Functional roles of the V3 hypervariable region of HIV-1 gp160 in the processing of gp160 and in the formation of syncytia in CD4+ cells

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143

165

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,...

Section: Resultsmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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

Binley

Sanders

Master

et al. 2002

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143

165

“…The cysteine residues at the base of the V3 loop are important for proper gp160 processing, at least in the context of envelope glycoproteins from the T-cell-line-adapted isolate LAI (12,21,63,67). Substitutions within and around the small intramolecular disulfide-bonded loop in the gp41 ectodomain also impair the efficiency of gp160 cleavage (15,60), especially in primaryisolate envelope glycoproteins (33).…”

Section: Discussionmentioning

confidence: 99%

Variable-Loop-Deleted Variants of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Can Be Stabilized by an Intermolecular Disulfide Bond between the gp120 and gp41 Subunits

Sanders

Schiffner

Master

et al. 2000

109

We have described an oligomeric gp140 envelope glycoprotein from human immunodeficiency virus type 1 that is stabilized by an intermolecular disulfide bond between gp120 and the gp41 ectodomain . In this protein, the protease cleavage site between gp120 and gp41 is fully utilized. Here we report the characterization of gp140 variants that have deletions in the first, second, and/or third variable loop (V1, V2, and V3 loops). The SOS disulfide bond formed efficiently in gp140s containing a single loop deletion or a combination deletion of the V1 and V2 loops. However, deletion of all three variable loops prevented formation of the SOS disulfide bond. Some variableloop-deleted gp140s were not fully processed to their gp120 and gp41 constituents even when the furin protease was cotransfected. The exposure of the gp120-gp41 cleavage site is probably affected in these proteins, even though the disabling change is in a region of gp120 distal from the cleavage site. Antigenic characterization of the variable-loop-deleted SOS gp140 proteins revealed that deletion of the variable loops uncovers cryptic, conserved neutralization epitopes near the coreceptor-binding site on gp120. These modified, disulfide-stabilized glycoproteins might be useful as immunogens.

“…Considerable efforts have therefore been made to generate and characterize forms of Env with loop deletions. Initial functional studies have shown that the deletion of V3 or V4 abrogated Env function and viral infectivity (15,52,70,79). Several constructs have been described lacking V1 or V2 that were compatible with minimal Env function and viral replication, indicating that they are not absolutely required for function (35,66,78).…”

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confidence: 99%

Optimization of Human Immunodeficiency Virus Type 1 Envelope Glycoproteins with V1/V2 Deleted, Using Virus Evolution

Bontjer

Land

Eggink

et al. 2009

The human immunodeficiency virus type 1 envelope glycoprotein (Env) complex is the principal focus of neutralizing antibody-based vaccines. The functional Env complex is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The individual subunits have proven unsuccessful as vaccines presumably because they do not resemble the functional Env complex. Variable domains and carbohydrates shield vulnerable neutralization epitopes on the functional Env complex. The deletion of variable loops has been shown to improve gp120's immunogenicity; however, problems have been encountered when introducing such modifications in stabilized Env trimer constructs. To address these issues, we have created a set of V1/V2 and V3 loop deletion variants in the context of complete virus to allow optimization by forced virus evolution. Compensatory second-site substitutions included the addition and/or removal of specific carbohydrates, changes in the disulfide-bonded architecture of the V1/V2 stem, the replacement of hydrophobic residues by hydrophilic and charged residues, and changes in distal parts of gp120 and gp41. These viruses displayed increased sensitivity to neutralizing antibodies, demonstrating the improved exposure of conserved domains. The results show that we can select for functionally improved Env variants with loop deletions through forced virus evolution. Selected evolved Env variants were transferred to stabilized Env trimer constructs and were shown to improve trimer expression and secretion. Based on these findings, we can make recommendations on how to delete the V1/V2 domain from recombinant Env trimers for vaccine and X-ray crystallography studies. In general, virus evolution may provide a powerful tool to optimize Env vaccine antigens.The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein complex (Env) is the principal target of vaccine research aimed at raising an antiviral humoral immune response. The isolation of a small number of broadly active neutralizing antibodies from HIV-infected individuals serves as a rationale for the search for vaccines that elicit such antibodies. Although many Env-based HIV-1 vaccines have been tested in preclinical and clinical studies, so far none has raised broadly reactive antibodies that could neutralize diverse primary virus isolates. Thus, relatively straightforward vaccine strategies that worked for other pathogens-for example, the use of unmodified surface antigens-have been explored without satisfactory results for HIV-1, emphasizing the necessity for more-sophisticated vaccine design.The functional HIV-1 Env complex, which mediates viral entry into CD4 ϩ host cells, is a trimer consisting of six individual subunits: three gp120 molecules and three gp41 molecules. The attachment of HIV-1 to a target cell is followed by the fusion of the viral and cellular membranes. First, gp120 binds to the CD4 receptor, a process that induces conformational changes to create and expose the coreceptor binding site (71, 76). The...