RESUMO
The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.
Assuntos
Citomegalovirus , Domínios Proteicos , Proteínas do Envelope Viral , Internalização do Vírus , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Citomegalovirus/genética , Citomegalovirus/fisiologia , Infecções por Citomegalovirus/virologia , Células Gigantes/virologia , Linhagem Celular , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Proteínas Virais de Fusão/química , Mutagênese Sítio-Dirigida , Fusão CelularRESUMO
Human cytomegalovirus (HCMV) glycoprotein B (gB) is a class III membrane fusion protein required for viral entry. HCMV vaccine candidates containing gB have demonstrated moderate clinical efficacy, but no HCMV vaccine has been approved. Here, we used structure-based design to identify and characterize amino acid substitutions that stabilize gB in its metastable prefusion conformation. One variant containing two engineered interprotomer disulfide bonds and two cavity-filling substitutions (gB-C7), displayed increased expression and thermostability. A 2.8 Å resolution cryoelectron microscopy structure shows that gB-C7 adopts a prefusion-like conformation, revealing additional structural elements at the membrane-distal apex. Unlike previous observations for several class I viral fusion proteins, mice immunized with postfusion or prefusion-stabilized forms of soluble gB protein displayed similar neutralizing antibody titers, here specifically against an HCMV laboratory strain on fibroblasts. Collectively, these results identify initial strategies to stabilize class III viral fusion proteins and provide tools to probe gB-directed antibody responses.
Assuntos
Citomegalovirus , Proteínas do Envelope Viral , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Citomegalovirus/imunologia , Humanos , Animais , Camundongos , Microscopia Crioeletrônica , Conformação Proteica , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Internalização do Vírus , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/virologia , Estabilidade Proteica , Vacinas contra Citomegalovirus/imunologia , Substituição de Aminoácidos , Modelos MolecularesRESUMO
Viral glycoproteins drive membrane fusion in enveloped viruses and determine host range, tissue tropism and pathogenesis1. Despite their importance, there is a fragmentary understanding of glycoproteins within the Flaviviridae2, a large virus family that include pathogens such as hepatitis C, dengue and Zika viruses, and numerous other human, animal and emergent viruses. For many flaviviruses the glycoproteins have not yet been identified, for others, such as the hepaciviruses, the molecular mechanisms of membrane fusion remain uncharacterized3. Here we combine phylogenetic analyses with protein structure prediction to survey glycoproteins across the entire Flaviviridae. We find class II fusion systems, homologous to the Orthoflavivirus E glycoprotein in most species, including highly divergent jingmenviruses and large genome flaviviruses. However, the E1E2 glycoproteins of the hepaciviruses, pegiviruses and pestiviruses are structurally distinct, may represent a novel class of fusion mechanism, and are strictly associated with infection of vertebrate hosts. By mapping glycoprotein distribution onto the underlying phylogeny, we reveal a complex evolutionary history marked by the capture of bacterial genes and potentially inter-genus recombination. These insights, made possible through protein structure prediction, refine our understanding of viral fusion mechanisms and reveal the events that have shaped the diverse virology and ecology of the Flaviviridae.
Assuntos
Evolução Molecular , Flaviviridae , Glicoproteínas , Filogenia , Proteínas do Envelope Viral , Animais , Humanos , Flaviviridae/química , Flaviviridae/classificação , Glicoproteínas/química , Glicoproteínas/classificação , Glicoproteínas/metabolismo , Modelos Moleculares , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/classificação , Proteínas do Envelope Viral/metabolismoRESUMO
Fifty-eight million individuals worldwide are affected by chronic hepatitis C virus (HCV) infection, a primary driver of liver cancer for which no vaccine is available1. The HCV envelope proteins E1 and E2 form a heterodimer (E1/E2), which is the target for neutralizing antibodies2. However, the higher-order organization of these E1/E2 heterodimers, as well as that of any Hepacivirus envelope protein complex, remains unknown. Here we determined the cryo-electron microscopy structure of two E1/E2 heterodimers in a homodimeric arrangement. We reveal how the homodimer is established at the molecular level and provide insights into neutralizing antibody evasion and membrane fusion by HCV, as orchestrated by E2 motifs such as hypervariable region 1 and antigenic site 412, as well as the organization of the transmembrane helices, including two internal to E1. This study addresses long-standing questions on the higher-order oligomeric arrangement of Hepacivirus envelope proteins and provides a critical framework in the design of novel HCV vaccine antigens.
