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Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association.

ABSTRACT: Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The available gp41 crystal structures lack the conserved disulfide-bonded loop region which, in human T-lymphotropic virus type 1 (HTLV-1) and murine leukemia virus TM proteins, mediates a chain reversal, connecting the antiparallel N- and C-terminal regions. Mutations in the HTLV-1 TM protein gp21 disulfide-bonded loop/chain reversal region adversely affected fusion activity without abolishing SU-TM association (A. L. Maerz, R. J. Center, B. E. Kemp, B. Kobe, and P. Poumbourios, J. Virol. 74:6614-6621, 2000). We now report that in contrast to our findings with HTLV-1, conservative substitutions in the HIV-1 gp41 disulfide-bonded loop/chain reversal region abolished association with gp120. While the mutations affecting gp120-gp41 association also affected cell-cell fusion activity, HIV-1 glycoprotein maturation appeared normal. The mutant glycoproteins were processed, expressed at the cell surface, and efficiently immunoprecipitated by conformation-dependent monoclonal antibodies. The gp120 association site includes aromatic and hydrophobic residues on either side of the gp41 disulfide-bonded loop and a basic residue within the loop. The HIV-1 gp41 disulfide-bonded loop/chain reversal region is a critical gp120 contact site; therefore, it is also likely to play a central role in fusion activation by linking CD4 plus chemokine receptor-induced conformational changes in gp120 to gp41 fusogenicity. These gp120 contact residues are present in diverse primate lentiviruses, suggesting conservation of function.

SUBMITTER: Maerz AL

PROVIDER: S-EPMC114387 | biostudies-literature | 2001 Jul

REPOSITORIES: biostudies-literature

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Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association.

Maerz A L AL Drummer H E HE Wilson K A KA Poumbourios P P

Journal of virology 20010701 14

Human immunodeficiency virus type 1 (HIV-1) entry into cells is mediated by the surface-exposed envelope protein (SU) gp120, which binds to cellular CD4 and chemokine receptors, triggering the membrane fusion activity of the transmembrane (TM) protein gp41. The core of gp41 comprises an N-terminal triple-stranded coiled coil and an antiparallel C-terminal helical segment which is packed against the exterior of the coiled coil and is thought to correspond to a fusion-activated conformation. The a ...[more]

PMID: 11413331

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Project description:BACKGROUND: The disulfide-bonded region (DSR) of HIV-1 gp41 mediates association with gp120 and plays a role in transmission of receptor-induced conformational changes in gp120 to gp41 that activate membrane fusion function. In this study, forced viral evolution of a DSR mutant that sheds gp120 was employed to identify domains within gp120-gp41 that are functionally linked to the glycoprotein association site. RESULTS: The HIV-1AD8 mutant, W596L/K601D, was serially passaged in U87.CD4.CCR5 cells until replication was restored. Whereas the W596L mutation persisted throughout the cultures, a D601H pseudoreversion in the DSR partially restored cell-free virus infectivity and virion gp120-gp41 association, with further improvements to cell-free virus infectivity following a 2nd-site D674E mutation in the membrane-proximal external region (MPER) of gp41. In an independent culture, D601H appeared with a deletion in V4 (Thr-394-Trp-395) and a D674N substitution in the MPER, however this MPER mutation was inhibitory to W596L/K601H cell-free virus infectivity. While cell-free virus infectivity was not fully restored for the revertant genotypes, their cell-to-cell transmission approached the levels observed for WT. Interestingly, the functional boost associated with the addition of D674E to W596L/K601H was not observed for cell-cell fusion where the cell-surface expressed glycoproteins function independently of virion assembly. The W596L/K601H and W596L/K601H/D674E viruses exhibited greater sensitivity to neutralization by the broadly reactive MPER directed monoclonal antibodies, 2F5 and 4E10, indicating that the reverting mutations increase the availability of conserved neutralization epitopes in the MPER. CONCLUSIONS: The data indicate for the first time that functional crosstalk between the DSR and MPER operates in the context of assembled virions, with the Leu-596-His-601-Glu-674 combination optimizing viral spread via the cell-to-cell route. Our data also indicate that changes in the gp120-gp41 association site may increase the exposure of conserved MPER neutralization epitopes in virus.

