[Show abstract][Hide abstract] ABSTRACT: We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1) infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII) of DENV-1 envelope (E) protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.
[Show abstract][Hide abstract] ABSTRACT: This protocol describes protein production in mammalian cells by transient transfection. It assumes the expression construct contains either a 6-HIS or Fc fusion tag to allow recovery of the protein by affinity chromatography. The method is one of the simplest available for protein expression in eukaryotic cells, requires little specialized equipment, and has a reasonably high rate of success.
[Show abstract][Hide abstract] ABSTRACT: This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.
[Show abstract][Hide abstract] ABSTRACT: St. Louis encephalitis virus (SLEV) is a mosquito borne flavivirus responsible for several human encephalitis outbreaks over the last 80 years. Mature flavivirus virions are coated with dimeric envelope (E) proteins that mediate attachment and fusion with host cells. E is a class II fusion protein, the hallmark of which is a distinct dimer-to-trimer rearrangement that occurs upon endosomal acidification and insertion of hydrophobic fusion peptides into the endosomal membrane. Herein, we report the crystal structure of SLEV E in the posfusion trimer conformation. The structure revealed specific features that differentiate SLEV E from trimers of related flavi- and alphaviruses. SLEV E fusion loops have distinct intermediate spacing such that they are positioned further apart than previously observed in flaviviruses but closer together than Semliki Forest Virus, an alphavirus. Domains II (DII) and III (DIII) of SLEV E also adopt different angles relative to domain I (DI) which suggests the DI-DII joint may accommodate spheroidal motions. However, trimer interfaces are well conserved amongst flaviviruses, so it is likely the differences observed represent structural features specific to SLEV function. Analysis of surface potentials revealed a basic platform underneath flavivirus fusion loops that may interact with the anionic lipid head groups found in membranes. Taken together, these results highlight variation in E structure and assembly that may direct virus-specific interactions with host determinants to influence pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Osteoprotegerin (OPG) and receptor activator of nuclear factor κB (RANK) are members of the tumor necrosis factor receptor (TNFR) superfamily that regulate osteoclast formation and function by competing for RANK ligand (RANKL). RANKL promotes osteoclast development through RANK activation, while OPG inhibits this process by sequestering RANKL. For comparison, we solved crystal structures of RANKL with RANK and RANKL with OPG. Complementary biochemical and functional studies reveal that the monomeric cytokine-binding region of OPG binds RANKL with ∼500-fold higher affinity than RANK and inhibits RANKL-stimulated osteoclastogenesis ∼150 times more effectively, in part because the binding cleft of RANKL makes unique contacts with OPG. Several side chains as well as the C-D and D-E loops of RANKL occupy different orientations when bound to OPG versus RANK. High affinity OPG binding requires a 90s loop Phe residue that is mutated in juvenile Paget's disease. These results suggest cytokine plasticity may help to fine-tune specific tumor necrosis factor (TNF)-family cytokine/receptor pair selectivity.
[Show abstract][Hide abstract] ABSTRACT: We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC' loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC' loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development.
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV) is the leading global cause of viral encephalitis. The JEV envelope protein (E) facilitates cellular attachment and membrane fusion and is the primary target of neutralizing antibodies. We have determined the 2.1-Å resolution crystal structure of the JEV E ectodomain refolded from bacterial inclusion bodies. The E protein possesses the three domains characteristic of flavivirus envelopes and epitope mapping of neutralizing antibodies onto the structure reveals determinants that correspond to the domain I lateral ridge, fusion loop, domain III lateral ridge, and domain I-II hinge. While monomeric in solution, JEV E assembles as an antiparallel dimer in the crystal lattice organized in a highly similar fashion as seen in cryo-electron microscopy models of mature flavivirus virions. The dimer interface, however, is remarkably small and lacks many of the domain II contacts observed in other flavivirus E homodimers. In addition, uniquely conserved histidines within the JEV serocomplex suggest that pH-mediated structural transitions may be aided by lateral interactions outside the dimer interface in the icosahedral virion. Our results suggest that variation in dimer structure and stability may significantly influence the assembly, receptor interaction, and uncoating of virions.
