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A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM). [ABSTRACT FROM AUTHOR]

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs. [Display omitted] • LIBRA-seq identifies 54042-4, a potently neutralizing SARS-CoV-2 antibody • 54042-4 maintains potent neutralization against Alpha, Beta, Gamma, and Delta VOCs • The epitope of 54042-4 is highly conserved among current SARS-CoV-2 isolates Kramer et al. demonstrate that antibody 54042-4 recognizes residues highly conserved across global SARS-CoV-2 isolates. Antibody 54042-4 potently neutralizes all known circulating variants of concern (VOCs) and could be developed as a clinical candidate to treat COVID-19 infection. [ABSTRACT FROM AUTHOR]

Unrelated individuals can produce genetically similar clones of antibodies, known as public clonotypes, which have been seen in responses to different infectious diseases, as well as healthy individuals. Here we identify 37 public clonotypes in memory B cells from convalescent survivors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection or in plasmablasts from an individual after vaccination with mRNA-encoded spike protein. We identify 29 public clonotypes, including clones recognizing the receptor-binding domain (RBD) in the spike protein S1 subunit (including a neutralizing, angiotensin-converting enzyme 2 [ACE2]-blocking clone that protects in vivo) and others recognizing non-RBD epitopes that bind the S2 domain. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting these common germline-encoded antibodies are preconfigured for avid recognition. Identification of large numbers of public clonotypes provides insight into the molecular basis of efficacy of SARS-CoV-2 vaccines and sheds light on the immune pressures driving the selection of common viral escape mutants. [Display omitted] • Public clonotypes are shared among SARS-CoV-2-vaccinated and -infected individuals • Group 3 antibodies share the same binding site as CR3022 but neutralize variants • Germline-revertant forms of public clonotypes bind efficiently to spike protein • Public clonotype responses inform research on new viral variants of concern Chen et al. describe neutralizing and non-neutralizing public antibodies elicited to SARS-CoV-2 spike in vaccinated and naturally infected individuals. Germline-revertant forms of some public clonotypes bind efficiently to spike protein, suggesting common germline-encoded properties for recognition. [ABSTRACT FROM AUTHOR]

The original version of this Article contained an error in the Acknowledgements, which incorrectly listed the NIH grant "AI142759". This has been corrected in both the PDF and HTML versions of the Article. [Extracted from the article]

Antibodies that target the glycan cap epitope on the ebolavirus glycoprotein (GP) are common in the adaptive response of survivors. A subset is known to be broadly neutralizing, but the details of their epitopes and basis for neutralization are not well understood. Here, we present cryoelectron microscopy (cryo-EM) structures of diverse glycan cap antibodies that variably synergize with GP base-binding antibodies. These structures describe a conserved site of vulnerability that anchors the mucin-like domains (MLDs) to the glycan cap, which we call the MLD anchor and cradle. Antibodies that bind to the MLD cradle share common features, including use of IGHV1-69 and IGHJ6 germline genes, which exploit hydrophobic residues and form β-hairpin structures to mimic the MLD anchor, disrupt MLD attachment, destabilize GP quaternary structure, and block cleavage events required for receptor binding. Our results provide a molecular basis for ebolavirus neutralization by broadly reactive glycan cap antibodies. [Display omitted] • Glycan cap antibody-mediated GP destabilization correlates with synergy • Cryo-EM structures reveal antibodies target a highly conserved epitope • Antibodies use long CDRH3 loops to displace and mimic portions of the glycan cap • Glycan cap antibodies block cleavage events required for viral entry A rare subset of ebolavirus antibodies targeting the glycan cap are broadly neutralizing. Murin et al. report cryo-EM structures and custom in vitro assays identifying a conserved site of vulnerability in the glycan cap and detail mechanisms of action, including structural mimicry, trimer instability, and blocking cleavage. [ABSTRACT FROM AUTHOR]

