RESUMEN
Hepatitis C virus (HCV) is a major cause of chronic liver disease and hepatocellular carcinoma. Antibody development efforts mainly revolve around HCV envelope glycoprotein 2 (E2), which mediates host cell entry by interacting with several cell surface receptors, including CD81. We still have limited knowledge about the structural ensembles and the dynamic behavior of both the CD81 binding sites and the glycans on E2. Here, multiple microsecond-long, all-atom molecular dynamics (MD) simulations, as well as a Markov state model (MSM), were performed to provide an atomistic perspective on the dynamic nature of E2 and its glycans. End-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of E2, which may be exploited in therapeutic efforts. Additionally, the Markov state model built from the simulation maps four metastable states for AS412 and three metastable states for the front layer in CD81 binding sites, while binding with HEPC3 would induce a conformation selection for both of them. Overall, this work presents hitherto unseen functional and structural insights into E2 and its glycan coat, providing a new theoretical foundation to control the conformational plasticity of E2 that could be harnessed for vaccine development.
Asunto(s)
Simulación de Dinámica Molecular , Polisacáridos , Conformación Proteica , Proteínas del Envoltorio Viral , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismo , Polisacáridos/química , Polisacáridos/metabolismo , Hepacivirus/química , Cadenas de Markov , Humanos , Sitios de UniónRESUMEN
Hepatitis C virus (HCV) is a major medical health burden and the leading cause of chronic liver disease and cancer worldwide. More than 58 million people are chronically infected with HCV, with 1.5 million new infections occurring each year. An effective HCV vaccine is a major public health and medical need as recognized by the World Health Organization. However, due to the high variability of the virus and its ability to escape the immune response, HCV rapidly accumulates mutations, making vaccine development a formidable challenge. An effective vaccine must elicit broadly neutralizing antibodies (bnAbs) in a consistent fashion. After decades of studies from basic research through clinical development, the antigen of choice is considered the E1E2 envelope glycoprotein due to conserved, broadly neutralizing antigenic domains located in the constituent subunits of E1, E2, and the E1E2 heterodimeric complex itself. The challenge has been elicitation of robust humoral and cellular responses leading to broad virus neutralization due to the relatively low immunogenicity of this antigen. In view of this challenge, structure-based vaccine design approaches to stabilize key antigenic domains have been hampered due to the lack of E1E2 atomic-level resolution structures to guide them. Another challenge has been the development of a delivery platform in which a multivalent form of the antigen can be presented in order to elicit a more robust anti-HCV immune response. Recent nanoparticle vaccines are gaining prominence in the field due to their ability to facilitate a controlled multivalent presentation and trafficking to lymph nodes, where they can interact with both the cellular and humoral components of the immune system. This review focuses on recent advances in understanding the E1E2 heterodimeric structure to facilitate a rational design approach and the potential for development of a multivalent nanoparticle-based HCV E1E2 vaccine. Both aspects are considered important in the development of an effective HCV vaccine that can effectively address viral diversity and escape.
Asunto(s)
Hepacivirus , Hepatitis C , Desarrollo de Vacunas , Proteínas del Envoltorio Viral , Vacunas contra Hepatitis Viral , Hepacivirus/inmunología , Hepacivirus/genética , Hepacivirus/química , Humanos , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Vacunas contra Hepatitis Viral/inmunología , Hepatitis C/prevención & control , Hepatitis C/inmunología , Hepatitis C/virología , Anticuerpos Neutralizantes/inmunología , Animales , Anticuerpos contra la Hepatitis C/inmunologíaRESUMEN
IMPORTANCE: The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.
