Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 Å reveals a compact architecture composed of a central Ig-fold β-sandwich flanked by two additional protein layers. The CD81 receptor-binding site was identified by EM and by site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and EM E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide invaluable information for HCV drug and vaccine design.

A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization.

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug-and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibodybound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded β-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate-and structurally plastic-layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated β-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a β-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.HIV-1 viral spike | molecular motion | protein architecture | receptortriggered entry | type 1 fusion protein T he viral spike (gp120/gp41) of HIV type 1 (HIV-1) uses substantial conformational changes to facilitate viral entry (reviewed in ref. 1). Receptor binding by gp120 triggers a series of conformational changes in gp41, which in the unliganded envelope spike possesses a high potential energy that will ultimately be used to fuse the viral and target cell membranes. Binding of the initial receptor, CD4, induces changes in gp120 conformation that allow high-affinity interaction with the coreceptor, CCR5 or CXCR4, and the formation of a gp41 prehairpin intermediate. Subsequent engagement of coreceptor is thought to promote additional conformational changes in gp41 that create an energetically stable sixhelix bundle coincident with the fusion of viral and cell membranes.The gp120 and gp41 glycoproteins are not linked by disulfide bonds, and the noncovalent association of these spike subunits presents significant challenges. The gp120 glycoprotein must be flexible to allow conformational change, yet retain sufficient contact with gp41 to maintain the integrity of the unliganded trimer and, after CD4 binding, to...

SUMMARY The high mannose patch on the HIV-1 Envelope (Env) glycoprotein is the epicenter for binding of the potent broadly neutralizing PGT121 family of antibodies, but strategies for generating such antibodies by vaccination have not been defined. We generated structures of inferred antibody intermediates by X-ray crystallography and electron microscopy to elucidate the molecular events that occurred during evolution of this family. Binding analyses revealed that affinity maturation was primarily focused on avoiding, accommodating, or binding the N137 glycan. The overall antibody approach angle to Env was defined very early in the maturation process, yet differences evolved in the PGT121 family branches that led to differences in glycan specificities in their respective epitopes. Furthermore, we determined a crystal structure of the recombinant BG505 SOSIP.664 HIV-1 trimer with a PGT121 family member at 3.0 Å that, in concert with these antibody intermediate structures, provide insights to advance design of HIV vaccine candidates.