Journal of Virology 12/2011; 86(4):2337-46. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gammaherpesviruses encode numerous immunomodulatory molecules that contribute to their ability to evade the host immune response and establish persistent, lifelong infections. As the human gammaherpesviruses are strictly species specific, small animal models of gammaherpesvirus infection, such as murine gammaherpesvirus 68 (γHV68) infection, are important for studying the roles of gammaherpesvirus immune evasion genes in in vivo infection and pathogenesis. We report here the genome sequence and characterization of a novel rodent gammaherpesvirus, designated rodent herpesvirus Peru (RHVP), that shares conserved genes and genome organization with γHV68 and the primate gammaherpesviruses but is phylogenetically distinct from γHV68. RHVP establishes acute and latent infection in laboratory mice. Additionally, RHVP contains multiple open reading frames (ORFs) not present in γHV68 that have sequence similarity to primate gammaherpesvirus immunomodulatory genes or cellular genes. These include ORFs with similarity to major histocompatibility complex class I (MHC-I), C-type lectins, and the mouse mammary tumor virus and herpesvirus saimiri superantigens. As these ORFs may function as immunomodulatory or virulence factors, RHVP presents new opportunities for the study of mechanisms of immune evasion by gammaherpesviruses.
Journal of Virology 03/2011; 85(6):2642-56. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antibody protection against flaviviruses is associated with the development of neutralizing antibodies against the viral envelope (E) protein. Prior studies with West Nile virus (WNV) identified therapeutic mouse and human monoclonal antibodies (MAbs) that recognized epitopes on domain III (DIII) of the E protein. To identify an analogous panel of neutralizing antibodies against DENV type-1 (DENV-1), we immunized mice with a genotype 2 strain of DENV-1 virus and generated 79 new MAbs, 16 of which strongly inhibited infection by the homologous virus and localized to DIII. Surprisingly, only two MAbs, DENV1-E105 and DENV1-E106, retained strong binding and neutralizing activity against all five DENV-1 genotypes. In an immunocompromised mouse model of infection, DENV1-E105 and DENV1-E106 exhibited therapeutic activity even when administered as a single dose four days after inoculation with a heterologous genotype 4 strain of DENV-1. Using epitope mapping and X-ray crystallographic analyses, we localized the neutralizing determinants for the strongly inhibitory MAbs to distinct regions on DIII. Interestingly, sequence variation in DIII alone failed to explain disparities in neutralizing potential of MAbs among different genotypes. Overall, our experiments define a complex structural epitope on DIII of DENV-1 that can be recognized by protective antibodies with therapeutic potential.
[Show abstract][Hide abstract] ABSTRACT: Flaviviruses are a group of human pathogens causing severe encephalitic or hemorrhagic diseases that include West Nile, dengue and yellow fever viruses. Here, using X-ray crystallography we have defined the structure of the flavivirus cross-reactive antibody E53 that engages the highly conserved fusion loop of the West Nile virus envelope glycoprotein. Using cryo-electron microscopy, we also determined that E53 Fab binds preferentially to spikes in noninfectious, immature flavivirions but is unable to bind significantly to mature virions, consistent with the limited solvent exposure of the epitope. We conclude that the neutralizing impact of E53 and likely similar fusion-loop-specific antibodies depends on its binding to the frequently observed immature component of flavivirus particles. Our results elucidate how fusion-loop antibodies, which comprise a significant fraction of the humoral response against flaviviruses, can function to control infection without appreciably recognizing mature virions. As these highly cross-reactive antibodies are often weakly neutralizing they also may contribute to antibody-dependent enhancement and flavi virus pathogenesis thereby complicating development of safe and effective vaccines.