Assuntos
Hepacivirus , Multimerização Proteica , Proteínas do Envelope Viral , Humanos , Motivos de Aminoácidos , Anticorpos Neutralizantes/imunologia , Microscopia Crioeletrônica , Hepacivirus/química , Hepacivirus/imunologia , Hepacivirus/metabolismo , Hepacivirus/ultraestrutura , Evasão da Resposta Imune/imunologia , Fusão de Membrana , Modelos Moleculares , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/ultraestrutura , Internalização do Vírus , Vacinas contra Hepatite Viral/química , Vacinas contra Hepatite Viral/imunologiaRESUMO
Nipah virus infection, one of the top priority diseases recognized by the World Health Organization, underscores the urgent need to develop effective countermeasures against potential epidemics and pandemics. Here, we identify a fully human single-domain antibody that targets a highly conserved cryptic epitope situated at the dimeric interface of the Nipah virus G protein (receptor binding protein, RBP), as elucidated through structures by high-resolution cryo-electron microscopy (cryo-EM). This unique binding mode disrupts the tetramerization of the G protein, consequently obstructing the activation of the F protein and inhibiting viral membrane fusion. Furthermore, our investigations reveal that this compact antibody displays enhanced permeability across the blood-brain barrier (BBB) and demonstrates superior efficacy in eliminating pseudovirus within the brain in a murine model of Nipah virus infection, particularly compared to the well-characterized antibody m102.4 in an IgG1 format. Consequently, this single-domain antibody holds promise as a therapeutic candidate to prevent Nipah virus infections and has potential implications for vaccine development.
Assuntos
Anticorpos Antivirais , Microscopia Crioeletrônica , Epitopos , Infecções por Henipavirus , Vírus Nipah , Anticorpos de Domínio Único , Vírus Nipah/imunologia , Humanos , Animais , Infecções por Henipavirus/imunologia , Infecções por Henipavirus/prevenção & controle , Infecções por Henipavirus/virologia , Epitopos/imunologia , Camundongos , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/química , Anticorpos Antivirais/imunologia , Barreira Hematoencefálica/metabolismo , Barreira Hematoencefálica/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Feminino , Células HEK293RESUMO
Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.
Assuntos
Espectrometria de Massas em Tandem , Glicosilação , Animais , Vacinas contra Influenza/metabolismo , Neuraminidase/metabolismo , Humanos , Lisina/metabolismo , Embrião de Galinha , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/química , Proteômica/métodos , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Cromatografia LíquidaRESUMO
The white spot syndrome virus (WSSV), a rapidly replicating and highly lethal pathogen that targets Penaeid shrimp, has emerged as one of the most widespread viruses globally due to its high virulence. With effective chemotherapeutics still unavailable, the pursuit of novel and viable strategies against WSSV remains a crucial focus in the field of shrimp farming. The envelope proteins of WSSV are essential for virus entry, serving as excellent targets for the development of antiviral therapeutics. Novel strategies in the design of inhibitory peptides, especially those targeting envelope protein (VP28) located on the surface of the virus particle, play a critical role as a significant virulence factor during the early stages of inherent WSSV infection in shrimp. In this direction, the current computational study focused on identifying self-inhibitory peptides from the hydrophobic membrane regions of the VP28 protein, employing peptide docking and molecular dynamics simulation (MDS) approaches. Such inhibitory peptides could be useful building blocks for the rational engineering of inhibitory therapeutics since they imitate the mechanism of binding to homologous partners used by their origin domain to interact with other molecules. The N-terminal sequence of VP28 has been reported as the potential site for membrane interactions during the virus entry. Moreover, drug delivery systems mediated by chitosan and gold nanoparticles are being developed to enhance the therapeutic efficacy of anti-viral peptides. These systems can increase the solubility, stability, and selectivity of peptides, possessing better qualities than conventional delivery methods. This computational study on self-inhibitory peptides could be a valuable resource for further in vitro and in vivo studies on anti-viral therapeutics in the aquaculture industry.