Asunto(s)
Antivirales , Hepacivirus , Proteínas del Núcleo Viral , Humanos , Antivirales/metabolismo , Antivirales/farmacología , Hepacivirus/química , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Nucleocápside/antagonistas & inhibidores , Nucleocápside/química , Nucleocápside/metabolismo , Proteínas del Núcleo Viral/antagonistas & inhibidores , Proteínas del Núcleo Viral/metabolismo , Ensamble de Virus , Replicación Viral , Imagen Individual de Molécula/métodos , Unión ProteicaRESUMEN
Viral proteins interact with lipid membranes during various stages in the viral life cycle to propagate infection. p7 is an ion channel forming protein of Hepatitis C virus (HCV) that participates in viral assembly. Studies show that it has close ties to lipid metabolism in the cell and anionic phosphatidylserine (PS) lipids are suggested to be key for its permeabilizing function, but the mechanism of its interaction with the lipid environment is largely unknown. To begin unraveling the molecular processes of the protein, we evaluated the impact of lipid environment on the binding and insertion mechanism of p7 prior to channel formation and viral assembly using molecular dynamics simulations. It is seen that p7 is sensitive to its lipid environment and results in different remodeling patterns in membranes. Helix 1 (H1) is especially important for peptide insertion, with deeper entry taking place when the membrane contains phosphatidylserine (PS). Helix 2 (H2) and the adjacent loop connecting to Helix 3 (H3) prompts recruitment of phosphatidylethanolamine (PE) lipids to the protein binding site in membrane models with lower surface charge. This work provides perspectives on the interplay between protein-lipid dynamics and membrane composition, and insights on membrane reorganization in mechanisms of disease.
Asunto(s)
Fosfatidilserinas , Proteínas Viroporinas , Proteínas Viroporinas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas Virales/química , Hepacivirus/química , Hepacivirus/metabolismo , Simulación de Dinámica MolecularRESUMEN
Biochemical processes in cells, including enzyme-catalyzed reactions, occur in crowded conditions with various background macromolecules occupying up to 40% of cytoplasm's volume. Viral enzymes in the host cell also encounter such crowded conditions as they often function at the endoplasmic reticulum membranes. We focus on an enzyme encoded by the hepatitis C virus, the NS3/4A protease, which is crucial for viral replication. We have previously found experimentally that synthetic crowders, polyethylene glycol (PEG) and branched polysucrose (Ficoll), differently affect the kinetic parameters of peptide hydrolysis catalyzed by NS3/4A. To gain understanding of the reasons for such behavior, we perform atomistic molecular dynamics simulations of NS3/4A in the presence of either PEG or Ficoll crowders and with and without the peptide substrates. We find that both crowder types make nanosecond long contacts with the protease and slow down its diffusion. However, they also affect the enzyme structural dynamics; crowders induce functionally relevant helical structures in the disordered parts of the protease cofactor, NS4A, with the PEG effect being more pronounced. Overall, PEG interactions with NS3/4A are slightly stronger but Ficoll forms more hydrogen bonds with NS3. The crowders also interact with substrates; we find that the substrate diffusion is reduced much more in the presence of PEG than Ficoll. However, contrary to NS3, the substrate interacts more strongly with Ficoll than with PEG crowders, with the substrate diffusion being similar to crowder diffusion. Importantly, crowders also affect the substrate-enzyme interactions. We observe that both PEG and Ficoll enhance the presence of substrates near the active site, especially near catalytic H57 but Ficoll crowders increase substrate binding more than PEG molecules.
Asunto(s)
Péptido Hidrolasas , Proteínas no Estructurales Virales , Ficoll , Proteínas no Estructurales Virales/química , Péptidos , Hepacivirus/química , Proteasas ViralesRESUMEN
An array of computational approaches DFT/QSAR/POM methods has been used for a better understanding of drug properties regarding 13 inhibitor derivatives containing either P2 cyclopentane P1 carboxylic acid moiety (1-9) or a P1 cyclopropyl acyl sulfonamide (10-13). To further recognize binding interactions and their activity trends, molecular docking studies were carried out with the use of HCV, which can be used to accurately predict the interactions of ligands with the receptor. The QSAR models are developed through the use of Multiple Linear Regression (MLR) together with Principal Component Analysis (PCA) methods. The statistical results indicate the multiple correlation coefficient R2 = 0.840, which shows favorable estimation stability, as well as showing a significant correlation between the HCV NS3 protease of the studied compounds and their electron-accepting ability. The POM analysis of the Physico-chemical properties of compounds 1-13, shows that they are bearing (O1, O2) and/or (O1, O2, O3) antiviral pockets, whereby all oxygen atoms are Osp2 and bearing negative charges. Similar to the reference ligand (F9K), the most active compound 10 was bound deeply into the binding cavity of NS3 protease making interactions with the residues Gly137, His57, Ala157, and His528. The anti-hepatitis pharmacophore site is similar to the anti-HIV pharmacophore site.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Antivirales , Hepatitis C , Humanos , Antivirales/química , Péptido Hidrolasas , Simulación del Acoplamiento Molecular , Farmacóforo , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/química , Endopeptidasas , Hepacivirus/químicaRESUMEN
Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo-electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies-AR4A, AT1209, and IGH505-at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.
Asunto(s)
Hepacivirus , Hepatitis C , Proteínas del Envoltorio Viral , Humanos , Antivirales/química , Anticuerpos ampliamente neutralizantes , Microscopía por Crioelectrón , Hepacivirus/química , Hepacivirus/inmunología , Hepatitis C/virología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Multimerización de Proteína , Vacunas contra Hepatitis Viral/química , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
microRNAs (miRNAs) form regulatory networks in metazoans. Viruses engage miRNA networks in numerous ways, with Flaviviridae members exploiting direct interactions of their RNA genomes with host miRNAs. For hepatitis C virus (HCV), binding of liver-abundant miR-122 stabilizes the viral RNA and regulates viral translation. Here, we investigate the structural basis for these activities, taking into consideration that miRNAs function in complex with Argonaute (Ago) proteins. The crystal structure of the Ago2:miR-122:HCV complex reveals a structured RNA motif that traps Ago2 on the viral RNA, masking its 5' end from enzymatic attack. The trapped Ago2 can recruit host factor PCBP2, implicated in viral translation, while binding of a second Ago2:miR-122 competes with PCBP2, creating a potential molecular switch for translational control. Combined results reveal a viral RNA structure that modulates Ago2:miR-122 dynamics and repurposes host proteins to generate a functional analog of the mRNA cap-binding complex.
Asunto(s)
Proteínas Argonautas/química , Genoma Viral/genética , Hepacivirus/genética , MicroARNs/química , Regiones no Traducidas 5' , Proteínas Argonautas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Exorribonucleasas/metabolismo , Hepacivirus/química , Hepacivirus/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/metabolismo , Motivos de Nucleótidos , Monoéster Fosfórico Hidrolasas/metabolismo , Conformación Proteica , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismoRESUMEN
Hepatitis C virus (HCV) infection is a causal agent of chronic liver disease, cirrhosis and hepatocellular carcinoma in humans, and afflicts more than 70 million people worldwide. The HCV envelope glycoproteins E1 and E2 are responsible for the binding of the virus to the host cell, but the exact entry process remains undetermined1. The majority of broadly neutralizing antibodies block interaction between HCV E2 and the large extracellular loop (LEL) of the cellular receptor CD81 (CD81-LEL)2. Here we show that low pH enhances the binding of CD81-LEL to E2, and we determine the crystal structure of E2 in complex with an antigen-binding fragment (2A12) and CD81-LEL (E2-2A12-CD81-LEL); E2 in complex with 2A12 (E2-2A12); and CD81-LEL alone. After binding CD81, residues 418-422 in E2 are displaced, which allows for the extension of an internal loop consisting of residues 520-539. Docking of the E2-CD81-LEL complex onto a membrane-embedded, full-length CD81 places the residues Tyr529 and Trp531 of E2 proximal to the membrane. Liposome flotation assays show that low pH and CD81-LEL increase the interaction of E2 with membranes, whereas structure-based mutants of Tyr529, Trp531 and Ile422 in the amino terminus of E2 abolish membrane binding. These data support a model in which acidification and receptor binding result in a conformational change in E2 in preparation for membrane fusion.
Asunto(s)
Hepacivirus/metabolismo , Unión Proteica , Receptores Virales/química , Receptores Virales/metabolismo , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Internalización del Virus , Animales , Anticuerpos Neutralizantes/inmunología , Membrana Celular/química , Membrana Celular/metabolismo , Células HEK293 , Hepacivirus/química , Hepacivirus/genética , Humanos , Leontopithecus , Fusión de Membrana , Modelos Moleculares , Receptores Virales/inmunología , Tetraspanina 28/inmunología , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Early diagnosis of hepatitis C virus (HCV) infection is essential to prevent disease from spreading and progression. Herein, a novel electrochemical biosensor was developed for ultrasensitive detection of HCV core antigen (HCVcAg) based on terminal deoxynucleotidyl transferase (TdT) amplification and DNA nanowires (DNW). After sandwich-type antibody-antigen recognition, the antibody-conjugated DNA was pulled to the electrode surface and further extended into a long DNA sequence by robust TdT reaction. Then, large numbers of methylene blue-loaded DNW (MB@DNW) as signal labels are linked to the extended DNA sequence. This results in an amplified electrochemical signal for HCVcAg determination, typically measured at around -0.25 V (Ag/AgCl). Under the optimum conditions, the proposed biosensor achieved a wide linear range for HCVcAg from 0.1 to 312.5 pg/mL with a low limit of detection of 32 fg/mL. The good practicality of the biosensor was demonstrated by recovery experiment (recoveries from 98 to 104% with RSD of 2.5-4.4%) and comparison with enzyme-linked immunosorbent assay (ELISA). Given the highlighted performance, the biosensor is expected to act as a reliable sensing tool for HCVcAg determination in clinics. Schematic representation of the ultrasensitive electrochemical biosensor based on terminal deoxynucleotidyl transferase (TdT) amplification linked with methylene blue-loaded DNA nanowires (MB@DNW), which can be applied to the determination of hepatitis C virus core antigen (HCVcAg) in clinical samples. dTTPs, 2'-deoxythymidine 5'-triphosphate.
Asunto(s)
Técnicas Biosensibles/métodos , ADN Nucleotidilexotransferasa/química , ADN/química , Hepacivirus/química , Nanocables/química , Proteínas del Núcleo Viral/sangre , Técnicas Electroquímicas/métodos , Hepatitis C/sangre , Hepatitis C/diagnóstico , Humanos , Límite de Detección , Azul de Metileno/química , Oxidación-ReducciónRESUMEN
An effective vaccine for the hepatitis C virus (HCV) is a major unmet medical and public health need, and it requires an antigen that elicits immune responses to multiple key conserved epitopes. Decades of research have generated a number of vaccine candidates; based on these data and research through clinical development, a vaccine antigen based on the E1E2 glycoprotein complex appears to be the best choice. One bottleneck in the development of an E1E2-based vaccine is that the antigen is challenging to produce in large quantities and at high levels of purity and antigenic/functional integrity. This review describes the production and characterization of E1E2-based vaccine antigens, both membrane-associated and a novel secreted form of E1E2, with a particular emphasis on the major challenges facing the field and how those challenges can be addressed.
Asunto(s)
Hepacivirus/química , Hepatitis C/prevención & control , Proteínas del Envoltorio Viral/química , Vacunas contra Hepatitis Viral/química , Animales , Epítopos/inmunología , Células HEK293 , Hepacivirus/genética , Hepacivirus/inmunología , Hepatitis C/virología , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Multimerización de Proteína , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
A hepatitis C virus (HCV) vaccine is a critical yet unfulfilled step in addressing the global disease burden of HCV. While decades of research have led to numerous clinical and pre-clinical vaccine candidates, these efforts have been hindered by factors including HCV antigenic variability and immune evasion. Structure-based and rational vaccine design approaches have capitalized on insights regarding the immune response to HCV and the structures of antibody-bound envelope glycoproteins. Despite successes with other viruses, designing an immunogen based on HCV glycoproteins that can elicit broadly protective immunity against HCV infection is an ongoing challenge. Here, we describe HCV vaccine design approaches where immunogens were selected and optimized through analysis of available structures, identification of conserved epitopes targeted by neutralizing antibodies, or both. Several designs have elicited immune responses against HCV in vivo, revealing correlates of HCV antigen immunogenicity and breadth of induced responses. Recent studies have elucidated the functional, dynamic and immunological features of key regions of the viral envelope glycoproteins, which can inform next-generation immunogen design efforts. These insights and design strategies represent promising pathways to HCV vaccine development, which can be further informed by successful immunogen designs generated for other viruses.
Asunto(s)
Hepacivirus/química , Hepacivirus/inmunología , Antígenos de la Hepatitis C/química , Antígenos de la Hepatitis C/inmunología , Desarrollo de Vacunas/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Ensayos Clínicos como Asunto , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/inmunología , Vacunas contra Hepatitis Viral/inmunologíaRESUMEN
The molecular mechanisms of hepatitis C virus (HCV) persistence and pathogenesis are poorly understood. The design of an effective HCV vaccine is challenging despite a robust humoral immune response against closely related strains of HCV. This is primarily because of the huge genetic diversity of HCV and the molecular evolution of various virus escape mechanisms. These mechanisms are steered by the presence of a high mutational rate in HCV, structural plasticity of the immunodominant regions on the virion surface of diverse HCV genotypes, and constant amino acid substitutions on key structural components of HCV envelope glycoproteins. Here, we review the molecular basis of neutralizing antibody (nAb)-mediated immune response against diverse HCV variants, HCV-steered humoral immune evasion strategies and explore the essential structural elements to consider for designing a universal HCV vaccine. Structural perspectives on key escape pathways mediated by a point mutation within the epitope, allosteric modulation of the epitope by distant mutations and glycan shift on envelope glycoproteins will be highlighted (abstract graphic).
Asunto(s)
Hepacivirus/inmunología , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Evasión Inmune , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/química , Antígenos Virales/genética , Antígenos Virales/inmunología , Epítopos , Variación Genética , Hepacivirus/química , Hepacivirus/genética , Anticuerpos contra la Hepatitis C/inmunología , Humanos , Inmunidad Humoral , Epítopos Inmunodominantes , Mutación , Conformación Proteica , Dominios Proteicos , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Hepatitis C virus remains a global threat, despite the availability of highly effective direct-acting antiviral (DAA) drugs. With thousands of new infections annually, the need for a prophylactic vaccine is evident. However, traditional vaccine design has been unable to provide effective vaccines so far. Therefore, alternative strategies need to be investigated. In this work, a chemistry-based approach is explored towards fully synthetic peptide-based vaccines using epitope mimicry, by focusing on highly effective and conserved amino acid sequences in HCV, which, upon antibody binding, inhibit its bio-activity. Continuous and discontinuous epitope mimics were both chemically synthesized based on the HCV-E2 glycoprotein while using designed fully synthetic cyclic peptides. These cyclic epitope mimics were assembled on an orthogonally protected scaffold. The scaffolded epitope mimics have been assessed in immunization experiments to investigate the elicitation of anti-HCV-E2 glycoprotein antibodies. The neutralizing potential of the elicited antibodies was investigated, representing a first step in employing chemically synthesized epitope mimics as a novel strategy towards vaccine design.
Asunto(s)
Epítopos/química , Hepacivirus/inmunología , Hepatitis C/inmunología , Vacunas Sintéticas/química , Proteínas del Envoltorio Viral/síntesis química , Anticuerpos Antivirales/inmunología , Diseño de Fármacos , Epítopos/genética , Epítopos/inmunología , Hepacivirus/química , Hepacivirus/genética , Hepatitis C/prevención & control , Hepatitis C/virología , Humanos , Imitación Molecular , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/química , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas del Envoltorio Viral/administración & dosificación , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Early diagnosis remains key for effective prevention and treatment. Unfortunately, current screening with anti-hepatitis C virus antibody (anti-HCV Ab) test may have limited utility in the diagnosis of HCV infection and reinfection. This is of special concern to at-risk population, such as immunocompromised hosts and end-stage renal failure patients on hemodialysis. HCV antigen (Ag) could be useful in identifying the ongoing infection in such clinical scenarios. Hence, we aimed to study the utility of HCV Ag testing for the diagnosis of acute and chronic hepatitis C. Of 89 samples studied, 19 were from acute hepatitis C patients who were immunocompromised or were on hemodialysis, 43 were from active chronic hepatitis C patients and 27 were from patients treated for chronic hepatitis C. All samples were tested for HCV Ag using the Abbott ARCHITECT HCV Ag assay. HCV Ag was reactive in 19/19 samples from acute hepatitis C patients and 42/43 samples from active chronic hepatitis C patients. It was nonreactive in all samples from treated patients. The test showed a sensitivity and specificity of 98.4% and 100.0%, respectively. The positive and negative predictive values were 100.0% and 96.4%, respectively. The HCV antigen test has high clinical sensitivity and specificity and is useful for the diagnosis of acute and chronic hepatitis C infection in at-risk and immunocompromised patients. Its short turnaround time and relatively low cost are advantageous for use in patients on hemodialysis and other at-risk patients who require monitoring of HCV infection and reinfection.
