Owen Pornillos

Tsg101 and the vacuolar protein sorting pathway are essential for HIV-1 budding.

Like other enveloped viruses, HIV-1 uses cellular machinery to bud from infected cells. We now show that Tsg101 protein, which functions in vacuolar protein sorting (Vps), is required for HIV-1 budding. The UEV domain of Tsg101 binds to an essential tetrapeptide (PTAP) motif within the p6 domain of the structural Gag protein and also to ubiquitin. Depletion of cellular Tsg101 by small interfering RNA arrests HIV-1 budding at a late stage, and budding is rescued by reintroduction of Tsg101. Dominant negative mutant Vps4 proteins that inhibit vacuolar protein sorting also arrest HIV-1 and MLV budding. These observations suggest that retroviruses bud by appropriating cellular machinery normally used in the Vps pathway to form multivesicular bodies.

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.

Mechanisms of enveloped RNA virus budding

To spread infection, enveloped viruses must bud from infected host cells. Recent research indicates that HIV and other enveloped RNA viruses bud by appropriating the cellular machinery that is normally used to create vesicles that bud into late endosomal compartments called multivesicular bodies. This new model of virus budding has many potential implications for cell biology and viral pathogenesis.

Structure and functional interactions of the Tsg101 UEV domain.

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide ‘late domain’ motif located within the viral Gag protein. These interactions are mediated by the N‐terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the struc ture of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N‐terminal helix, an extended β‐hairpin that links strands 1 and 2, and lacks the two C‐terminal helices normally found in E2 enzymes. PTAP‐containing peptides bind in a hydrophobic cleft exposed by the absence of the C‐terminal helices, whereas ubiquitin binds in a novel site surrounding the β‐hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein–protein interactions required for HIV budding and VPS.

X-ray structure of EmrE supports dual topology model.

EmrE, a multidrug transporter from Escherichia coli, functions as a homodimer of a small four-transmembrane protein. The membrane insertion topology of the two monomers is controversial. Although the EmrE protein was reported to have a unique orientation in the membrane, models based on electron microscopy and now defunct x-ray structures, as well as recent biochemical studies, posit an antiparallel dimer. We have now reanalyzed our x-ray data on EmrE. The corrected structures in complex with a transport substrate are highly similar to the electron microscopy structure. The first three transmembrane helices from each monomer surround the substrate binding chamber, whereas the fourth helices participate only in dimer formation. Selenomethionine markers clearly indicate an antiparallel orientation for the monomers, supporting a “dual topology” model.

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.

HIV Gag mimics the Tsg101-recruiting activity of the human Hrs protein

The HIV-1 Gag protein recruits the cellular factor Tsg101 to facilitate the final stages of virus budding. A conserved P(S/T)AP tetrapeptide motif within Gag (the “late domain”) binds directly to the NH2-terminal ubiquitin E2 variant (UEV) domain of Tsg101. In the cell, Tsg101 is required for biogenesis of vesicles that bud into the lumen of late endosomal compartments called multivesicular bodies (MVBs). However, the mechanism by which Tsg101 is recruited from the cytoplasm onto the endosomal membrane has not been known. Now, we report that Tsg101 binds the COOH-terminal region of the endosomal protein hepatocyte growth factor–regulated tyrosine kinase substrate (Hrs; residues 222–777). This interaction is mediated, in part, by binding of the Tsg101 UEV domain to the Hrs 348PSAP351 motif. Importantly, Hrs222–777 can recruit Tsg101 and rescue the budding of virus-like Gag particles that are missing native late domains. These observations indicate that Hrs normally functions to recruit Tsg101 to the endosomal membrane. HIV-1 Gag apparently mimics this Hrs activity, and thereby usurps Tsg101 and other components of the MVB vesicle fission machinery to facilitate viral budding.

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.

X-ray structure of the EmrE multidrug transporter in complex with a substrate.

EmrE is a prototype of the Small Multidrug Resistance family of efflux transporters and actively expels positively charged hydrophobic drugs across the inner membrane of Escherichia coli. Here, we report the x-ray crystal structure, at 3.7 angstrom resolution, of one conformational state of the EmrE transporter in complex with a translocation substrate, tetraphenylphosphonium. Two EmrE polypeptides form a homodimeric transporter that binds substrate at the dimerization interface. The two subunits have opposite orientations in the membrane and adopt slightly different folds, forming an asymmetric antiparallel dimer. This unusual architecture likely confers unidirectionality to transport by creating an asymmetric substrate translocation pathway. On the basis of available structural data, we propose a model for the proton-dependent drug efflux mechanism of EmrE.

X-ray crystal structures of native HIV-1 capsid protein reveal conformational variability

Retroviral capsids in their native form Capsid proteins of retroviruses form protective lattices around viral RNA molecules. The precise molecular details of how individual, full-length capsid proteins assemble to shield the viral genome; however, are not well understood. Obal et al. and Gres et al. now report high resolution crystal structures of the full length capsid proteins from Bovine Leukemia Virus and HIV-1, respectively. The two studies complement each other to reveal the dynamic nature of capsid protein assembly and of how individual capsid proteins interact in the lattice. The findings may have relevance for drug design. Science, this issue p. 95; see also p. 99 Crystal structures of native retroviral capsid proteins reveal how these large protein structures assemble and interact. The detailed molecular interactions between native HIV-1 capsid protein (CA) hexamers that shield the viral genome and proteins have been elusive. We report crystal structures describing interactions between CA monomers related by sixfold symmetry within hexamers (intrahexamer) and threefold and twofold symmetry between neighboring hexamers (interhexamer). The structures describe how CA builds hexagonal lattices, the foundation of mature capsids. Lattice structure depends on an adaptable hydration layer modulating interactions among CA molecules. Disruption of this layer alters interhexamer interfaces, highlighting an inherent structural variability. A CA-targeting antiviral affects capsid stability by binding across CA molecules and subtly altering interhexamer interfaces remote to the ligand-binding site. Inherent structural plasticity, hydration layer rearrangement, and effector binding affect capsid stability and have functional implications for the retroviral life cycle.