63.6.2674-2679.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Richardson C. D., Scheid A., Choppin P. W. Specific inhibition of paramyxovirus and myxovirus replication by oligopeptides with amino acid sequences similar to those at the N-termini of the F1 or HA2 viral polypeptides. Virology. 1980 Aug;105(1):205–222. doi: 10.1016/0042-6822(80)90168-3. [DOI] [PubMed] [Google Scholar]
  47. Rosen C. A., Terwilliger E., Dayton A., Sodroski J. G., Haseltine W. A. Intragenic cis-acting art gene-responsive sequences of the human immunodeficiency virus. Proc Natl Acad Sci U S A. 1988 Apr;85(7):2071–2075. doi: 10.1073/pnas.85.7.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  49. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schawaller M., Smith G. E., Skehel J. J., Wiley D. C. Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV. Virology. 1989 Sep;172(1):367–369. doi: 10.1016/0042-6822(89)90142-6. [DOI] [PubMed] [Google Scholar]
  51. Server A. C., Smith J. A., Waxham M. N., Wolinsky J. S., Goodman H. M. Purification and amino-terminal protein sequence analysis of the mumps virus fusion protein. Virology. 1985 Jul 30;144(2):373–383. doi: 10.1016/0042-6822(85)90279-x. [DOI] [PubMed] [Google Scholar]
  52. Skehel J. J., Waterfield M. D. Studies on the primary structure of the influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 1975 Jan;72(1):93–97. doi: 10.1073/pnas.72.1.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Slepushkin V. A., Kornilaeva G. V., Andreev S. M., Sidorova M. V., Petrukhina A. O., Matsevich G. R., Raduk S. V., Grigoriev V. B., Makarova T. V., Lukashov V. V. Inhibition of human immunodeficiency virus type 1 (HIV-1) penetration into target cells by synthetic peptides mimicking the N-terminus of the HIV-1 transmembrane glycoprotein. Virology. 1993 May;194(1):294–301. doi: 10.1006/viro.1993.1260. [DOI] [PubMed] [Google Scholar]
  54. Sodroski J., Goh W. C., Rosen C., Dayton A., Terwilliger E., Haseltine W. A second post-transcriptional trans-activator gene required for HTLV-III replication. Nature. 1986 May 22;321(6068):412–417. doi: 10.1038/321412a0. [DOI] [PubMed] [Google Scholar]
  55. Stein B. S., Engleman E. G. Intracellular processing of the gp160 HIV-1 envelope precursor. Endoproteolytic cleavage occurs in a cis or medial compartment of the Golgi complex. J Biol Chem. 1990 Feb 15;265(5):2640–2649. [PubMed] [Google Scholar]
  56. Strebel K., Daugherty D., Clouse K., Cohen D., Folks T., Martin M. A. The HIV 'A' (sor) gene product is essential for virus infectivity. Nature. 1987 Aug 20;328(6132):728–730. doi: 10.1038/328728a0. [DOI] [PubMed] [Google Scholar]
  57. Vonèche V., Callebaut I., Lambrecht B., Brasseur R., Burny A., Portetelle D. Enhancement of bovine leukemia virus-induced syncytia formation by di- and tripeptides. Virology. 1993 Jan;192(1):307–311. doi: 10.1006/viro.1993.1034. [DOI] [PubMed] [Google Scholar]
  58. Vonèche V., Portetelle D., Kettmann R., Willems L., Limbach K., Paoletti E., Ruysschaert J. M., Burny A., Brasseur R. Fusogenic segments of bovine leukemia virus and simian immunodeficiency virus are interchangeable and mediate fusion by means of oblique insertion in the lipid bilayer of their target cells. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3810–3814. doi: 10.1073/pnas.89.9.3810. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES