1887

Abstract

The transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus type 1 possesses four consensus sites (Asn-X-Ser/Thr) for the incorporation of -linked sugars situated on the extracellular domain of the molecule. The purpose of this investigation was to determine the significance of each of these sites in relation to the structure and function of the viral envelope glycoprotein. Each of the four sites was removed by mutagenesis of gp160 sequence in the non-infectious viral clone pEVdl443, so that amino acids 616, 621, 642 and 679 were each changed from asparagine to serine. The effects of mutagenesis were assessed by syncytium assay after wild-type or mutant envelope clones had been transfected into CD4 HeLa cells. Removal of the glycosylation site at position 642 resulted in the synthesis of precursor gp160 that was neither cleaved, to give gp120 and gp41, nor transported to the plasma membrane of transfected cells. A consequence of these events was that envelope mutant 642 failed to induce syncytia between neighbouring cells in which it had been expressed. The results of this study indicate that -linked glycosylation of Asn-642 in the glycoprotein produced by the pEVdl443 expression system is necessary for the correct intracellular processing of gp160 to yield surface-expressed, fusogenic gp41.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-75-6-1389
1994-06-01
2022-01-21
Loading full text...

Full text loading...

/deliver/fulltext/jgv/75/6/JV0750061389.html?itemId=/content/journal/jgv/10.1099/0022-1317-75-6-1389&mimeType=html&fmt=ahah