Integral membrane proteins are important drug targets, and monoclonal antibodies (mAbs) against them are highly sought for therapeutic and diagnostic purposes. However, the complex structure of multipass membrane proteins makes discovery of these mAbs particularly challenging. By employing strategies such as sequential immunization with DNA (prime) and virus-like particles (boost), and using divergent species (chickens) as immunization hosts, we routinely isolate diverse panels of mAbs against structurally complex and highly conserved membrane protein targets. Here, we report the isolation and characterization of mAbs recognizing two distinct metabolically relevant targets: the 12-transmembrane insulin-responsive glucose transporter GLUT4 and the 7-transmembrane GPCR cannabinoid receptor-1 (CB1). In both cases, we isolated diverse panels of mAbs against native conformational epitopes. Affinities ranged from 2.6 nM (anti-CB1 IM-102) to 1 pM (anti-GLUT4 LM048). Two anti-GLUT4 mAbs discriminated between inward-open and outward-open conformational states, including one mAb (LM048) that inhibited transporter function. Similarly, we isolated several anti-CB1 mAbs that antagonized CB1-mediated calcium flux. Lead mAbs were confirmed to be specific for their target proteins, based on reactivity profiling in the Membrane Proteome Array of >5,600 human membrane proteins. Identified mAbs represent novel and valuable molecular tools for understanding and potentially treating common metabolic diseases, such as type 2 diabetes mellitus and nonalcoholic steatohepatitis/fatty liver disease (NASH/NAFLD). Furthermore, the technologies underpinning their discovery can be rapidly and routinely employed to facilitate discovery of mAbs against other therapeutically relevant membrane protein targets. Disclosure: D. Tucker: None. T. Charpentier: None. N. Molino: None. E. Rosenberg: None. B. Screnci: None. L.J. Stafford: None. C. Sulli: None. R. Chambers: None. B.J. Doranz: None. J.B. Rucker: None. Funding: National Institutes of Health [ABSTRACT FROM AUTHOR]

Sequential dengue virus (DENV) infections often generate neutralizing antibodies against all four DENV serotypes and sometimes, Zika virus. Characterizing cross-flavivirus broadly neutralizing antibody (bnAb) responses can inform countermeasures that avoid enhancement of infection associated with non-neutralizing antibodies. Here, we used single cell transcriptomics to mine the bnAb repertoire following repeated DENV infections. We identified several new bnAbs with comparable or superior breadth and potency to known bnAbs, and with distinct recognition determinants. Unlike all known flavivirus bnAbs, which are IgG1, one newly identified cross-flavivirus bnAb (F25.S02) was derived from IgA1. Both IgG1 and IgA1 versions of F25.S02 and known bnAbs displayed neutralizing activity, but only IgG1 enhanced infection in monocytes expressing IgG and IgA Fc receptors. Moreover, IgG-mediated enhancement of infection was inhibited by IgA1 versions of bnAbs. We demonstrate a role for IgA in flavivirus infection and immunity with implications for vaccine and therapeutic strategies. Author summary: A central challenge for developing clinical interventions for dengue virus or the closely related Zika virus is the ability of IgG antibodies to enhance, rather than neutralize infection under certain conditions. When present prior to infection, as in the case of vaccination, these antibodies can worsen disease outcome. In this study, we analyzed B cells of individuals who experienced dengue or Zika infection to identify those expressing antibodies that can potently neutralize these viruses with minimal potential to enhance infection. We used a method that captured a larger number and wider variety of antibodies than previous approaches. We discovered several potent antibodies that simultaneously neutralized dengue and Zika viruses, including those of IgG isotype, which are common, and one of IgA isotype, which had never been described against this group of viruses. Although IgG antibodies enhanced infection in certain cases, the IgA antibody did not. We further showed that modifying a region of IgG antibodies to convert them to IgA antibodies eliminated their ability to enhance infection. Moreover, the modified IgA versions inhibited the ability of IgG versions to enhance infection. These results suggest that inducing IgA antibodies may be an attractive goal for safe and effective vaccines. [ABSTRACT FROM AUTHOR]

Novel therapeutic monoclonal antibodies (MAbs) must accommodate comprehensive breadth of activity against diverse sarbecoviruses and high neutralization potency to overcome emerging variants. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain (RBD) in complex with MAb WRAIR-2063, a moderate-potency neutralizing antibody with exceptional sarbecovirus breadth, that targets the highly conserved cryptic class V epitope. This epitope overlaps substantially with the spike protein N-terminal domain (NTD) -interacting region and is exposed only when the spike is in the open conformation, with one or more RBDs accessible. WRAIR-2063 binds the RBD of SARS-CoV-2 WA-1, all variants of concern (VoCs), and clade 1 to 4 sarbecoviruses with high affinity, demonstrating the conservation of this epitope and potential resiliency against variation. We compare structural features of additional class V antibodies with their reported neutralization capacity to further explore the utility of the class V epitope as a pan-sarbecovirus vaccine and therapeutic target. [ABSTRACT FROM AUTHOR]

The protective human antibody response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) focuses on the spike (S) protein, which decorates the virion surface and mediates cell binding and entry. Most SARS-CoV-2 protective antibodies target the receptor-binding domain or a single dominant epitope ("supersite") on the N-terminal domain (NTD). Using the single B cell technology called linking B cell receptor to antigen specificity through sequencing (LIBRA-Seq), we isolated a large panel of NTD-reactive and SARS-CoV-2-neutralizing antibodies from an individual who had recovered from COVID-19. We found that neutralizing antibodies against the NTD supersite were commonly encoded by the IGHV1-24 gene, forming a genetic cluster representing a public B cell clonotype. However, we also discovered a rare human antibody, COV2-3434, that recognizes a site of vulnerability on the SARS-CoV-2 S protein in the trimer interface (TI) and possesses a distinct class of functional activity. COV2-3434 disrupted the integrity of S protein trimers, inhibited the cell-to-cell spread of the virus in culture, and conferred protection in human angiotensin-converting enzyme 2-transgenic (ACE2-transgenic) mice against the SARS-CoV-2 challenge. This study provides insight into antibody targeting of the S protein TI region, suggesting this region may be a site of virus vulnerability. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) is a global public health concern due to its ability to cause congenital Zika syndrome and lack of approved vaccine, therapeutic, or other control measures. We discovered eight novel rabbit monoclonal antibodies (MAbs) that bind to distinct ZIKV envelope protein epitopes. The majority of the MAbs were ZIKV specific and targeted the lateral ridge of the envelope (E) protein domain III, while the MAb with the highest neutralizing activity recognized a putative quaternary epitope spanning E protein domains I and III. One of the non-neutralizing MAbs specifically recognized ZIKV precursor membrane protein (prM). Somatic hypermutation of immunoglobulin variable regions increases antibody affinity maturation and triggers antibody class switching. Negative correlations were observed between the somatic hypermutation rate of the immunoglobulin heavy-chain variable region and antibody binding parameters such as equilibrium dissociation constant, dissociation constant, and half-maximal effective concentration value of MAb binding to ZIKV virus-like particles. Complementarity-determining regions recognize the antigen epitopes and are scaffolded by canonical framework regions. Reversion of framework region amino acids to the rabbit germ line sequence decreased anti-ZIKV MAb binding activity of some MAbs. Thus, antibody affinity maturation, including somatic hypermutation and framework region mutations, contributed to the binding and function of these anti-ZIKV MAbs. IMPORTANCE ZIKV is a global health concern against which no vaccine or therapeutics are available. We characterized eight novel rabbit monoclonal antibodies recognizing ZIKV envelope and prM proteins and studied the relationship between somatic hypermutation of complementarity-determining regions, framework regions, mutations, antibody specificity, binding, and neutralizing activity. The results contribute to understanding structural features and somatic mutation pathways by which potent Zika virus-neutralizing antibodies can evolve, including the role of antibody framework regions. [ABSTRACT FROM AUTHOR]

Influenza B viruses (IBVs) comprise a substantial portion of the circulating seasonal human influenza viruses. Here, we describe the isolation of human monoclonal antibodies (mAbs) that recognized the IBV neuraminidase (NA) glycoprotein from an individual following seasonal vaccination. Competition-binding experiments suggested the antibodies recognized two major antigenic sites. One group, which included mAb FluB-393, broadly inhibited IBV NA sialidase activity, protected prophylactically in vivo , and bound to the lateral corner of NA. The second group contained an active site mAb, FluB-400, that broadly inhibited IBV NA sialidase activity and virus replication in vitro in primary human respiratory epithelial cell cultures and protected against IBV in vivo when administered systemically or intranasally. Overall, the findings described here shape our mechanistic understanding of the human immune response to the IBV NA glycoprotein through the demonstration of two mAb delivery routes for protection against IBV and the identification of potential IBV therapeutic candidates. [Display omitted] • Isolation of cross-reactive human antibodies to two distinct IBV NA epitopes • FluB-393 binds at the lateral corner of NA and FluB-400 to the active site • FluB-400 inhibits virus on ALI cell cultures and is protective with intranasal delivery Although influenza B viruses (IBVs) comprise a substantial portion of seasonal flu cases, research focus and treatment options remain limited. Wolters et al. isolate two clonal families of antibodies recognizing distinct epitopes of IBV neuraminidase protein and reveal mechanisms of protection involving enzymatic inhibition, mediating egress, neutralization, and Fc function. [ABSTRACT FROM AUTHOR]

Eastern equine encephalitis virus (EEEV), western equine encephalitis virus (WEEV) and Venezuelan equine encephalitis virus (VEEV) can cause fatal encephalitis in humans and equids. Some MAbs to the E1 glycoprotein are known to be cross-reactive, weakly neutralizing in vitro but can protect from disease in animal models. We investigated the mechanism of neutralization of VEEV infection by the broadly cross-reactive E1-specific MAb 1A4B-6. 1A4B-6 protected 3-week-old Swiss Webster mice prophylactically from lethal VEEV challenge. Likewise, 1A4B-6 inhibited virus growth in vitro at a pre-attachment step after virions were incubated at 37 °C and inhibited virus-mediated cell fusion. Amino acid residue N100 in the fusion loop of E1 protein was identified as critical for binding. The potential to elicit broadly cross-reactive MAbs with limited virus neutralizing activity in vitro but that can inhibit virus entry and protect animals from infection merits further exploration for vaccine and therapeutic developmental research. • Cross-reactive monoclonal antibody 1A4B-6 protects mice prophylactically from lethal Venezuelan equine encephalitis virus challenge. • 1A4B-6 inhibits virus growth in vitro at a pre-attachment step after virions are incubated at 37oC and inhibits virus-mediated cell fusion. • Amino acid residue N100 in the fusion loop of E1 protein is critical for binding of 1A4B-6. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV), from the genus flavivirus of the family flaviviridae, causes serious health problems globally. Human monoclonal antibodies (HuMAb) can be used to elucidate the mechanisms of neutralization and antibody-dependent enhancement (ADE) of DENV infections, leading to the development of a vaccine or therapeutic antibodies. Here, we generated eight HuMAb clones from an Indonesian patient infected with DENV. These HuMAbs exhibited the typical characteristics of weak neutralizing antibodies including high cross-reactivity with other flaviviruses and targeting of the fusion loop epitope (FLE). However, one of the HuMAbs, 3G9, exhibited strong neutralization (NT50 < 0.1 μg/ml) and possessed a high somatic hyper-mutation rate of the variable region, indicating affinity-maturation. Administration of this antibody significantly prolonged the survival of interferon-α/β/γ receptor knockout C57BL/6 mice after a lethal DENV challenge. Additionally, Fc-modified 3G9 that had lost their in vitro ADE activity showed enhanced therapeutic potency in vivo and competed strongly with an ADE-prone antibody in vitro. Taken together, the affinity-matured FLE-targeting antibody 3G9 exhibits promising features for therapeutic application including a low NT50 value, potential for treatment of various kinds of mosquito-borne flavivirus infection, and suppression of ADE. This study demonstrates the therapeutic potency of affinity-matured FLE-targeting antibodies. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV), a mosquito-transmitted flavivirus, caused a large epidemic in Latin America between 2015 and 2017. Effective ZIKV vaccines and treatments are urgently needed to prevent future epidemics and severe disease sequelae. People infected with ZIKV develop strongly neutralizing antibodies linked to viral clearance and durable protective immunity. To understand the mechanisms of protective immunity and to support the development of ZIKV vaccines, we characterize here a strongly neutralizing antibody, B11F, isolated from a patient who recovered from ZIKV. Our results indicate that B11F targets a complex epitope on the virus that spans domains I and III of the envelope glycoprotein. While previous studies point to quaternary epitopes centered on domain II of the ZIKV E glycoprotein as targets of strongly neutralizing and protective human antibodies, we uncover a new site spanning domains I and III as a target of strongly neutralizing human antibodies. [ABSTRACT FROM AUTHOR]

The development of vaccines against flaviviruses, including Zika virus (ZIKV) and dengue virus (DENV), continues to be a major challenge, hindered by the lack of efficient and reliable methods for screening neutralizing activity of sera or antibodies. To address this need, we previously developed a plasmid-based, replication-incompetent DENV reporter virus particle (RVP) production system as an efficient and safe alternative to the Plaque Reduction Neutralization Test (PRNT). As part of the response to the 2015–2016 ZIKV outbreak, we developed pseudo-infectious ZIKV RVPs by modifying our DENV RVP system. The use of ZIKV RVPs as critical reagents in human clinical trials requires their further validation using stability and reproducibility metrics for large-scale applications. In the current study, we validated ZIKV RVPs using infectivity, neutralization, and enhancement assays with monoclonal antibodies (MAbs) and human ZIKV-positive patient serum. ZIKV RVPs are antigenically equivalent to live virus based on binding ELISA and neutralization results and are nonreplicating based on the results of live virus replication assays. We demonstrate reproducible neutralization titer data (NT50 values) across different RVP production lots, volumes, time frames, and laboratories. We also show RVP stability across experimentally relevant time intervals and temperatures. Our results demonstrate that ZIKV RVPs provide a safe, high-throughput, and reproducible reagent for large-scale, long-term studies of neutralizing antibodies and sera, which can facilitate large-scale screening and epidemiological studies to help expedite ZIKV vaccine development. Author summary: ZIKV is a mosquito-borne virus that can cause severe birth defects and other disorders. Large outbreaks of ZIKV occurred in 2015 and 2016 and there are still no drugs or vaccines available to protect against ZIKV infection. Vaccine development has been hindered by the lack of safe and efficient methods to screen ZIKV neutralizing properties of patient sera or antibodies, especially in the context of large clinical trials. To address this unmet need, we developed and validated the use of ZIKV reporter virus particles (RVPs), a safe, high-throughput, and quantitative alternative to using live virus for neutralization studies. We show that ZIKV RVPs are stable, show lot-to-lot consistency, and provide reproducible neutralization data that is suitable for the large-scale studies needed for development of a ZIKV vaccine, epidemiologic surveillance, and high-throughput screening. [ABSTRACT FROM AUTHOR]

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes. [ABSTRACT FROM AUTHOR]

Half of the world's population is exposed to the risk of dengue virus infection. Although a vaccine for dengue virus is now available in a few countries, its reported overall efficacy of about 60% is not ideal. Protective immune correlates following natural dengue virus infection remain undefined, which makes it difficult to predict the efficacy of new vaccines. In this study, we address the protective capacity of dengue virus-specific antibodies that are produced by plasmablasts a few days after natural secondary infection. Among a panel of 18 dengue virus-reactive human monoclonal antibodies, four groups of antibodies were identified based on their binding properties. While antibodies targeting the fusion loop of the glycoprotein of dengue virus dominated the antibody response, two smaller groups of antibodies bound to previously undescribed epitopes in domain II of the E protein. The latter, largely serotype-cross-reactive antibodies, demonstrated increased stability of binding at pH 5. These antibodies possessed weak to moderate neutralization capacity in vitro but were the most efficacious in promoting the survival of infected mice. Our data suggest that the cross-reactive anamnestic antibody response has a protective capacity despite moderate neutralization in vitro and a moderate decrease of viremia in vivo. [ABSTRACT FROM AUTHOR]

