Mark Yeager

The structural biology of HIV assembly

HIV assembly and replication proceed through the formation of morphologically distinct immature and mature viral capsids that are organized by the Gag polyprotein (immature) and by the fully processed CA protein (mature). The Gag polyprotein is composed of three folded polypeptides (MA, CA, and NC) and three smaller peptides (SP1, SP2, and p6) that function together to coordinate membrane binding and Gag-Gag lattice interactions in immature virions. Following budding, HIV maturation is initiated by proteolytic processing of Gag, which induces conformational changes in the CA domain and results in the assembly of the distinctive conical capsid. Retroviral capsids are organized following the principles of fullerene cones, and the hexagonal CA lattice is stabilized by three distinct interfaces. Recently identified inhibitors of viral maturation act by disrupting the final stage of Gag processing, or by inhibiting the formation of a critical intermolecular CA-CA interface in the mature capsid. Following release into a new host cell, the capsid disassembles and host cell factors can potently restrict this stage of retroviral replication. Here, we review the structures of immature and mature HIV virions, focusing on recent studies that have defined the global organization of the immature Gag lattice, identified sites likely to undergo conformational changes during maturation, revealed the molecular structure of the mature capsid lattice, demonstrated that capsid architectures are conserved, identified the first capsid assembly inhibitors, and begun to uncover the remarkable biology of the mature capsid.

X-ray structures of the hexameric building block of the HIV capsid.

The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.

Three-dimensional organization of a human water channel

Aquaporins (AQP) are members of the major intrinsic protein (MIP) superfamily of integral membrane proteins and facilitate water transport in various eukaryotes and prokaryotes. The archetypal aquaporin AQP1 is a partly glycosylated water-selective channel that is widely expressed in the plasma membranes of several water-permeable epithelial and endothelial cells,. Here we report the three-dimensional structure of deglycosylated, human erythrocyte AQP1, determined at 7Å resolution in the membrane plane by electron crystallography of frozen-hydrated two-dimensional crystals. The structure has an in-plane, intramolecular 2-fold axis of symmetry located in the hydrophobic core of the bilayer. The AQP1 monomer is composed of six membrane-spanning, tilted α-helices. These helices form a barrel that encloses a vestibular region leading to the water-selective channel, which is outlined by densities attributed to the functionally important NPA boxes and their bridges to the surrounding helices. The intramolecular symmetry within the AQP1 molecule represents a new motif for the topology and design of membrane protein channels, and is a simple and elegant solution to the problem of bidirectional transport across the bilayer.

Structure of Full-Length HIV-1 CA: A Model for the Mature Capsid Lattice

The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. These capsids are modeled as fullerene structures composed of closed hexameric arrays of the viral CA protein, but a high-resolution structure of the lattice has remained elusive. A three-dimensional map of two-dimensional crystals of the R18L mutant of HIV-1 CA was derived by electron cryocrystallography. The docking of high-resolution domain structures into the map yielded the first unambiguous model for full-length HIV-1 CA. Three important protein-protein assembly interfaces are required for capsid formation. Each CA hexamer is composed of an inner ring of six N-terminal domains and an outer ring of C-terminal domains that form dimeric linkers connecting neighboring hexamers. Interactions between the two domains of CA further stabilize the hexamer and provide a structural explanation for the mechanism of action of known HIV-1 assembly inhibitors.

Type IV Pilin Structure and Assembly X-Ray and EM Analyses of Vibrio cholerae Toxin-Coregulated Pilus and Pseudomonas aeruginosa PAK Pilin

Pilin assembly into type IV pili is required for virulence by bacterial pathogens that cause diseases such as cholera, pneumonia, gonorrhea, and meningitis. Crystal structures of soluble, N-terminally truncated pilin from Vibrio cholera toxin-coregulated pilus (TCP) and full-length PAK pilin from Pseudomonas aeruginosa reveal a novel TCP fold, yet a shared architecture for the type IV pilins. In each pilin subunit a conserved, extended, N-terminal alpha helix wrapped by beta strands anchors the structurally variable globular head. Inside the assembled pilus, characterized by cryo-electron microscopy and crystallography, the extended hydrophobic alpha helices make multisubunit contacts to provide mechanical strength and flexibility. Outside, distinct interactions of adaptable heads contribute surface variation for specificity of pilus function in antigenicity, motility, adhesion, and colony formation.

Atomic-level modelling of the HIV capsid

The mature capsids of human immunodeficiency virus type 1 (HIV-1) and other retroviruses are fullerene shells, composed of the viral CA protein, that enclose the viral genome and facilitate its delivery into new host cells. Retroviral CA proteins contain independently folded amino (N)- and carboxy (C)-terminal domains (NTD and CTD) that are connected by a flexible linker. The NTD forms either hexameric or pentameric rings, whereas the CTD forms symmetric homodimers that connect the rings into a hexagonal lattice. We previously used a disulphide crosslinking strategy to enable isolation and crystallization of soluble HIV-1 CA hexamers. Here we use the same approach to solve the X-ray structure of the HIV-1 CA pentamer at 2.5 Å resolution. Two mutant CA proteins with engineered disulphides at different positions (P17C/T19C and N21C/A22C) converged onto the same quaternary structure, indicating that the disulphide-crosslinked proteins recapitulate the structure of the native pentamer. Assembly of the quasi-equivalent hexamers and pentamers requires remarkably subtle rearrangements in subunit interactions, and appears to be controlled by an electrostatic switch that favours hexamers over pentamers. This study completes the gallery of substructures describing the components of the HIV-1 capsid and enables atomic-level modelling of the complete capsid. Rigid-body rotations around two assembly interfaces appear sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone.