The EMBO Journal 09/2009; 28(20):3269-76. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The B and T lymphocyte attenuator (BTLA) appears to act as a negative regulator of T cell activation and growth. BTLA specifically interacts with herpesvirus entry mediator (HVEM), a member of the TNFR family. Herein, we have undertaken surface plasmon resonance studies to quantitatively assess BTLA and HVEM ectodomain interactions. We find that soluble BALB/cJ BTLA engages HVEM with an equilibrium affinity of 0.97+/-0.19 microM while the C57BL/6 BTLA binds slightly better with an equilibrium affinity of 0.42+/-0.06 microM. Despite its lower affinity for HVEM, the kinetic half-life of BALB/cJ BTLA complexes are twice as long as observed for C57BL/6 BTLA (4 vs 2 s). To further explore these interactions, we solved the crystal structure of a murine BTLA (BALB/cJ) ectodomain at 1.8-A resolution, revealing a beta sandwich fold with strong similarity to I-set members of the Ig superfamily. Using a structure-based mutagenesis strategy, we then examined the individual contributions of 26 BTLA surface-exposed residues toward HVEM binding. Four single-site substitutions were identified that decrease HVEM binding below detectable levels and two that decrease binding by more than half. All six of these cluster at the edge of the beta sandwich in a membrane distal patch formed primarily from the A and G strands. This patch falls within the contacting surface recently revealed in the crystal structure of the human BTLA-HVEM cocomplex. The critical binding residues identified here are highly conserved across species, suggesting that BTLA employs a conserved binding mode for HVEM recognition.
The Journal of Immunology 02/2008; 180(2):940-7. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The envelope glycoprotein (E) of West Nile virus (WNV) undergoes a conformational rearrangement triggered by low pH that results in a class II fusion event required for viral entry. Herein we present the 3.0-A crystal structure of the ectodomain of WNV E, which reveals insights into the flavivirus life cycle. We found that WNV E adopts a three-domain architecture that is shared by the E proteins from dengue and tick-borne encephalitis viruses and forms a rod-shaped configuration similar to that observed in immature flavivirus particles. Interestingly, the single N-linked glycosylation site on WNV E is displaced by a novel alpha-helix, which could potentially alter lectin-mediated attachment. The localization of histidines within the hinge regions of E implicates these residues in pH-induced conformational transitions. Most strikingly, the WNV E ectodomain crystallized as a monomer, in contrast to other flavivirus E proteins, which have crystallized as antiparallel dimers. WNV E assembles in a crystalline lattice of perpendicular molecules, with the fusion loop of one E protein buried in a hydrophobic pocket at the DI-DIII interface of another. Dimeric E proteins pack their fusion loops into analogous pockets at the dimer interface. We speculate that E proteins could pivot around the fusion loop-pocket junction, allowing virion conformational transitions while minimizing fusion loop exposure.
Journal of Virology 01/2007; 80(23):11467-74. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral surface of domain III (DIII) of the West Nile virus (WNV) envelope (E) strongly protect against infection in animals. Herein, we observed significantly less efficient neutralization by 89 MAbs that recognized domain I (DI) or II (DII) of WNV E protein. Moreover, in cells expressing Fc gamma receptors, many of the DI- and DII-specific MAbs enhanced infection over a broad range of concentrations. Using yeast surface display of E protein variants, we identified 25 E protein residues to be critical for recognition by DI- or DII-specific neutralizing MAbs. These residues cluster into six novel and one previously characterized epitope located on the lateral ridge of DI, the linker region between DI and DIII, the hinge interface between DI and DII, and the lateral ridge, central interface, dimer interface, and fusion loop of DII. Approximately 45% of DI-DII-specific MAbs showed reduced binding with mutations in the highly conserved fusion loop in DII: 85% of these (34 of 40) cross-reacted with the distantly related dengue virus (DENV). In contrast, MAbs that bound the other neutralizing epitopes in DI and DII showed no apparent cross-reactivity with DENV E protein. Surprisingly, several of the neutralizing epitopes were located in solvent-inaccessible positions in the context of the available pseudoatomic model of WNV. Nonetheless, DI and DII MAbs protect against WNV infection in mice, albeit with lower efficiency than DIII-specific neutralizing MAbs.