Assuntos
Antivirais , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Penaeidae , Peptídeos , Vírus da Síndrome da Mancha Branca 1 , Vírus da Síndrome da Mancha Branca 1/efeitos dos fármacos , Vírus da Síndrome da Mancha Branca 1/genética , Antivirais/farmacologia , Animais , Peptídeos/farmacologia , Peptídeos/química , Penaeidae/virologia , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Internalização do Vírus/efeitos dos fármacosRESUMO
Eastern equine encephalitis virus (EEEV) is the most virulent alphavirus that infects humans, and many survivors develop neurological sequelae, including paralysis and intellectual disability. Alphavirus spike proteins comprise trimers of heterodimers of glycoproteins E2 and E1 that mediate binding to cellular receptors and fusion of virus and host cell membranes during entry. We recently identified very-low density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2) as cellular receptors for EEEV and a distantly related alphavirus, Semliki Forest virus (SFV). Here, we use single-particle cryo-electron microscopy (cryo-EM) to determine structures of the EEEV and SFV spike glycoproteins bound to the VLDLR ligand-binding domain and found that EEEV and SFV interact with the same cellular receptor through divergent binding modes. Our studies suggest that the ability of LDLR-related proteins to interact with viral spike proteins through very small footprints with flexible binding modes results in a low evolutionary barrier to the acquisition of LDLR-related proteins as cellular receptors for diverse sets of viruses.
Assuntos
Microscopia Crioeletrônica , Vírus da Encefalite Equina do Leste , Receptores de LDL , Receptores de LDL/metabolismo , Receptores de LDL/química , Vírus da Encefalite Equina do Leste/metabolismo , Vírus da Encefalite Equina do Leste/ultraestrutura , Humanos , Animais , Vírus da Floresta de Semliki/metabolismo , Ligação Proteica , Receptores Virais/metabolismo , Receptores Virais/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/ultraestrutura , Modelos MolecularesRESUMO
The World Health Organization (WHO) has declared the monkeypox outbreak a public health emergency, as there is no specific therapeutics for monkeypox virus (MPXV) disease. This study focused on docking various commercial drugs and plant-derived compounds against the E8 envelope protein crucial for MPXV attachment and pathogenesis. The target protein structure was modeled based on the vaccinia virus D8L protein. Notably, maraviroc and punicalagin emerged as potential ligands, with punicalagin exhibiting higher binding affinity (- 9.1 kcal/mol) than maraviroc (- 7.8 kcal/mol). Validation through 100 ns molecular dynamics (MD) simulations demonstrated increased stability of the E8-punicalagin complex, with lower RMSD, RMSF, and Rg compared to maraviroc. Enhanced hydrogen bonding, lower solvent accessibility, and compact motions also attributed to higher binding affinity and stability of the complex. MM-PBSA calculations revealed van der Waals, electrostatic, and non-polar solvation as principal stabilizing energies. The binding energy decomposition per residue favored stable interactions between punicalagin and the protein's active site residues (Arg20, Phe56, Glu228, Tyr232) compared to maraviroc. Overall study suggests that punicalagin can act as a potent inhibitor against MPXV. Further research and experimental investigations are warranted to validate its efficacy and safety.