Palivizumab was the first antiviral monoclonal antibody (mAb) approved for therapeutic use in humans, and remains a prophylactic treatment for infants at risk for severe disease because of respiratory syncytial virus (RSV). Palivizumab is an engineered humanized version of a murine mAb targeting antigenic site II of the RSV fusion (F) protein, a key target in vaccine development. There are limited reported naturally occurring human mAbs to site II; therefore, the structural basis for human antibody recognition of this major antigenic site is poorly understood. Here, we describe a nonneutralizing class of site II-specific mAbs that competed for binding with palivizumab to postfusion RSV F protein. We also describe two classes of site II-specific neutralizing mAbs, one of which escaped competition with nonneutralizing mAbs. An X-ray crystal structure of the neutralizing mAb 14N4 in complex with F protein showed that the binding angle at which human neutralizing mAbs interact with antigenic site II determines whether or not nonneutralizing antibodies compete with their binding. Fine-mapping studies determined that nonneutralizing mAbs that interfere with binding of neutralizing mAbs recognize site II with a pose that facilitates binding to an epitope containing F surface residues on a neighboring protomer. Neutralizing antibodies, like motavizumab and a new mAb designated 3J20 that escape interference by the inhibiting mAbs, avoid such contact by binding at an angle that is shifted away from the nonneutralizing site. Furthermore, binding to rationally and computationally designed site II helix- loop-helix epitope-scaffold vaccines distinguished neutralizing from nonneutralizing site II antibodies. [ABSTRACT FROM AUTHOR]

The atomic-level mechanisms by which G protein-coupled receptors (GPCRs) transmit extracellular ligand binding events through their transmembrane helices to activate intracellular G proteins remain unclear. Using a comprehensive library of mutations covering all 352 residues of the GPCR CXC chemokine receptor 4 (CXCR4), we identified 41 amino acids that are required for signaling induced by the chemokine ligand CXCL12 (stromal cell-derived factor 1). CXCR4 variants with each of these mutations do not signal properly but remain folded, based on receptor surface trafficking, reactivity to conformationally sensitive monoclonal antibodies, and ligand binding. When visualized on the structure of CXCR4, the majority of these residues form a continuous intramolecular signaling chain through the transmembrane helices; this chain connects chemokine binding residues on the extracellular side of CXCR4 to G protein-coupling residues on its intracellular side. Integrated into a cohesive model of signal transmission, these CXCR4 residues cluster into five functional groups that mediate (i) chemokine engagement, (ii) signal initiation, (iii) signal propagation, (iv) microswitch activation, and (v) G protein coupling. Propagation of the signal passes through a "hydrophobic bridge" on helix VI that coordinates with nearly every known GPCR signaling motif. Our results agree with known conserved mechanisms of GPCR activation and significantly expand on understanding the structural principles of CXCR4 signaling. [ABSTRACT FROM AUTHOR]

The lack of structural information on hepatitis C virus (HCV) surface proteins has so far hampered the development of effective vaccines. Recently, two crystallographic structures have described the core portion (E2c) of E2 surface glycoprotein, the primary mediator of HCV entry. Despite the importance of these studies, the E2 overall structure is still unknown and, most importantly, several biochemical and functional studies are in disagreement with E2c structures. Here, the main literature will be discussed and an alternative disulfide bridge pattern will be proposed, based on unpublished human monoclonal antibody reactivity. A modeling strategy aiming at recapitulating the available structural and functional studies of E2 will also be proposed. [ABSTRACT FROM AUTHOR]

A number of structures have been solved for the Envelope (E) protein from dengue virus and closely related flaviviruses, providing detailed pictures of the conformational states of the protein at different stages of infectivity. However, the key functional residues responsible for mediating the dynamic changes between these structures remain largely unknown. Using a comprehensive library of functional point mutations covering all 390 residues of the dengue virus E protein ectodomain, we identified residues that are critical for virus infectivity, but that do not affect E protein expression, folding, virion assembly, or budding. The locations and atomic interactions of these critical residues within different structures representing distinct fusogenic conformations help to explain how E protein (i) regulates fusion-loop exposure by shielding, tethering, and triggering its release; (ii) enables hinge movements between E domain interfaces during triggered structural transformations; and (iii) drives membrane fusion through late-stage zipper contacts with stem. These results provide structural targets for drug and vaccine development and integrate the findings from structural studies and isolated mutagenesis efforts into a cohesive model that explains how specific residues in this class II viral fusion protein enable virus infectivity. [ABSTRACT FROM AUTHOR]