Journal of Virology 01/2007; 80(24):12149-59. · 5.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interaction between B- and T-lymphocyte attenuator (BTLA), an inhibitory receptor whose extracellular domain belongs to the immunoglobulin superfamily, and herpesvirus-entry mediator (HVEM), a co-stimulatory tumour-necrosis factor receptor, is unique in that it is the only receptor-ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and what downstream signalling events might occur as a result of receptor ligation. As we discuss, recent studies show that engagement of HVEM with its endogenous ligand (LIGHT) from the tumour-necrosis factor family induces a powerful immune response, whereas HVEM interactions with BTLA negatively regulate T-cell responses.
[Show abstract][Hide abstract] ABSTRACT: Recently a new inhibitory immunoglobulin domain-containing lymphocyte receptor was identified on the basis of its T helper 1 (T(H)1)-selective expression in murine T cell lines, which was named B and T lymphocyte attenuator (BTLA). Several groups have confirmed the initial characterization of BTLA as an inhibitory receptor, which was initially inferred from the mild increases in several parameters of BTLA-deficient mice. The initial expectation that BTLA would interact with a B7 family ligand, such as the B7x protein, was surprisingly overturned with the functional cloning of the actual BTLA ligand as herpesvirus entry mediator (HVEM). This was unexpected largely due to the fact that this interaction represents the convergence of two very different, although each quite extensive, families of receptors and ligands. The interaction of BTLA, which belongs to the CD28 family of the immunoglobulin superfamily, and HVEM, a costimulatory tumor-necrosis factor (TNF) receptor (TNFR), is quite unique in that it is the only receptor-ligand interaction that directly bridges these two families of receptors. This interaction has raised many questions about how receptors from two different families could interact and which are the signaling events downstream of receptor ligation. As we discuss here and recently demonstrated, HVEM interaction with BTLA serves to negatively regulate T cell responses, in contrast to the strong activation observed when HVEM engages its endogenous ligand from the TNF family. Finally, as studies of BTLA are just now beginning to extend beyond the initial characterizations, it is becoming clear that there are many complex issues remaining to be resolved, particularly potential polymorphisms that may engender disease susceptibility in the human.
Advances in Immunology 02/2006; 92:157-85. · 7.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The open reading frame (ORF) 7a of the SARS-associated coronavirus (SARS-CoV) encodes a unique type I transmembrane protein of unknown function. We have determined the 1.8 A resolution crystal structure of the N-terminal ectodomain of orf7a, revealing a compact seven-stranded beta sandwich unexpectedly similar in fold and topology to members of the Ig superfamily. We also demonstrate that, in SARS-CoV- infected cells, the orf7a protein is expressed and retained intracellularly. Confocal microscopy studies using orf7a and orf7a/CD4 chimeras implicate the short cytoplasmic tail and transmembrane domain in trafficking of the protein within the endoplasmic reticulum and Golgi network. Taken together, our findings provide a structural and cellular framework in which to explore the role of orf7a in SARS-CoV pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: The M3 protein encoded by murine gamma herpesvirus68 (gamma HV68) functions as an immune system saboteur by the engagement of chemoattractant cytokines, thereby altering host antiviral inflammatory responses. Here we report the crystal structures of M3 both alone and in complex with the CC chemokine MCP-1. M3 is a two-domain beta sandwich protein with a unique sequence and topology, forming a tightly packed anti-parallel dimer. The stoichiometry of the MCP-1:M3 complex is 2:2, with two monomeric chemokines embedded at distal ends of the preassociated M3 dimer. Conformational flexibility and electrostatic complementation are both used by M3 to achieve high-affinity and broad-spectrum chemokine engagement. M3 also employs structural mimicry to promiscuously sequester chemokines, engaging conservative structural elements associated with both chemokine homodimerization and binding to G protein-coupled receptors.