Assuntos
Maraviroc , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Proteínas do Envelope Viral , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/antagonistas & inibidores , Maraviroc/química , Maraviroc/farmacologia , Monkeypox virus/química , Monkeypox virus/metabolismo , Antivirais/química , Antivirais/farmacologia , Ligação de Hidrogênio , Ligação Proteica , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/metabolismoRESUMO
The West Nile virus (WNV) subtype Kunjin virus (WNVKUN) is endemic to Australia. Here, we characterized the classical WNVKUN strain, OR393. The original OR393 strain contained two types of viruses: small plaque-forming virus (SP) and large plaque-forming virus (LP). The amino acid residues at positions 156 and 332 in the E protein (E156 and E332) of SP were Ser and Lys (E156S/332K), respectively, whereas those in LP were Phe and Thr (E156F/332T). SP grew slightly faster than LP in vitro. The E protein of SP was N-glycosylated, whereas that of LP was not. Analysis using two recombinant single-mutant LP viruses, rKUNV-LP-EF156S and rKUNV-LP-ET332K, indicated that E156S enlarged plaques formed by LP, but E332K potently reduced them, regardless of the amino acid at E156. rKUNV-LP-EF156S showed significantly higher neuroinvasive ability than LP, SP, and rKUNV-LP-ET332K. Our results indicate that the low-pathogenic classical WNVKUN can easily change its pathogenicity through only a few amino acid substitutions in the E protein. It was also found that Phe at E156 of the rKUNV-LP-ET332K was easily changed to Ser during replication in vitro and in vivo, suggesting that E156S is advantageous for the propagation of WNVKUN in mammalian cells.
Assuntos
Proteínas do Envelope Viral , Ensaio de Placa Viral , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/patogenicidade , Vírus do Nilo Ocidental/fisiologia , Camundongos , Febre do Nilo Ocidental/virologia , Virulência , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Aminoácidos/metabolismo , Aminoácidos/genética , Replicação Viral , Chlorocebus aethiops , Substituição de Aminoácidos , Células Vero , Feminino , Humanos , Austrália , Linhagem CelularRESUMO
BACKGROUND AND OBJECTIVE: Dengue is a major infectious disease with potential for outbreaks and epidemics. A specific and sensitive diagnosis is a prerequisite for clinical management of the disease. We designed our study to identify epitopes on the Dengue virus (DENV) envelope (E) and non-structural protein 1 (NS1) with potential for diagnosis. METHODS: Serology and immunoinformatic approaches were employed. We collected DENV-positive, DENV-negative and Japanese encephalitis virus-positive samples from collaborating hospitals in 2019 and 2022-2023. Seropositive peptides in 15-18 mer peptide arrays of E and NS1 proteins of DENV2 were determined by an indirect enzyme-linked immunosorbent assay. B-cell linear and conformational epitopes were predicted using BepiPred2.0 and ElliPro, respectively. A consensus recombinant peptide was designed, synthesised and evaluated for its diagnostic potential using patient sera. RESULTS: Eight peptides of E protein and six peptides of NS1 protein were identified to be the most frequently recognised by Dengue-positive patients. These peptide sequences were compared with B-cell epitope regions and found to be overlapped with predicted B-cell linear and conformational epitopes. EP11 and NSP15 showed a 100% amino acid sequence overlap with B-cell epitopes. EP1 and NSP15 had 14 whereas EP28, EP31, EP60 16, NSP12 and NSP32 had more than 15 interacting interface residues with a neutralising antibody, suggesting a strength of interaction. Interestingly, potential epitopes identified were localised on the surface of proteins as visualised by PyMOL. Validation with a recombined synthetic peptide yielded 92.3% sensitivity and 91.42% specificity. CONCLUSIONS: Immunodominant regions identified by serology and computationally predicted epitopes overlapped, thereby showing the robustness of the methodology and the peptide designed for diagnosis.
