Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV

doi:10.1016/0042-6822(89)90142-6

Virology

Volume 172, Issue 1, September 1989, Pages 367-369

https://doi.org/10.1016/0042-6822(89)90142-6 Get rights and content

Abstract

The subunit composition of env glycoproteins isolated from HIV-infected cell membranes, virus particles, and baculovirus recombinant-infected insect cells has been investigated using a number of crosslinking reagents. The results obtained provide evidence that gp160 is a tetramer of identical subunits and that gp41 is also tetrameric.

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Cited by (151)

Analysis of HIV-1 envelope cytoplasmic tail effects on viral replication
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Trimers of the HIV-1 envelope (Env) protein perform receptor binding and virus-cell fusion functions during the virus life cycle. The cytoplasmic tail (CT) of Env forms an unusual baseplate structure, and is palmitoylated, rich in arginines, carries trafficking motifs, binds cholesterol, and interacts with host proteins. To dissect CT activities, we examined a panel of Env variants, including CT truncations, mutations, and an extension. We found that whereas all variants could replicate in permissive cells, viruses with CT truncations or baseplate mutations were defective in restrictive cells. We also identified a determinant in HIV-1 amphotericin sensitivity, and characterized variants that escape amphotericin inhibition via viral protease-mediated CT cleavage. Results additionally showed that full-length, his tagged Env can oligomerize and be co-assembled with CT truncations that delete portions of the baseplate, host protein binding sites, and trafficking signals. Our observations illuminate novel aspects of HIV-1 CT structure, interactions, and functions.
Replication of HIV-1 envelope protein cytoplasmic domain variants in permissive and restrictive cells
2019, Virology
Citation Excerpt :
Evidence indicates that full-length wild type (WT) HIV-1 Env proteins are translated and initially glycosylated at rough endoplasmic reticulum (RER) membranes to yield membrane-anchored 160 kDa glycoprotein precursor (gp160) proteins (Checkley et al., 2011). The gp160 monomers oligomerize in the ER, and traffic to the Golgi, where they acquire complex carbohydrates and are cleaved by furin or furin-like proteases to yield non-covalently associated surface (SU; gp120) and transmembrane (TM, gp41) proteins (Checkley et al., 2011; Pinter et al., 1989; Schawaller et al., 1989; Earl et al., 1990; Bernstein et al., 1994; Montefiori et al., 1988; Hallenberger et al., 1992). Trimers of associated gp120 and gp41 proteins then are delivered to cell plasma membranes (PMs), where they are subject to rapid endocytosis by virtue of YxxL AP-2 binding sites at the membrane proximal ends of CTs, and/or LL AP-1 binding sites at the CT C-termini (Qi et al., 2013, 2015; Kirschman et al., 2018; Ohno et al., 1997; Wyss et al., 2001).
Wild type (WT) HIV-1 envelope (Env) protein cytoplasmic tails (CTs) appear to be composed of membrane-proximal, N-terminal unstructured regions, and three C-terminal amphipathic helices. Previous studies have shown that WT and CT-deleted (ΔCT) Env proteins are incorporated into virus particles via different mechanisms. WT Env proteins traffic to cell plasma membranes (PMs), are rapidly internalized, recycle to PMs, and are incorporated into virions in permissive and restrictive cells in a Gag matrix (MA) protein-dependent fashion. In contrast, previously described ΔCT proteins do not appear to be internalized after their arrival to PMs, and do not require MA, but are only incorporated into virions in permissive cell lines. We have analyzed a new set of HIV-1 CT variants with respect to their replication in permissive and restrictive cells. Our results provide novel details as to how CT elements regulate HIV-1 Env protein function.
HIV-1 gp120 dimers decrease the overall affinity of gp120 preparations for CD4-induced ligands
2015, Journal of Virological Methods
Citation Excerpt :
It has previously been reported that producing either gp120 or gp140 in mammalian cells can lead to the formation of a large quantity of unusual dimers. Their formation is influenced by several residues and the variable region 2 of the gp120 (V2) (Schawaller et al., 1989; Doms et al., 1991; Hallenberger et al., 1993; Center et al., 2000; Finzi et al., 2010a). Importantly, it has been previously shown that these gp120 dimers do not fold properly and do not expose several epitopes present in monomeric gp120 (Finzi et al., 2010a).
