Forced virus evolution reveals functional crosstalk between the disulfide bonded region and membrane proximal ectodomain region of HIV-1 gp41

Abstract

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-1 AD8 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.

Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization

“…This would also explain why saturation of the mutant spike by 10E8 does not fully block 2F5 neutralization and why the effect also occurs with ligands that bind distal to 10E8. Flexibility of the MPER [19] would presumably allow propagation of conformational changes to other regions of Env upon antibody binding; functional links between the MPER, NHR and DSL regions have also been described [65] , [66] . MPER mutations might also lead to an exchange of the MPER between adjacent subunits and membrane.…”

Distinct functions for the membrane-proximal ectodomain region (MPER) of HIV-1 gp41 in cell-free and cell–cell viral transmission and cell–cell fusion

“…We have previously shown that loss of the V1 glycans at Asn 136 or Asn 141 /Asn 142 brings about changes in the gp120 -gp41 association site (96). We have also found that restoration of infectivity to a defective DSR double mutant (W596L and K601D) involves a D674E mutation in the MPER (58). These results suggest that the 136/142 glycans in V1 are allosterically linked to the function of the DSR, which is in turn linked to the function of the MPER.…”

“…1, C and D). By contrast, very little virus production was observed for the W596L/K601D (WLKD) mutant that lacks gp120 association (58), indicating that a fusion-competent glycoprotein complex is required for virus transmission in cultures initiated by cell-free and cell-associated virus. Because VSV G-pseudotyped WLKD virions are fully competent to initiate infection (see Fig.…”

“…Infection of U87.CD4.CCR5 cells (from H. Deng and D. Littman (54), AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health) was carried out as described (58). Briefly, virus stocks were prepared by transfecting 293T cell (American Type Culture Collection) monolayers with infectious clones using FuGENE HD (Roche Applied Science).…”

Studying Evolutionary Adaptation of MERS-CoV

“…While the majority of these mutations will have neutral or negative effects on viral fitness, a small subset of these mutations may prove beneficial and enhance the ability for certain variants to replicate despite selective pressures of interest such as the host immune response or an antiviral drug. Forced adaptation experiments have been used to determine viral mutations that facilitate escape from drugs [4][5][6], monoclonal antibodies [7,8], host restriction factors [9][10][11], and species variation in host receptors [12][13][14] and to elucidate various viral mechanisms of infection and replication [15][16][17].…”