Assuntos
Anticorpos Antivirais , Vírus da Dengue , Dengue , Epitopos de Linfócito B , Epitopos Imunodominantes , Peptídeos , Proteínas do Envelope Viral , Proteínas não Estruturais Virais , Proteínas não Estruturais Virais/imunologia , Humanos , Vírus da Dengue/imunologia , Epitopos Imunodominantes/imunologia , Dengue/diagnóstico , Dengue/imunologia , Dengue/virologia , Dengue/sangue , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Peptídeos/imunologia , Peptídeos/química , Ensaio de Imunoadsorção EnzimáticaRESUMO
CONTEXT: Flaviviruses cause severe encephalitic or hemorrhagic diseases in humans. Its members, Kyasanur forest disease virus (KFDV) and Alkhumra hemorrhagic fever virus (ALKV), cause hemorrhagic fever and are prevalent in India and Saudi Arabia, respectively, while the tick-borne encephalitis virus (TBEV) causes a dangerous encephalitic infection in Europe and Asia. However, little information is available about the targets of immune responses for these deadly viruses. Here, we predict potential antigenic peptide epitopes of viral envelope protein for inducing a cell-mediated and humoral immune response. METHODS: Using the Immune Epitope Database and Analysis Resource (IEDB-AR), we identified 13 MHC-I and two MHC-II dominant conserved epitopes in KFDV and ALKV and six MHC-I and three MHC-II epitopes in TBEV envelope proteins. Parallelly, we also predicted B-cell linear and discontinuous envelope protein epitopes for these viruses. Interestingly, the epitopes are conserved in all three viral envelope proteins. Further, the discontinuous epitopes are structurally compared with the available DENV, ZIKV, WNV, TBEV, and LIV envelope protein antibody structures. Overall structural comparison analyses highlight (i) lateral ridge epitope in the ED-III domain of E protein, and (ii) envelope dimer epitope (EDE) could be targeted for developing potent vaccine candidates as well as therapeutic antibody production. Moreover, existing structural and biochemical functions of the same epitopes in homologous viruses are predicted to have a reduced antibody-dependent enhancement (ADE) effect on flaviviral infection.
Assuntos
Flavivirus , Flavivirus/imunologia , Humanos , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Biologia Computacional , Sequência de Aminoácidos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/química , Homologia de Sequência de Aminoácidos , Epitopos/imunologia , Epitopos/química , Modelos Moleculares , Vírus da Encefalite Transmitidos por Carrapatos/imunologiaRESUMO
The Rift Valley fever virus (RVFV), transmitted through mosquito bites, leads to severe illness in humans and livestock throughout Africa and the Arabian Peninsula, causing significant morbidity and mortality. As of now, there are no verified and efficacious drugs or licensed vaccines accessible for the prevention or treatment of RVFV infections in both humans and livestock. The mature RVFV virion has two envelope proteins on its surface: glycoprotein N (GN) and glycoprotein C (GC). These proteins play a significant role in facilitating the virus's entry into the host cell, making them prominent targets for entry mechanism research as well as targets for drugs and vaccine development. The initial stage in obtaining atomic-resolution structural and mechanistic information on viral entry as well as developing biochemical and biophysical research tools involves recombinant protein production. In this chapter, we describe a simplified and scalable protocol facilitating the generation of high-quality, high-titer baculovirus virus for expression and purification of RVFV GC, utilizing the baculovirus-mediated expression system in insect cells.
