Deletion of a single N-linked glycosylation site from the transmembrane envelope protein of human immunodeficiency virus type 1 stops cleavage and transport of gp160 preventing env-mediated fusion.

The transmembrane envelope glycoprotein (gp41) of human immunodeficiency virus type 1 possesses four consensus sites (Asn-X-Ser/Thr) for the incorporation of N-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 in vitro mutagenesis of gp160 sequence in the non-infectious viral clone pEVd1443, 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 N-linked glycosylation of Asn-642 in the glycoprotein produced by the pEVd1443 expression system is necessary for the correct intracellular processing of gp160 to yield surface-expressed, fusogenic gp41.

[1]  John W. Mellors,et al.  Human retroviruses and AIDS 1996. A compilation and analysis of nucleic acid and amino acid sequences , 1997 .

[2]  I. Jones,et al.  Legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin , 1993, Journal of virology.

[3]  J. Sodroski,et al.  Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein , 1993, Journal of virology.

[4]  M. Kieny,et al.  Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain , 1993, Journal of virology.

[5]  L. Ratner,et al.  Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function. , 1992, Virology.

[6]  X. Yu,et al.  Mutational analysis of conserved N-linked glycosylation sites of human immunodeficiency virus type 1 gp41 , 1992, Journal of virology.

[7]  J. Skehel,et al.  Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity , 1992, Cell.

[8]  E. Freed,et al.  Characterization of the fusion domain of the human immunodeficiency virus type 1 envelope glycoprotein gp41. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[9]  M. Pawlita,et al.  Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site , 1990, Journal of virology.

[10]  J. Lippincott-Schwartz,et al.  Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin a suggests an ER recycling pathway , 1990, Cell.

[11]  J. Skehel,et al.  Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV. , 1989, Virology.

[12]  T. Klimkait,et al.  A syncytia assay for human immunodeficiency virus type I (HIV-I) envelope protein and its use in studying HIV-I mutations. , 1989, Virology.

[13]  R. Pal,et al.  Role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type 1. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Bonifacino,et al.  Biosynthesis, cleavage, and degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[15]  I. Weissman,et al.  Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus , 1988, Cell.

[16]  P. Earl,et al.  In vitro mutagenesis identifies a region within the envelope gene of the human immunodeficiency virus that is critical for infectivity , 1988, Journal of virology.

[17]  P. Earl,et al.  Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes , 1987, Molecular and cellular biology.

[18]  Robin A. Weiss,et al.  The T4 gene encodes the AIDS virus receptor and is expressed in the immune system and the brain , 1986, Cell.

[19]  H. Klenk,et al.  Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus. , 1986, The EMBO journal.

[20]  K. Steimer,et al.  Induction of CD4-dependent cell fusion by the HTLV-III/LAV envelope glycoprotein , 1986, Nature.

[21]  M. Martin,et al.  Identification of conserved and divergent domains within the envelope gene of the acquired immunodeficiency syndrome retrovirus. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Sodroski,et al.  Major glycoprotein antigens that induce antibodies in AIDS patients are encoded by HTLV-III. , 1985, Science.

[23]  H. Lodish,et al.  Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex , 1984, The Journal of cell biology.

[24]  W. N. Burnette,et al.  "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. , 1981, Analytical biochemistry.

[25]  S. Singer,et al.  Antibody-induced linkages of plasma membrane proteins to intracellular actomyosin-containing filaments in cultured fibroblasts. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[26]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[27]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[28]  J. T. Syverton,et al.  STUDIES ON THE PROPAGATION IN VITRO OF POLIOMYELITIS VIRUSES , 1952, The Journal of experimental medicine.

[29]  P. Earl,et al.  Oligomeric structure of the human immunodeficiency virus type 1 envelope glycoprotein. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[30]  R. Pal,et al.  Processing and secretion of envelope glycoproteins of human immunodeficiency virus type 1 in the presence of trimming glucosidase inhibitor deoxynojirimycin. , 1989, Intervirology.

[31]  J. Sodroski,et al.  Role of the HTLV-III/LAV envelope in syncytium formation and cytopathicity , 1986, Nature.

[32]  S. Kornfeld,et al.  Assembly of asparagine-linked oligosaccharides. , 1985, Annual review of biochemistry.

[33]  Thomas A. Kunkel,et al.  Rapid and efficient site-specific mutagenesis without phenotypic selection. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[34]  M. Greaves,et al.  The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus , 1984, Nature.