Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: Functional complementation of specific defects in gp120 and gp41

Abstract

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120͞gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.T he envelope glycoprotein (Env) of HIV-1 mediates virus entry into target cells by catalyzing a complex series of receptor-binding events and associated conformational changes that result ultimately in fusion between the membranes of the virion and target cell. The biochemical outline of the fusion reaction has been established (reviewed in refs. 1-4). The external gp120 subunit must bind to two distinct receptors on the target cell: CD4 (the ''primary receptor'') and a specific chemokine receptor (the coreceptor, usually CCR5 or CXCR4). These receptor interactions trigger the transmembrane gp41 subunit to promote fusion via a process that is presumed to involve insertion of its N-terminal fusion peptide into the membrane of the target cell. A specific sequence of receptor interactions is required to activate the fusion reaction, as revealed by binding analyses with soluble gp120 (5-7), fusion and infectivity experiments with soluble CD4 (8, 9), high-resolution x-ray crystallographic structural determinations (10), and sitedirected mutagenesis studies (11). In the favored model, CD4 binding exposes, creates, or stabilizes the coreceptor binding determinants on gp120; interaction with coreceptor triggers additional conformational changes, leading to gp41 activation and consequent fusion.Env is first synthesized as a high molecular weight precursor designated gp160. The protein forms a homo-oligomer in the endoplasmic reticulum and is transported to the Golgi apparatus where processing by a host cell protease(s) occurs; the functional Env on the surface of both the infected cell and the virion is a homo-oligomer of multiple gp120͞gp41 noncova...

Stoichiometry of Murine Leukemia Virus Envelope Protein-Mediated Fusion and Its Neutralization

“…The existence of complementation by itself argues that trimers do not need to have two wild-type monomers to function, since complementing Envs have either only one wild-type SU or only one wild-type TM. Yang et al found no complementation in their system, but this contrasts with the results reported here as well as several other reports (32,35,47,48). Previous reports on complementation do not distinguish whether one or both heterotrimers are functional.…”

“…The mechanism of complementation is not yet clear, though it is usually considered in terms of SU and TM from different monomers being able to work together to induce fusion (32,35,47,48). Our results support another mechanism that may contribute to complementation in some cases, namely, heterotrimerization increasing steady-state levels and transport to the plasma membrane of mutant Env species that misfold as homotrimers.…”

“…Thus, some Env mutants form functional heterotrimers with wild-type Env (44), and some Envs with lethal mutations in SU can complement Envs with lethal mutations in TM (35,47). Two kinds of heterotrimers may be formed, which can be designated X2Y1 and X1Y2, where X and Y stand for the different monomers and 1 and 2 stand for the number of monomers of each type in a trimer.…”

Intraprotomer masking of third variable loop (V3) epitopes by the first and second variable loops (V1V2) within the native HIV-1 envelope glycoprotein trimer

“…Our previous analyses of subunit complementation (i.e., generation of functional mixed trimers upon coexpression of two Envs with different inactivating mutations) used the Env-mediated cell fusion assay (14,15), which is highly sensitive for detecting Env function. Here we wished to apply the Env pseudotype virion infection system to test for complementation with virus particles.…”

“…We devised a functional strategy based on a previously described Env complementation system (14,15) to distinguish between cis versus trans mechanisms of epitope masking. The results are discussed in terms of various proposed trimer models based on structural analyses and molecular modeling.…”