Structural Investigation and Kinetic Characterization of Potential Cleavage Sites of HIV Gp160 by Human Furin and Pc1

Brakch, Noureddine; Dettin, Monica; Scarinci, C; Seidah, Nabil G.; DiBello, Carlo

doi:10.1006/bbrc.1995.2137

Cited by 20 publications

(26 citation statements)

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“…For the wild-type HIV-1 gp160 with two separated furin motifs, the effect of sequence duplication on cleavage efficiency is not known. Nevertheless, cleavage of HIV-1 gp160 by furin occurred preferentially at the cleavage-proximal physiological site both in vivo (21,58) and in vitro (8,18), as was the case for the influenza virus HA 0 precursor. This cleavage site preference was further demonstrated in vitro with two other proprotein convertases for the influenza virus HA 0 precursor (5) and HIV-1 gp160 (8,18).…”

Section: Discussionmentioning

confidence: 89%

“…Nevertheless, cleavage of HIV-1 gp160 by furin occurred preferentially at the cleavage-proximal physiological site both in vivo (21,58) and in vitro (8,18), as was the case for the influenza virus HA 0 precursor. This cleavage site preference was further demonstrated in vitro with two other proprotein convertases for the influenza virus HA 0 precursor (5) and HIV-1 gp160 (8,18). The pronounced cleavage of prM in JEVpr/16681 is consistent with this role of duplicated furin motifs in enhancing the cleavability of the physiologic cleavage site, as documented for the influenza virus HA 0 precursor and possibly HIV-1 gp160.…”

Section: Discussionmentioning

confidence: 89%

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Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Keelapang

Sriburi

Supasa

et al. 2004

J Virol

102

114

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During the export of flavivirus particles through the secretory pathway, a viral envelope glycoprotein, prM, is cleaved by the proprotein convertase furin; this cleavage is required for the subsequent rearrangement of receptor-binding E glycoprotein and for virus infectivity. Similar to many furin substrates, prM in vector-borne flaviviruses contains basic residues at positions P1, P2, and P4 proximal to the cleavage site; in addition, a number of charged residues are found at position P3 and between positions P5 and P13 that are conserved for each flavivirus antigenic complex. The influence of additional charged residues on pr-M cleavage and virus replication was investigated by replacing the 13-amino-acid, cleavage-proximal region of a dengue virus (strain 16681) with those of tick-borne encephalitis virus (TBEV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV) and by comparing the resultant chimeric viruses generated from RNA-transfected mosquito cells. Among the three chimeric viruses, cleavage of prM was enhanced to a larger extent in JEVpr/16681 than in YFVpr/16681 but was slightly reduced in TBEVpr/16681. Unexpectedly, JEVpr/16681 exhibited decreased focus size, reduced peak titer, and depressed replication in C6/36, PS, and Vero cell lines. The reduction of JEVpr/16681 multiplication correlated with delayed export of infectious virions out of infected cells but not with changes in specific infectivity. Binding of JEVpr/16681 to immobilized heparin and the heparin-inhibitable infection of cells were not altered. Thus, diverse pr-M junction-proximal sequences of flaviviruses differentially influence pr-M cleavage when tested in a dengue virus prM background. More importantly, greatly enhanced prM cleavability adversely affects dengue virus export while exerting a minimal effect on infectivity. Because extensive changes of charged residues at the pr-M junction, as in JEVpr/16681, were not observed among a large number of dengue virus isolates, these results provide a possible mechanism by which the sequence conservation of the pr-M junction of dengue virus is maintained in nature.The genus Flavivirus within the family Flaviviridae comprises about 73 enveloped RNA viruses that are transmitted by either mosquitoes or ticks or without a known vector (11). For these viruses, a single-stranded RNA genome of about 11 kb encodes a polyprotein, which is cleaved by cellular and viral enzymes into three structural proteins (C, prM/M, and E) and seven nonstructural proteins (54). Virions consist of two envelope proteins, E and prM/M, and an internal C protein, which binds genomic RNA. Differences in antigenicities of E allow the subdivision of flaviviruses into eight antigenic complexes and a number of unclassified viruses, which include the prototype yellow fever virus (YFV) (12). More recent assignments based on nucleotide sequence variations of the nonstructural gene NS5 generally agree with antigenic classifications (49).The assembly of flaviviruses in the endoplasmic reticulum is followed by modif...

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Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 89%

Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Keelapang

Sriburi

Supasa

et al. 2004

J Virol

102

114

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“…All cleavage reactions were performed in 100 l of 50 mM Tris acetate buffer, 1% Triton X-100, 1 mM CaCl 2 , pH 7.5, except for PC1, where 5 mM CaCl 2 were added to the pH 6 buffer. All reactions contained 10 l of 35 Slabeled gp160 (10,000 cpm) and several dilutions of partially purified enzymes. After overnight digestion at 25°C, separation of the proteins by SDS-polyacrylamide gel electrophoresis was carried out on an 8% polyacrylamide gel.…”

Section: Endoproteolytic Cleavage Of Gp160 and Inhibitor Assays-[mentioning

confidence: 99%

“…To determine which are the convertases potentially involved in the processing of gp160, we first compared, in vitro, the ability of each convertase to cleave gp160 into gp120 and gp41. 35 S-Radiolabeled gp160 was expressed in the constitutively secreting CV-1 cells using a recombinant vaccinia virus. The protein gp160 was partially purified by an affinity column, taking advantage of the strong binding of its carbohydrate chains to lentil-lectin (17).…”

Section: Determination Of Calcium and Ph Dependence Of Recombinant Comentioning

confidence: 99%

“…In this work, four (PC1, furin, PACE4, and PC5/6-A as well as its isoform PC5/6B) of the seven known convertases were tested as gp160 convertases in vitro. This involved the comparison of their relative activities in the in vitro processing of gp160 and of a model 19-amino acid peptide spanning the junction between gp120 and gp41 (35). This synthetic peptide allowed us to measure the kinetics of the REKR 512 2AVGI cleavage.…”

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confidence: 99%

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Identification of the Paired Basic Convertases Implicated in HIV gp160 Processing Based on in Vitro Assays and Expression in CD4+ Cell Lines

Decroly

Wouters

Bello

et al. 1996

Journal of Biological Chemistry

113

130

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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 HIVinfected cells. 3) In an effort to evaluate specific convertase inhibitors, we showed that the ␣ 1 -antitrypsin variant, ␣ 1 -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.

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Structural Investigation of the HIV-1 Envelope Glycoprotein gp160 Cleavage Site

Oliva¹,

Leone

Falcigno³

et al. 2002

Chem. Eur. J.

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The selective proteolytic activation of the HIV‐1 envelope glycoprotein gp160 by furin and other precursor convertases (PCs) occurs at the carboxyl side of the sequence Arg508‐Glu‐Lys‐Arg511 (site 1), in spite of the presence of another consensus sequence: Lys500‐Ala‐Lys‐Arg503 (site 2). We report on the solution structural analysis of a 19‐residue synthetic peptide, p498, which spans the two gp160‐processing sites 1 and 2, and is properly digested by furin at site 1. A molecular model is obtained for p498, by means of molecular dynamics simulations, from NMR data collected in trifluoroethanol/water. The peptide N‐terminal side presents a 9‐residue helical segment, enclosing the processing site 2; the C‐terminal segment can be described as a loop exposing the processing site 1. A hypothesis for the docking of p498 onto the catalytic domain of human furin, modeled by homology and fitting previous site‐directed mutagenesis studies, is also presented. p498 site 1 is shown to have easy access to the furin catalytic site, unlike the nonphysiological site 2. Finally, on the basis of available data, we suggest a possible structural motif required for the gp160–PCs recognition.

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Structural Investigation and Kinetic Characterization of Potential Cleavage Sites of HIV Gp160 by Human Furin and Pc1

Cited by 20 publications

References 0 publications

Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Alterations of pr-M Cleavage and Virus Export in pr-M Junction Chimeric Dengue Viruses

Identification of the Paired Basic Convertases Implicated in HIV gp160 Processing Based on in Vitro Assays and Expression in CD4+ Cell Lines

Structural Investigation of the HIV-1 Envelope Glycoprotein gp160 Cleavage Site

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