References

  1. Allan J. S., Colligan J. E., Barin F., Mcclane M. F., Sodroski J. G., Rosen C. A., Haseltine W. A., Lee T. H., Essex M. 1985; Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. Science 228:1091–1094
    [Google Scholar]
  2. Ash J., Louvard D., Singer S. J. 1977; Antibody-induced linkages of plasma membrane proteins to intracellular actinomyosin- containing filaments in cultured fibroblasts. Proceedings of the National Academy of Sciences U.S.A.: 745584–5588
    [Google Scholar]
  3. Bosch V., Pawilta M. 1990; Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site. Journal of Virology 64:2337–2344
    [Google Scholar]
  4. Burnette W. N. 1981; “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated Protein A. Analytical Biochemistry 112:195–203
    [Google Scholar]
  5. Cao J., Bergeron L., Helseth E., Thali M., Repke H., Sodroski J. 1993; Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein. Journal of Virology 67:2747–2755
    [Google Scholar]
  6. Dalgleish A. G., Beverley P. C. L., Clapham P. R., Crawford D. H., Greaves M. F., Weiss R. A. 1984; The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus. Nature; London: 312763–767
    [Google Scholar]
  7. Dedera D. A., Gu R., Ratner L. 1992; Role of asparagine linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function. Virology 187:377–382
    [Google Scholar]
  8. Earl P. L., Doms R. W., Moss B. 1990; Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. Proceedings of the National Academy of Sciences U.S.A.: 87648–652
    [Google Scholar]
  9. Felser J. M., Klimkait T., Silver J. 1989; A syncytia assay for human immunodeficiency virus type 1 (HIV-1) envelope protein and its use in studying HIV-1 mutations. Virology 170:566–570
    [Google Scholar]
  10. Fenouillet E., Jones I., Powell B., Schmitt D., Kieny M. P., Gluckman J. C. 1993; Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain. Journal of Virology 67:150–160
    [Google Scholar]
  11. Freed E. O., Myers D. J., Risser R. 1990; Characterisation of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. Proceedings of the National Academy of Sciences U.S.A.: 874650–4654
    [Google Scholar]
  12. Fuerst T. R., Earl P. L., Moss B. 1987; Use of a hybrid vaccinia virus-T7 rNA polymerase system for expression of target genes. Molecular and Cellular Biology 7:2538–2544
    [Google Scholar]
  13. Godley L., Pfeifer J., Steinhauser D., Ely B., Shaw G., Kaufmann R., Suchanek E., Pabo C., Skehel J. J., Wiley D. C., Wharton S. 1992; Induction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity. Cell 68:635–645
    [Google Scholar]
  14. Kornfeld R., Kornfeld S. 1985; Assembly of asparagine-linked oligosaccharides. Annual Review of Biochemistry 54:631–664
    [Google Scholar]
  15. Kunkel T. A., Roberts J. D., Zakour R. A. 1987; Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods in Enzvmology 154:367–382
    [Google Scholar]
  16. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
    [Google Scholar]
  17. Lee W. R., Yu X. F., Syu W. J., Essex M., Lee T. H. 1992; Mutational analysis of conserved N-linked glycosylation sites of human immunodeficiency virus type 1 gp41. Journal of Virology 66:1799–1803
    [Google Scholar]
  18. Lifson J. D., Feinberg M. B., Reyes G. R., Rabin L., Banapour B., Chakrabarti S., Moss B., Wong-Staal F., Steimer K. S., Engleman E. G. 1986; Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein. Nature; London: 323725–728
    [Google Scholar]
  19. Lippincott-Schwartz J., Donaldson J. G., Schweizer A., Berger E. G., Hauri H. P., Yuan L. C., Klausner R. D. 1990; Microtubule dependent retrograde transport of proteins into the ER in the presence of Brefeldin A suggests an ER recycling pathway. Cell 60:821–836
    [Google Scholar]
  20. Lodish F., Kong N. 1984; Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex. Journal of Cell Biology 98:1720–1729
    [Google Scholar]
  21. McCune J. M., Rabin L. B., Feinberg M. B., Lieberman M., Kosec J. C., Reyes G. R., Weissman I. L. 1988; Endo- proteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell 53:55–67
    [Google Scholar]
  22. Maddon P. J., Dalgleish A. G., Mcdougal J. S., Clapham P. R., Weiss R. A., Axel R. 1986; The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain. Cell 47:333–348
    [Google Scholar]
  23. Morikawa Y., Barsov E., Jones I. 1993; Legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin. Journal of Virology 67:3601–3604
    [Google Scholar]
  24. Myers G., Rabson A. B., Berzofsky J. A., Smith T. F., Wong-Staal F. 1990 Human Retroviruses and AIDS 1990: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequence Los Alamos National Laboratory, Los Alamos, New Mexico:
    [Google Scholar]
  25. Pal R., Hoke G. M., Sarngadharan M. G. 1989a; Role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences U.S.A.: 863384–3388
    [Google Scholar]
  26. Pal R., Kalyanaraman V. S., Hoke G. M., Sarngadharan M. G. 1989b; Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin. Intervirology 30:27–35
    [Google Scholar]
  27. Sanger F., Nicklen S., Coulson A. R. 1977; DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences U.S.A.: 745463–5467
    [Google Scholar]
  28. Schawaller M., Smith G. E., Skehel J. J., Wiley D. C. 1989; Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIY. Virology 172:367–369
    [Google Scholar]
  29. Scherer W. F., Syverton J. T., Gey G. O. 1953; Studies on the propagation in vitro of poliomyelitis viruses. Journal of Experimental Medicine 97:695–709
    [Google Scholar]
  30. Schuy W., Will C., Kuroda K., Scholtissek C., Garten W., Klenk H. 1986; Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus. EM BO Journal 5:2831–2836
    [Google Scholar]
  31. Sodroski J., Goh W., Rosen C., Campbell K., Haseltine W. 1986; Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity. Nature; London: 322470–474
    [Google Scholar]
  32. Willey R. L., Rutledge R. A., Dias S., Folks T., Theodore T., Buckler C. E., Martin M. A. 1986; Identification of conserved and divergent domains within the envelope gene of the acquired immunodeficiency syndrome retrovirus. Proceedings of the National Academy of Sciences U.S.A.: 835038–5042
    [Google Scholar]
  33. Willey R. L., Bonifacino J. S., Potts B. J., Martin M. A., Klausner R. D. 1988a; Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proceedings of the National Academy of Sciences U.S.A.: 859580–9584
    [Google Scholar]
  34. Willey R. L., Smith D. H., Laskey L. A., Theodore T. S., Earl P. L., Moss B., Capon D. J., Martin M. A. 1988b; In vitro mutagenesis identifies a region within the envelope gene of human immunodeficiency virus that is critical for infectivity. Journal of Virology 62:139–147
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/0022-1317-75-6-1389
Loading
/content/journal/jgv/10.1099/0022-1317-75-6-1389
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error