Assuntos
Baculoviridae , Proteínas Recombinantes , Vírus da Febre do Vale do Rift , Proteínas do Envelope Viral , Baculoviridae/genética , Animais , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/isolamento & purificação , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Vírus da Febre do Vale do Rift/genética , Células Sf9 , Expressão Gênica , Humanos , Vetores Genéticos/genética , Clonagem Molecular/métodosRESUMO
Foamy viruses (FVs) are an ancient lineage of retroviruses, with an evolutionary history spanning over 450 million years. Vector systems based on Prototype Foamy Virus (PFV) are promising candidates for gene and oncolytic therapies. Structural studies of PFV contribute to the understanding of the mechanisms of FV replication, cell entry and infection, and retroviral evolution. Here we combine cryoEM and cryoET to determine high-resolution in situ structures of the PFV icosahedral capsid (CA) and envelope glycoprotein (Env), including its type III transmembrane anchor and membrane-proximal external region (MPER), and show how they are organized in an integrated structure of assembled PFV particles. The atomic models reveal an ancient retroviral capsid architecture and an unexpected relationship between Env and other class 1 fusion proteins of the Mononegavirales. Our results represent the de novo structure determination of an assembled retrovirus particle.
Assuntos
Microscopia Crioeletrônica , Spumavirus , Montagem de Vírus , Internalização do Vírus , Spumavirus/genética , Capsídeo/metabolismo , Capsídeo/química , Capsídeo/ultraestrutura , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Humanos , Evolução Molecular , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/genética , Modelos MolecularesRESUMO
Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.
Assuntos
Anticorpos Neutralizantes , Bombyx , Vírus da Dengue , Camundongos Endogâmicos BALB C , Proteínas do Envelope Viral , Animais , Bombyx/genética , Bombyx/virologia , Bombyx/metabolismo , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Camundongos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/biossíntese , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/biossíntese , Vacinas contra Dengue/imunologia , Vacinas contra Dengue/genética , Anticorpos Antivirais/imunologia , Dengue/imunologia , Dengue/virologia , Sorogrupo , Domínios Proteicos , FemininoRESUMO
We present structures of three immature tick-borne encephalitis virus (TBEV) isolates. Our atomic models of the major viral components, the E and prM proteins, indicate that the pr domains of prM have a critical role in holding the heterohexameric prM3E3 spikes in a metastable conformation. Destabilization of the prM furin-sensitive loop at acidic pH facilitates its processing. The prM topology and domain assignment in TBEV is similar to the mosquito-borne Binjari virus, but is in contrast to other immature flavivirus models. These results support that prM cleavage, the collapse of E protein ectodomains onto the virion surface, the large movement of the membrane domains of both E and M, and the release of the pr fragment from the particle render the virus mature and infectious. Our work favors the collapse model of flavivirus maturation warranting further studies of immature flaviviruses to determine the sequence of events and mechanistic details driving flavivirus maturation.
Assuntos
Vírus da Encefalite Transmitidos por Carrapatos , Proteínas do Envelope Viral , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Modelos Moleculares , Flavivirus/fisiologia , Animais , Vírion , Encefalite Transmitida por Carrapatos/virologia , HumanosRESUMO
Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive.
Assuntos
Motivos de Aminoácidos , Retículo Endoplasmático , Equartevirus , Equartevirus/genética , Equartevirus/metabolismo , Animais , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Arginina/metabolismo , Arginina/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas do Envelope Viral/química , Glicosilação , Linhagem Celular , Cavalos , HumanosRESUMO
The demand for Lentiviral Vector (LV) drug substance is increasing. However, primary capture using convective anion-exchange chromatography remains a significant manufacturing challenge. This stems from a poor understanding of the complex adsorption behaviors linked to LVs intricate and variable structure, such as high binding heterogeneity which is typically characterized by a gradient elution profile consisting of two peaks. Understanding which LV structural components drive these phenomena is therefore crucial for rational process design. This work identifies the key LV envelope components responsible for binding to quaternary-amine membrane adsorbents. Eliminating the pseudotype protein (Vesicular Stomatitis Virus G glycoprotein [VSV-G]) did not impact the heterogenous two-peak elution profile, suggesting it is not a major binding species. Digestion of envelope glycosaminoglycans (GAGs), present on proteoglycans, leads to a dramatic reduction in the proportion of vector eluted in peak 2, decreasing from 50% to 3.1%, and a threefold increase in peak 1 maximum. Data from reinjection experiments point towards interparticle envelope heterogeneity from discrete LV populations, where the two-peak profile emerges from a subpopulation of LVs interacting via highly charged GAGs (peak 2) along with a weaker binding population likely interacting through the phospholipid membrane and envelope protein (peak 1).
