The entry of human immunodeficiency virus (HIV) into cells requires the sequential interaction of the viral exterior envelope glycoprotein, gp120, with the CD4 glycoprotein and a chemokine receptor on the cell surface. These interactions initiate a fusion of the viral and cellular membranes. Although gp120 can elicit virus-neutralizing antibodies, HIV eludes the immune system. We have solved the X-ray crystal structure at 2.5 A resolution of an HIV-1 gp120 core complexed with a two-domain fragment of human CD4 and an antigen-binding fragment of a neutralizing antibody that blocks chemokine-receptor binding. The structure reveals a cavity-laden CD4-gp120 interface, a conserved binding site for the chemokine receptor, evidence for a conformational change upon CD4 binding, the nature of a CD4-induced antibody epitope, and specific mechanisms for immune evasion. Our results provide a framework for understanding the complex biology of HIV entry into cells and should guide efforts to intervene.

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Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody

Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.

Receptor Binding and Membrane Fusion in Virus Entry: The Influenza Hemagglutinin

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution

SNAREpins: Minimal Machinery for Membrane Fusion

In addition to CD4, the human immunodeficiency virus (HIV) requires a coreceptor for entry into target cells. The chemokine receptors CXCR4 and CCR5, members of the G protein-coupled receptor superfamily, have been identified as the principal coreceptors for T cell line-tropic and macrophage-tropic HIV-1 isolates, respectively. The updated coreceptor repertoire includes numerous members, mostly chemokine receptors and related orphans. These discoveries provide a new framework for understanding critical features of the basic biology of HIV-1, including the selective tropism of individual viral variants for different CD4 C target cells and the membrane fusion mechanism governing virus entry. The coreceptors also provide molecular perspectives on central puzzles of HIV-1 disease, including the selective transmission of macrophage-tropic variants, the appearance of T cell line-tropic variants in many infected persons during progression to AIDS, and differing susceptibilities of individuals to infection and disease progression. Genetic findings have yielded major insights into the in vivo roles of individual coreceptors and their ligands; of particular importance is the discovery of an inactivating mutation in the CCR5 gene which, in homozygous form, confers strong resistance to HIV-1 infection. Beyond providing new perspectives on fundamental aspects of HIV-1 transmission and pathogenesis, the coreceptors suggest new avenues for developing novel therapeutic and preventative strategies to combat the AIDS epidemic.

CHEMOKINE RECEPTORS AS HIV-1 CORECEPTORS: Roles in Viral Entry, Tropism, and Disease

https://www.cell.com/cell/pdf/S0092-8674(00)80205-6.pdf

Core structure of GP41 from the HIV envelope glycoprotein

Infection with HIV-1 is initiated by fusion of cellular and viral membranes. The gp41 subunit of the HIV-1 envelope plays a major role in this process, but the structure of gp41 is unknown. We have identified a stable, proteinase-resistant structure comprising two peptides, N-51 and C-43, derived from a recombinant protein fragment of the gp41 ectodomain. In isolation, N-51 is predominantly aggregated and C-43 is unfolded. When mixed, however, these peptides associate to form a stable, α-helical, discrete trimer of heterodimers. Proteolysis experiments indicate that the relative orientation of the N-51 and C-43 helices in the complex is antiparallel. We propose that N-51 forms an interior, parallel, homotrimeric, coiled-coil core, against which three C-43 helices pack in an antiparallel fashion. We suggest that this α-helical, trimeric complex is the core of the fusion-competent state of the HIV-1 envelope.

A trimeric structural domain of the HIV-1 transmembrane glycoprotein.

Previous attempts to define the oligomeric state of the HIV and SIV envelope glycoproteins have yielded conflicting results. We have produced in Escherichia coli a recombinant model for the ectodomain of the SIV envelope protein gp41 and have identified a small, trimeric subdomain by proteolytic digestion of this gp41 fragment. The subdomain assembles from two peptide fragments, spanning residues 28-80 (N28-80) and residues 107-149 (C107-149) of SIV gp41. Each of these peptides contains a 4,3-hydrophobic repeat, the hallmark of coiled-coil sequences. Upon mixing, the peptides form a highly helical, trimeric complex [3(N+C)] that resists proteolysis and has a melting temperature (Tm) above 90 degrees C in physiological buffer. The N- and C-terminal fragments are antiparallel to each other in the complex, as judged by the observation that digestion of a variant recombinant protein truncated at the amino terminus yields a C-terminal fragment shortened at its carboxy terminus. The N28-80 peptide contains more positions within the heptad repeat than C107-149 that are predominantly hydrophobic, suggesting that N28-80 is buried in the interior of the complex. We propose that the complex consists of a parallel, trimeric coiled-coil of the N-terminal peptide, encircled by three C-terminal peptide helices arranged in an antiparallel fashion, and that this complex forms a core within the gp41 extracellular domain.

A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein.

HIV-1 infection is initiated by the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 transmembrane envelope glycoprotein gp41 plays a major role in this membrane fusion process. Previous studies have shown that a stable, alpha-helical, trimeric structural domain of gp41 consists of N-terminal 51-residue (N-51) and C-terminal 43-residue (C-43) extraviral segments. This alpha-helical, trimeric complex has been proposed to form the core of the membrane fusion-active conformation of the HIV-1 envelope. We show here that a stable subdomain can be formed by shorter 36-residue (N-36) and 34-residue (C-34) peptides corresponding to the central regions of N-51 and C-43, respectively. In isolation, N-36 is predominantly aggregated, while C-34 is unfolded. Upon mixing, however, these peptides form a stable, alpha-helical, discrete trimer of heterodimers (the melting temperature of a 10 microM solution is 64 degrees C at pH 7). Thus, this subdomain displays the salient features of the stable core structure of the isolated gp41 protein. Our results also provide strong support for the notion that short peptides can form unique, cooperatively folded subdomains, in which elements of secondary structure are stabilized by native-like tertiary interactions.

Min Lu

Papers

Core structure of GP41 from the HIV envelope glycoprotein

A trimeric structural domain of the HIV-1 transmembrane glycoprotein.

A trimeric subdomain of the simian immunodeficiency virus envelope glycoprotein.

A Trimeric Structural Subdomain of the HIV-1 Transmembrane Glycoprotein