Project description:The HIV-1 gp120-gp41 complex, which mediates viral fusion and cellular entry, undergoes rapid evolution within its external glycan shield to enable escape from neutralizing antibody (NAb). Understanding how conserved protein determinants retain functionality in the context of such evolution is important for their evaluation and exploitation as potential drug and/or vaccine targets. In this study, we examined how the conserved gp120-gp41 association site, formed by the N- and C-terminal segments of gp120 and the disulfide-bonded region (DSR) of gp41, adapts to glycan changes that are linked to neutralization sensitivity. To this end, a DSR mutant virus (K601D) with defective gp120-association was sequentially passaged in peripheral blood mononuclear cells to select suppressor mutations. We reasoned that the locations of suppressors point to structural elements that are functionally linked to the gp120-gp41 association site. In culture 1, gp120 association and viral replication was restored by loss of the conserved glycan at Asn¹³? in V1 (T138N mutation) in conjunction with the L494I substitution in C5 within the association site. In culture 2, replication was restored with deletion of the N¹³?INN sequence, which ablates the overlapping Asn¹?¹-Asn¹?²-Ser-Ser potential N-linked glycosylation sequons in V1, in conjunction with D601N in the DSR. The 136 and 142 glycan mutations appeared to exert their suppressive effects by altering the dependence of gp120-gp41 interactions on the DSR residues, Leu??³, Trp??? and Lys??¹. The 136 and/or 142 glycan mutations increased the sensitivity of HIV-1 pseudovirions to the glycan-dependent NAbs 2G12 and PG16, and also pooled IgG obtained from HIV-1-infected individuals. Thus adjacent V1 glycans allosterically modulate the distal gp120-gp41 association site. We propose that this represents a mechanism for functional adaptation of the gp120-gp41 association site to an evolving glycan shield in a setting of NAb selection.

Project description:HIV-1 enters cells through binding between viral envelope glycoprotein (Env) and cellular receptors to initiate virus and cell fusion. HIV-1 Env precursor (gp160) is cleaved into two units noncovalently bound to form a trimer on virions, including a surface unit (gp120) and a transmembrane unit (gp41) responsible for virus binding and membrane fusion, respectively. The polar region (PR) at the N terminus of gp41 comprises 17 residues, including 7 polar amino acids. Previous studies suggested that the PR contributes to HIV-1 membrane fusion and infectivity; however, the precise role of the PR in Env-mediated viral entry and the underlying mechanisms remain unknown. Here, we show that the PR is critical for HIV-1 fusion and infectivity by stabilizing Env trimers. Through analyzing the PR sequences of 57,645 HIV-1 isolates, we performed targeted mutagenesis and functional studies of three highly conserved polar residues in the PR (S532P, T534A, and T536A) which have not been characterized previously. We found that single or combined mutations of these three residues abolished or significantly decreased HIV-1 infectivity without affecting viral production. These PR mutations abolished or significantly reduced HIV-1 fusion with target cells and also Env-mediated cell-cell fusion. Three PR mutations containing S532P substantially reduced gp120 and gp41 association, Env trimer stability, and increased gp120 shedding. Furthermore, S532A mutation significantly reduced HIV-1 infectivity and fusogenicity but not Env expression and cleavage. Our findings suggest that the PR of gp41, particularly the key residue S532, is structurally essential for maintaining HIV-1 Env trimer, viral fusogenicity, and infectivity.IMPORTANCE Although extensive studies of the transmembrane unit (gp41) of HIV-1 Env have led to a fusion inhibitor clinically used to block viral entry, the functions of different domains of gp41 in HIV-1 fusion and infectivity are not fully elucidated. The polar region (PR) of gp41 has been proposed to participate in HIV-1 membrane fusion in biochemical analyses, but its role in viral entry and infectivity remain unclear. In our effort to characterize three nucleotide mutations of an HIV-1 RNA element that partially overlaps the PR coding sequence, we identified a novel function of the PR that determines viral fusion and infectivity. We further demonstrated the structural and functional impact of six PR mutations on HIV-1 Env stability, viral fusion, and infectivity. Our findings reveal the previously unappreciated function of the PR and the underlying mechanisms, highlighting the important role of the PR in regulating HIV-1 fusion and infectivity.

Dataset Information

Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association.

Publications

Functional analysis of the disulfide-bonded loop/chain reversal region of human immunodeficiency virus type 1 gp41 reveals a critical role in gp120-gp41 association.

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