Three-dimensional EM structure of the ectodomain of integrin αVβ3 in a complex with fibronectin

Integrins are αβ heterodimeric cell surface receptors that mediate transmembrane signaling by binding extracellular and cytoplasmic ligands. The ectodomain of integrin αVβ3 crystallizes in a bent, genuflexed conformation considered to be inactive (unable to bind physiological ligands in solution) unless it is fully extended by activating stimuli. We generated a stable, soluble complex of the Mn2+-bound αVβ3 ectodomain with a fragment of fibronectin (FN) containing type III domains 7 to 10 and the EDB domain (FN7-EDB-10). Transmission electron microscopy and single particle image analysis were used to determine the three-dimensional structure of this complex. Most αVβ3 particles, whether unliganded or FN-bound, displayed compact, triangular shapes. A difference map comparing ligand-free and FN-bound αVβ3 revealed density that could accommodate the RGD-containing FN10 in proximity to the ligand-binding site of β3, with FN9 just adjacent to the synergy site binding region of αV. We conclude that the ectodomain of αVβ3 manifests a bent conformation that is capable of stably binding a physiological ligand in solution.

Hexagonal assembly of a restricting TRIM5α protein

TRIM5α proteins are restriction factors that protect mammalian cells from retroviral infections by binding incoming viral capsids, accelerating their dissociation, and preventing reverse transcription of the viral genome. Individual TRIM5 isoforms can often protect cells against a broad range of retroviruses, as exemplified by rhesus monkey TRIM5α and its variant, TRIM5-21R, which recognize HIV-1 as well as several distantly related retroviruses. Although capsid recognition is not yet fully understood, previous work has shown that the C-terminal SPRY/B30.2 domain of dimeric TRIM5α binds directly to viral capsids, and that higher-order TRIM5α oligomerization appears to contribute to the efficiency of capsid recognition. Here, we report that recombinant TRIM5-21R spontaneously assembled into two-dimensional paracrystalline hexagonal lattices comprising open, six-sided rings. TRIM5-21R assembly did not require the C-terminal SPRY domain, but did require both protein dimerization and a B-box 2 residue (Arg121) previously implicated in TRIM5α restriction and higher-order assembly. Furthermore, TRIM5-21R assembly was promoted by binding to hexagonal arrays of the HIV-1 CA protein that mimic the surface of the viral capsid. We therefore propose that TRIM5α proteins have evolved to restrict a range of different retroviruses by assembling a deformable hexagonal scaffold that positions the capsid-binding domains to match the symmetry and spacing of the capsid surface lattice. Capsid recognition therefore involves a synergistic combination of direct binding interactions, avidity effects, templated assembly, and lattice complementarity.

Ultrastructural and Biophysical Characterization of Hepatitis C Virus Particles Produced in Cell Culture

ABSTRACT We analyzed the biochemical and ultrastructural properties of hepatitis C virus (HCV) particles produced in cell culture. Negative-stain electron microscopy revealed that the particles were spherical (∼40- to 75-nm diameter) and pleomorphic and that some of them contain HCV E2 protein and apolipoprotein E on their surfaces. Electron cryomicroscopy revealed two major particle populations of ∼60 and ∼45 nm in diameter. The ∼60-nm particles were characterized by a membrane bilayer (presumably an envelope) that is spatially separated from an internal structure (presumably a capsid), and they were enriched in fractions that displayed a high infectivity-to-HCV RNA ratio. The ∼45-nm particles lacked a membrane bilayer and displayed a higher buoyant density and a lower infectivity-to-HCV RNA ratio. We also observed a minor population of very-low-density, >100-nm-diameter vesicular particles that resemble exosomes. This study provides low-resolution ultrastructural information of particle populations displaying differential biophysical properties and specific infectivity. Correlative analysis of the abundance of the different particle populations with infectivity, HCV RNA, and viral antigens suggests that infectious particles are likely to be present in the large ∼60-nm HCV particle populations displaying a visible bilayer. Our study constitutes an initial approach toward understanding the structural characteristics of infectious HCV particles.

Three-dimensional model of the human platelet integrin αIIbβ3 based on electron cryomicroscopy and x-ray crystallography

Integrins are a large family of heterodimeric transmembrane signaling proteins that affect diverse biological processes such as development, angiogenesis, wound healing, neoplastic transformation, and thrombosis. We report here the three-dimensional structure at 20-Å resolution of the unliganded, low-affinity state of the human platelet integrin αIIbβ3 derived by electron cryomicroscopy and single particle image reconstruction. The large ectodomain and small cytoplasmic domains are connected by a rod of density that we interpret as two parallel transmembrane α-helices. The docking of the x-ray structure of the αVβ3 ectodomain into the electron cryomicroscopy map of αIIbβ3 requires hinge movements at linker regions between domains in the crystal structure. Comparison of the putative high- and low-affinity conformations reveals dramatic conformational changes associated with integrin activation.