The function of coreceptor as a basis for the kinetic dissection of HIV type 1 envelope protein‐mediated cell fusion

Abstract

The function of HIV-1 HXB2 envelope (Env) glycoprotein (gp) was investigated by surface plasmon resonance and fluorescence imaging techniques. Strikingly, it was found that gp120 shedding requires the presence of the X4 coreceptor. A similar coreceptor requirement was observed for the membrane mixing and the Env recruitment on the cell surface. However, exposure and membrane penetration of the fusion peptide do not require X4 and occur within the first minute after incubation of Env with CD4 and/or X4. Analogously X4 was not required but enhanced binding of the fusion inhibitor. In contrast, bundle formation of the gp41 ectodomain, as monitored by NC-1, was accelerated by the presence of X4. The kinetics of these key post-Env binding events as determined in real time by fluorescence microscopic imaging, coupled with the differential coreceptor requirement, led to the proposition that gp120 shedding, which takes place from 1 to 10 min after engagement of receptor and coreceptor to Env, is a primary function of the coreceptor. The shedding of the surface subunits is needed for the subsequent processes including hemifusion, full fusion, and Env recruitment. The temporal order of these fusogenic steps allows construction of a refined model on the Env-mediated cell fusion event.

The fusion peptide domain is the primary membrane-inserted region and enhances membrane interaction of the ectodomain of HIV-1 gp41

“…The finding is compatible with our previous observation that the coreceptor-induced gp120 shedding from gp41 is a gradual process spanning 10 minutes after engagement of effector and target cells (Chien et al 2008). As FP refolds at approximately 1 min after virus-receptor (CD4) engagement, it may trigger the conformational change of gp120 on its way to the target membrane, since complete gp120 dislodgment has not occurred.…”

HIV Entry and Envelope Glycoprotein-mediated Fusion

“…Specific lipids in the viral and target cell membranes are not indicated. B, docking of the virus via gp120 to the cell surface receptor gives rise to Env conformational changes (72,73) and aggregation of Env proteins (1,43). C, further conformational changes lead to lipid mixing (47) and redistribution of small aqueous dyes (53,100).…”

“…In the absence of complete structural information, some of the details of the HIV-1 Env-mediated fusion reaction have been inferred from immunochemical, biochemical, and mutagenic analyses. Conformational changes in gp120-gp41 expressed on cells have been monitored as a function of time by analytical and quantitative video microscopy following interactions of Env-expressing cells with target cells using nonspecific probes that report on hydrophobicity changes (72,73), as well as reagents that are specific for the different conformations of triggered HIV-1 gp120/gp41 (74,75). After the interaction with target cell surface-bound CD4, a substantial increase in immunoreactivity to certain antibodies is observed, reflecting conformational changes in gp120, resulting in exposure of the CR-binding site (gp120 CR ), and in gp41, resulting in the prehairpin conformation (gp41 PHP ) (Fig.…”

Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10

“…HIV-1 enters the host by fusion of its membrane to the host cell membrane in a process initiated by binding of gp120 to CD4 and then to co-receptors CCR5 or CXCR4 (Chien et al, 2008). Receptor binding promotes Env conformational rearrangements leading to exposure of the gp41 hydrophobic N-terminal fusion peptide (Chien et al, 2008), which then inserts into the host cell membrane (Harrison, 2008).…”

“…Receptor binding promotes Env conformational rearrangements leading to exposure of the gp41 hydrophobic N-terminal fusion peptide (Chien et al, 2008), which then inserts into the host cell membrane (Harrison, 2008). Gp41 is thought to initially adopt a metastable conformation that eventually collapses into the six-helix bundle post-fusion conformation after receptor and co-receptor engagement (Buzon et al, 2010), thereby bringing the viral and host membranes together to form the hemifusion stalk and fusion pore (Harrison, 2008).…”