Asunto(s)
Hepacivirus/genética , Antígenos de la Hepatitis C/análisis , Hepatitis C Crónica/diagnóstico , Hepatitis C/diagnóstico , Huésped Inmunocomprometido , Pruebas Inmunológicas/métodos , Adulto , Diagnóstico Precoz , Femenino , Hepacivirus/química , Hepatitis C/sangre , Hepatitis C/prevención & control , Antígenos de la Hepatitis C/sangre , Antígenos de la Hepatitis C/inmunología , Hepatitis C Crónica/sangre , Hepatitis C Crónica/prevención & control , Humanos , Pruebas Inmunológicas/economía , Pruebas Inmunológicas/normas , Masculino , Tamizaje Masivo , Persona de Mediana Edad , Valor Predictivo de las Pruebas , ARN Viral/sangre , ARN Viral/genética , Sensibilidad y EspecificidadRESUMEN
Many viruses destabilize cellular membranous compartments to form their replication complexes, but the mechanism(s) underlying membrane perturbation remains unknown. Expression in eukaryotic cells of NS4B, a protein of the hepatitis C virus (HCV), alters membranous complexes and induces structures similar to the so-called membranous web that appears crucial to the formation of the HCV replication complex. As over-expression of the protein is lethal to both prokaryotic and eukaryotic cells, NS4B was produced in large quantities in a "cell-free" system in the presence of detergent, after which it was inserted into lipid membranes. X-ray diffraction revealed that NS4B modifies the phase diagram of synthetic lipid aqueous phases considerably, perturbing the transition temperature and cooperativity. Cryo-electron microscopy demonstrated that NS4B introduces significant disorder in the synthetic membrane as well as discontinuities that could be interpreted as due to the formation of pores and membrane merging events. C- and N-terminal fragments of NS4B are both able to destabilize liposomes. While most NS4B amphipathic peptides perforate membranes, one NS4B peptide induces membrane fusion. Cryo-electron microscopy reveals a particular structure that can be interpreted as arising from hemi-fusion-like events. Amphipathic domains are present in many proteins, and if exposed to the aqueous cytoplasmic medium are sufficient to destabilize membranes in order to form viral replication complexes. These domains have important functions in the viral replication cycle, and thus represent potential targets for the development of anti-viral molecules.
Asunto(s)
Hepacivirus/química , Membranas Artificiales , Péptidos/química , Proteínas no Estructurales Virales/química , Hepacivirus/metabolismo , Péptidos/metabolismo , Proteínas no Estructurales Virales/metabolismoRESUMEN
The Hepatitisâ C virus nonstructural protein 5A (NS5A) is a membrane-associated protein involved in multiple steps of the viral life cycle. Direct-acting antivirals (DAAs) targeting NS5A are a cornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure. Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes. We use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches. Spectra analyses disclose that both the AH membrane anchor and the linker are highly flexible. Paramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires a new type of NS5A self-interaction not reflected in previous crystal structures. In conclusion, we provide the first characterization of NS5A AH-linker-D1 in a lipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.
Asunto(s)
Hepacivirus/química , Membrana Dobles de Lípidos/metabolismo , Proteínas no Estructurales Virales/metabolismo , Membrana Dobles de Lípidos/química , Resonancia Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformación Proteica en Hélice alfa , Dominios Proteicos , Multimerización de Proteína , Espectroscopía de Protones por Resonancia Magnética , Proteínas no Estructurales Virales/químicaRESUMEN
Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years.
Asunto(s)
Cápside/ultraestructura , Resonancia Magnética Nuclear Biomolecular/instrumentación , Resonancia Magnética Nuclear Biomolecular/métodos , Fenómenos Fisiológicos de los Virus , Virus/ultraestructura , Cápside/química , VIH-1/química , Hepacivirus/química , Virus de la Hepatitis B/química , Virus de la Influenza A/química , Conformación Molecular , Proteínas Virales/química , Virus/químicaRESUMEN
Despite the approval of highly efficient direct-acting antivirals in the last decade Hepatitis C virus (HCV) remains a global health burden and the development of a vaccine would constitute an important step towards the control of HCV. The high genetic variability of the viral glycoproteins E1 and E2, which carry the main neutralizing determinants, together with their intrinsic structural flexibility, the high level of glycosylation that shields conserved neutralization epitopes and immune evasion using decoy epitopes renders the design of an efficient vaccine challenging. Recent structural and functional analyses have highlighted the role of the CD81 receptor binding site on E2, which overlaps with those neutralization epitopes within E2 that have been structurally characterized to date. This CD81 binding site consists of three distinct segments including "epitope I", "epitope II" and the "CD81 binding loop". In this review we summarize the structural features of the HCV glycoproteins that have been derived from X-ray structures of neutralizing and non-neutralizing antibody fragments complexed with either recombinant E2 or epitope-derived linear peptides. We focus on the current understanding how neutralizing antibodies interact with their cognate antigen, the structural features of the respective neutralization epitopes targeted by nAbs and discuss the implications for informed vaccine design.