Monash University
monash_83967.pdf (15.51 MB)

Receptor activated conformations of the gp120-gp41 glycoprotein complex of HIV-1

Download (15.51 MB)
posted on 2017-02-17, 04:01 authored by Bellamy-McIntyre, Anna
Human immunodeficiency virus (HIV) gains cellular entry through the action of two envelope glycoproteins, gp120 and gp41. gp120 mediates viral attachment to cellular receptors and activates the fusion protein, gp41 to refold from a metastable state into a stable six-helix bundle (6HB) leading to virus-cell membrane apposition and fusion. The aim of this study was to investigate the role of conserved gp41 regions outside of the conserved 6HB core, including the disulphide-bonded loop region (DSR), flexible polar linker, and membrane proximal external region (MPER) in viral fusion and entry. The DSR of gp41 mediates gp120 association. Tryptophan596-to-Leu and W610F mutations in the DSR of HIV-1QH1549.13 blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 prehairpin formation, indicating a decoupling of receptor-induced conformational changes in gp120 from gp41 activation. The data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation. The roles in membrane fusion of the flexible polar linker and MPER were examined by Ala substitution. The MPER mutations, W666A, W672A, F673A and I675A, reduced entry by up to 120-fold without affecting gp120-gp41 association or cell-cell fusion. The combination of individual MPER mutations with L537A in the flexible polar linker abolished entry with 50-80% reductions in cell-cell fusion. The gp120-gp41 complexes of fusion-defective double mutants were resistant to soluble CD4-induced shedding of gp120, suggesting their ability to undergo receptor-induced conformational changes was compromised. Consistent with this idea, the L537A W666A double mutant exhibited reduced hemifusion activity. The data indicate that the flexible polar linker and MPER act synergistically forming a fusion-competent gp120-gp41 complex. The paradoxical findings that W666A and I675A blocked viral entry without affecting cell-cell fusion were investigated further. The possibilities that the discrepant cell-cell fusion versus entry phenotypes were simple due to Env and/or receptor saturation in the fusion assay or subtle effects on fusion kinetics were ruled out. When the initial entry step was bypassed, by pseudotyping vesicular stomatitis virus glycoprotein G in trans, W666A and I675A exhibited WT levels of cell-cell spread, indicating the functional defect is manifested in the context of cell-free virus. Thus the MPER plays distinct roles in cell-free viral entry and cell-cell spread. The cell-free entry block due to W666A and I675A was overcome by truncation of the cytoplasmic tail indicating a functional link between the MPER and cytoplasmic tail in cell-free virus. Forced viral sequence evolution was used to identify structural elements in the gp120-gp41 complex that functionally interact with the MPER. Replication-defective W666A, W672A and I675A mutant viruses were serially passaged in PBMCs in order to select revertants with second- and third-site suppressor mutation(s) that overcome the initial attenuation. An Ala-to-Val pseudoreversion restored replication for I675A, whereas in W672A, replication was restored by the loss glycans at Asn136 or Asn141 within V1. A P369L mutation in the CD4 binding site plus D674E in the MPER and truncation of the cytoplasmic tail provided a single-cycle entry advantage to W666A These findings further our understanding of the HIV-1 membrane fusion mechanism and provide evidence for functional interactions between remote regions of gp120-gp41. That the MPER is critical for cell-free viral entry but not cell-cell spread has important implications for vaccine and drug development targeting this region.


Principal supervisor

Pantelis Poumbourios

Additional supervisor 1

Heidi Drummer

Year of Award


Department, School or Centre

School of Biomedical Sciences (Monash Biomedicine Discovery Institute)

Additional Institution or Organisation


Campus location



Doctor of Philosophy

Degree Type



Faculty of Medicine Nursing and Health Sciences