Assuntos
Vetores Genéticos , Lentivirus , Cromatografia por Troca Iônica/métodos , Lentivirus/genética , Vetores Genéticos/genética , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismoRESUMO
The envelope protein of dengue virus (DENV) is a primary target of the humoral immune response. The domain III of the DENV envelope protein (EDIII) is known to be the target of multiple potently neutralizing antibodies. One such antibody is 3H5, a mouse antibody that binds strongly to EDIII and potently neutralizes DENV serotype 2 (DENV-2) with unusually minimal antibody-dependent enhancement (ADE). To selectively display the binding epitope of 3H5, we strategically modified DENV-2 EDIII by shielding other known epitopes with engineered N-glycosylation sites. The modifications resulted in a glycosylated EDIII antigen termed "EDIII mutant N". This antigen was successfully used to sift through a dengue-immune scFv-phage library to select for scFv antibodies that bind to or closely surround the 3H5 epitope. The selected scFv antibodies were expressed as full-length human antibodies and showed potent neutralization activity to DENV-2 with low or negligible ADE resembling 3H5. These findings not only demonstrate the capability of the N-glycosylated EDIII mutant N as a tool to drive an epitope-directed antibody selection campaign but also highlight its potential as a dengue immunogen. This glycosylated antigen shows promise in focusing the antibody response toward a potently neutralizing epitope while reducing the risk of antibody-dependent enhancement.
Assuntos
Anticorpos Neutralizantes , Anticorpos Antivirais , Vírus da Dengue , Epitopos , Anticorpos de Cadeia Única , Proteínas do Envelope Viral , Anticorpos Neutralizantes/imunologia , Vírus da Dengue/imunologia , Vírus da Dengue/genética , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/química , Glicosilação , Epitopos/imunologia , Epitopos/química , Humanos , Animais , Anticorpos Antivirais/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/química , Camundongos , Dengue/imunologia , Dengue/prevenção & controle , Engenharia de Proteínas , Anticorpos Facilitadores , Domínios ProteicosRESUMO
Lassa virus (LASV) is an enveloped, negative-sense RNA virus that causes Lassa hemorrhagic fever. Successful entry of LASV requires the viral glycoprotein 1 (GP1) to undergo a receptor switch from its primary receptor alpha-dystroglycan (α-DG) to its endosomal receptor lysosome-associated membrane protein 1 (LAMP1). A conserved histidine triad in LASV GP1 has been reported to be responsible for receptor switch. To test the hypothesis that other non-conserved residues also contribute to receptor switch, we constructed a series of mutant LASV GP1 proteins and tested them for binding to LAMP1. Four residues, L84, K88, L107, and H170, were identified as critical for receptor switch. Substituting any of the four residues with the corresponding lymphocytic choriomeningitis virus (LCMV) residue (L84 âN, K88E, L10F, and H170S) reduced the binding affinity of LASV GP1 for LAMP1. Moreover, all mutations caused decreases in glycoprotein precursor (GPC)-mediated membrane fusion at both pH 4.5 and 5.2. The infectivity of pseudotyped viruses bearing either GPCL84N or GPCK88E decreased sharply in multiple cell types, while L107F and H170S had only mild effects on infectivity. Using biolayer light interferometry assay, we found that all four mutants had decreased binding affinity to LAMP1, in the order of binding affinity being L84 âN â> âL107F â> âK88E â> âH170S. The four amino acid loci identified for the first time in this study have important reference significance for the in-depth investigation of the mechanism of receptor switching and immune escape of LASV occurrence and the development of reserve anti-LASV infection drugs.