For several years, tools to study the conformational changes of HIV-1 envelope glycoproteins have been developed in order to comprehend those changes and their role in the fusion process and immunogenicity of HIV-1. To facilitate these studies, expression of the HIV-1 gp120 envelope glycoprotein has been done in several over-expression settings. However, over-expression of HIV-1 gp120 in mammalian cells leads to the formation of aberrant disulfide-linked dimers that can bias the results of experiments aimed at measuring gp120 affinity with different ligands. The presence of these gp120 dimers, generated in a widely used gp120 expression system, affects the affinity of gp120 for CD4-induced ligands, as evaluated by surface plasmon resonance. Upon monomeric gp120 purification, neither the removal of potential glycosylation sites on V4 nor the removal of the V5 variable region affect the overall affinity of gp120 for 17b and A32 CD4-induced ligands. Removal of these aberrant disulfide-linked gp120 dimers by standard size exclusion chromatography is sufficient to restore the overall affinity of gp120 preparations for these ligands.
HIV-1 envelope glycoprotein biosynthesis, trafficking, and incorporation
2011, Journal of Molecular Biology
Citation Excerpt :
Concomitant with translation, gp160 is glycosylated with N-linked (and some O-linked) oligosaccharide side chains.39–41 Monomers of gp160 oligomerize in the ER into predominantly trimers, although dimers and tetramers can also be observed.42–45 This oligomerization process is thought to facilitate the trafficking of gp160 to the Golgi complex.
The HIV-1 envelope (Env) glycoproteins play an essential role in the virus replication cycle by mediating the fusion between viral and cellular membranes during the entry process. The Env glycoproteins are synthesized as a polyprotein precursor (gp160) that is cleaved by cellular proteases to the mature surface glycoprotein gp120 and the transmembrane glycoprotein gp41. During virus assembly, the gp120/gp41 complex is incorporated as heterotrimeric spikes into the lipid bilayer of nascent virions. These gp120/gp41 complexes then initiate the infection process by binding receptor and coreceptor on the surface of target cells. Much is currently known about the HIV-1 Env glycoprotein trafficking pathway and the structure of gp120 and the extracellular domain of gp41. However, the mechanism by which the Env glycoprotein complex is incorporated into virus particles remains incompletely understood. Genetic data support a major role for the cytoplasmic tail of gp41 and the matrix domain of Gag in Env glycoprotein incorporation. Still to be defined are the identities of host cell factors that may promote Env incorporation and the role of specific membrane microdomains in this process. Here, we review our current understanding of HIV-1 Env glycoprotein trafficking and incorporation into virions.
Conformational characterization of aberrant disulfide-linked HIV-1 gp120 dimers secreted from overexpressing cells
2010, Journal of Virological Methods
The envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) mediate viral entry and are also the primary target of neutralizing antibodies. The gp160 envelope glycoprotein precursor undergoes proteolytic cleavage in the Golgi complex to produce the gp120 exterior glycoprotein and the gp41 transmembrane glycoprotein, which remain associated non-covalently in the trimeric Env complex. Monomeric soluble gp120 has been used extensively to investigate conformational states, structure, antigenicity and immunogenicity of the HIV-1 Env glycoproteins. Expression of gp120 alone (without gp41) leads to the accumulation not only of monomeric gp120 but also an aberrant dimeric form. The gp120 dimers were sensitive to reducing agents. The formation of gp120 dimers was disrupted by a single amino acid change in the inner domain, and was reduced by removal of the V1/V2 variable loops or the N and C termini. Epitopes on the gp120 inner domain and the chemokine receptor-binding surface were altered or occluded by gp120 dimerization. Awareness of the existence and properties of gp120 dimers should assist interpretation of studies of this key viral protein.
Introduction to Retroviruses
2004, AIDS and Other Manifestations of HIV Infection

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Short communicationStudies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV

Abstract

Cell

Lancet

Virology

FEBS Lett.

J. Biochem. Biophys. Methods

Short communication
Studies with crosslinking reagents on the oligomeric structure of the env glycoprotein of HIV