Identification of the paired basic convertases implicated in HIV gp160 processing based on in vitro assays and expression in CD4(+) cell lines.
The human immunodeficiency virus HIV envelope glycoprotein gp160 is synthesized as an inactive precursor, which is processed into its fusiogenic form gp120/gp41 by host cell proteinases during its intracellular trafficking. Kexin/subtilisin-related endoproteases have been proposed to be enzyme candidates for this maturation process. In the present study, 1) we examined the ability of partially purified precursor convertases and their isoforms to cleave gp160 in vitro. The data demonstrate that all the convertases tested specifically cleave the HIV envelope glycoprotein into gp120 and gp41. 2) We demonstrated that a 19-amino acid model peptide spanning the gp120/gp41 junction is cleaved by all convertases at the same gp160 site as that recognized in HIV-infected cells. 3) In an effort to evaluate specific convertase inhibitors, we showed that the alpha1-antitrypsin variant, alpha1-PDX, inhibits equally well the ability of the tested convertases to cleave gp160 in vitro. 4) Three lymphocyte cell lines were screened by reverse transcription polymerase chain reaction in an effort to identify which are the convertases expressed in the most common HIV target, the CD4(+) lymphocytes. The data demonstrate that furin, PC5/6, and the newly cloned PC7 are the main transcribed convertases, suggesting that these proteinases are the major gp160-converting enzymes in T4 lymphocytes.
Decroly
E
E
Laboratoire de Chimie Physique des Macromolécules aux Interfaces, CP206/2, Université libre de Bruxelles, 1050, Brussels, Belgium.
Wouters
S
S
Di Bello
C
C
Lazure
C
C
Ruysschaert
J M
JM
Seidah
N G
NG
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045683:Furin; D015870:Gene Expression; D019302:HIV Envelope Protein gp160 / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D045706:Proprotein Convertase 2; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D012697:Serine Endopeptidases / Q000378:metabolism*; D013381:Subtilisins / Q000378:metabolism*
D019302:HIV Envelope Protein gp160; D012333:RNA, Messenger; D011994:Recombinant Proteins; C080654:PCSK6 protein, human; C099101:PCSK7 protein, human; D045702:Proprotein Convertase 5; D043484:Proprotein Convertases; D012697:Serine Endopeptidases; D013381:Subtilisins; D045683:Furin; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases; D002118:Calcium