Folding, interaction with GRP78-BiP, assembly, and transport of the human immunodeficiency virus type 1 envelope protein.
A detailed kinetic and quantitative analysis of the early and late biosynthetic events undergone by the human immunodeficiency virus type 1 envelope protein expressed by a recombinant vaccinia virus was performed. Early folding events that occurred in the endoplasmic reticulum included disulfide bond formation (t1/2 approximately 10 min), folding of envelope protein into a form competent to bind CD4 (t1/2 approximately 15 min), and specific and transient association and dissociation with GRP78-BiP (t1/2 approximately 25 min). After initial folding, envelope protein monomers formed noncovalently associated dimers with high efficiency (t1/2 approximately 30 min). Studies with brefeldin A, a compound that inhibits endoplasmic reticulum-to-Golgi transport, suggested that assembly occurred in the endoplasmic reticulum while cleavage of gp160 into gp120/gp41 subunits occurred in a post-endoplasmic reticulum compartment. Transport to the Golgi was monitored by modification of N-linked sugars to forms partially resistant to endoglycosidase H. The kinetics of endoglycosidase H resistance were nearly identical to the kinetics of gp160 cleavage (t1/2 approximately 80 min). Cleavage efficiency was strongly cell type dependent, ranging from 13 to 70%. By contrast, approximately 50% of the gp120 generated by the cleavage event was shed (t1/2 approximately 120 min) regardless of the cell type used. The results are discussed in terms of the overall biosynthetic pathway of the envelope protein and provide a framework with which to assess the effects of mutations on structure and function.
Earl
P L
PL
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Moss
B
B
Doms
R W
RW
D000118:Acetylglucosaminidase / Q000494:pharmacology; D001692:Biological Transport; D015704:CD4 Antigens / Q000276:immunology*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004351:Drug Resistance; D004721:Endoplasmic Reticulum / Q000276:immunology / Q000382:microbiology; D000091342:Endoplasmic Reticulum Chaperone BiP; D015686:Gene Products, env / Q000235:genetics / Q000276:immunology; D006056:Golgi Apparatus / Q000276:immunology / Q000382:microbiology; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41 / Q000276:immunology; D015497:HIV-1 / Q000187:drug effects / Q000276:immunology*; D006360:Heat-Shock Proteins; D006801:Humans; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D018832:Molecular Chaperones; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics / Q000276:immunology; D014616:Vaccinia virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*
D015704:CD4 Antigens; D002352:Carrier Proteins; D004220:Disulfides; D000091342:Endoplasmic Reticulum Chaperone BiP; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D011498:Protein Precursors; D014759:Viral Envelope Proteins; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase