Retroviral Envelope Glycoprotein Processing:  Structural Investigation of the Cleavage Site

Moulard, Maxime; Chaloin, Laurent; Canarelli, Stéphane; Mabrouk, Kamel; Darbon, Hervé

doi:10.1021/bi972662f

Cited by 13 publications

(13 citation statements)

References 49 publications

(68 reference statements)

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“…The CD results suggest that HIV gp120 C5 is in a random coil conformation in aqueous solutions, which is in agreement with previous studies of similar C5 peptides [20,21]. In contrast the presence of helix is clearly indicated by CD when C5 is in the presence of the cosolvents trifluoroethanol or hexafluoroisopropanol.…”

Section: Discussionsupporting

confidence: 91%

Solution structure of the HIV gp120 C5 Domain

Guilhaudis

Jacobs

Caffrey

2002

European Journal of Biochemistry

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In HIV the viral envelope protein is processed by a host cell protease to form gp120 and gp41. The C1 and C5 domains of gp120 are thought to directly interact with gp41 but are largely missing from the available X-ray structure. Biophysical studies of the HIV gp120 C5 domain (residues 489-511 of HIV-1 strain HXB2), which corresponds to the carboxy terminal region of gp120, have been undertaken. CD studies of the C5 domain suggest that it is unstructured in aqueous solutions but partially helical in trifluoroethanol/ aqueous and hexafluoroisopropanol/aqueous buffers. The solution structure of the C5 peptide in 40% trifluoroethanol/ aqueous buffer was determined by NMR spectroscopy. The resulting structure is a turn helix structural motif, consistent with the CD results. Fluorescence titration experiments suggest that HIV C5 forms a 1 : 1 complex with the HIV gp41 ectodomain in the presence of cosolvent with an apparent K d of 1.0 lM. The absence of complex formation in the absence of cosolvent indicates that formation of the turn-helix structural motif of C5 is necessary for complex formation. Examination of the C5 structure provides insight into the interaction between gp120 and gp41 and provides a possible target site for future drug therapies designed to disrupt the gp120/gp41 complex. In addition, the C5 structure lends insight into the site of HIV envelope protein maturation by the host enzymes furin and PC7, which provides other possible targets for drug therapies.

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Section: Discussionsupporting

confidence: 91%

Solution structure of the HIV gp120 C5 Domain

Guilhaudis

Jacobs

Caffrey

2002

European Journal of Biochemistry

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“…The presence of a nearby carbohydrate moiety reduces cleavage efficiency, whereas a polybasic amino acid insertion within the loop or replacement of an uncharged amino acid with a basic residue serves to nullify the adverse influence of the carbohydrate, possibly by reversing the poor accessibility of the target sequence to protease (41,75). For HIV-1 gp160, a loop configuration for the furin cleavage motif was also proposed (60,63). If the dengue virus pr-M junction is located in a similar loop, the P5 deletion in TBEVpr/16681 may shorten or otherwise alter the local conformation of this loop in dengue virus prM in such a way that basic residues at more distal positions cannot compensate for the lack of P5 arginine for efficient cleavage.…”

Section: Discussionmentioning

confidence: 99%

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

Keelapang

Sriburi

Supasa

et al. 2004

J Virol

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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“…This renders its use impractical. Purified furin can also cleave secreted Env (17,18,38,56), albeit at low efficiency (17). Our own findings were similar: Even when furin digestion of gp140 SOS was performed at optimal pH with the enzyme in great excess, approximately 40% of the Env substrate remained uncleaved, suggesting that there may be a subpopulation of gp140 that is more resistant to cleavage.…”

Section: Discussionmentioning

confidence: 99%

“…It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid).…”

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confidence: 99%

Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

Binley

Sanders

Master

et al. 2002

J Virol

142

165

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In virus-infected cells, the envelope glycoprotein (Env) precursor, gp160, of human immunodeficiency virus type 1 is cleaved by cellular proteases into a fusion-competent gp120-gp41 heterodimer in which the two subunits are noncovalently associated. However, cleavage can be inefficient when recombinant Env is expressed at high levels, either as a full-length gp160 or as a soluble gp140 truncated immediately N-terminal to the transmembrane domain. We have explored several methods for obtaining fully cleaved Env for use as a vaccine antigen. We tested whether purified Env could be enzymatically digested with purified protease in vitro. Plasmin efficiently cleaved the Env precursor but also cut at a second site in gp120, most probably the V3 loop. In contrast, a soluble form of furin was specific for the gp120-gp41 cleavage site but cleaved inefficiently. Coexpression of Env with the full-length or soluble form of furin enhanced Env cleavage but also reduced Env expression. When the Env cleavage site (REKR) was mutated in order to see if its use by cellular proteases could be enhanced, several mutants were found to be processed more efficiently than the wild-type protein. The optimal cleavage site sequences were RRRRRR, RRRRKR, and RRRKKR. These mutations did not significantly alter the capacity of the Env protein to mediate fusion, so they have not radically perturbed Env structure. Furthermore, unlike that of wild-type Env, expression of the cleavage site mutants was not significantly reduced by furin coexpression. Coexpression of Env cleavage site mutants and furin is therefore a useful method for obtaining high-level expression of processed Env.The Env glycoprotein complex mediates receptor binding and membrane fusion during human immunodeficiency virus type 1 (HIV-1) infection of susceptible cells (66). It is synthesized as a polypeptide precursor (gp160) that oligomerizes to form a heavily glycosylated trimer (20,24). At a late stage of synthesis, most probably in the trans-Golgi network (TGN), gp160 is cleaved by furin (17,18,(55)(56)(57)(58) or other, related subtilisin-like proteases (17,18,28,38,58,90) into the surface (SU; gp120) and transmembrane (TM; gp41) subunits (34,43,(55)(56)(57)(58)82). Cleavage occurs at a motif at the gp120-gp41 juncture that contains a basic amino acid tetrad, R-X-(R/K)-R (where X is any amino acid). The gp120 and gp41 proteins then remain noncovalently associated, forming the functional, native gp120 3 -gp41 3 complex (20,24,66).During fusion, the gp120 protein interacts with the virus receptor and coreceptor on target cells. This triggers conformational changes that lead to the insertion of a hydrophobic fusion peptide, located at the N terminus of gp41, into the target cell membrane (66). Cleavage of gp160 is essential for fusion, since uncleaved gp160 is fusion incompetent (9,33,39,48). Generally, only cleaved Env is incorporated into virions (22), although uncleaved Env can be virion associated (39,48). By analogy with other enveloped viruses such as influenza A virus (5,...

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Retroviral Envelope Glycoprotein Processing: Structural Investigation of the Cleavage Site

Cited by 13 publications

References 49 publications

Solution structure of the HIV gp120 C5 Domain

Solution structure of the HIV gp120 C5 Domain

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

Enhancing the Proteolytic Maturation of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins

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