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Nature Communications Dec 2023Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the...
Zika virus (ZIKV) has emerged as a global health issue, yet neither antiviral therapy nor a vaccine are available. ZIKV is an enveloped RNA virus, replicating in the cytoplasm in close association with ER membranes. Here, we isolate ER membranes from ZIKV-infected cells and determine their proteome. Forty-six host cell factors are enriched in ZIKV remodeled membranes, several of these having a role in redox and methylation pathways. Four proteins are characterized in detail: thioredoxin reductase 1 (TXNRD1) contributing to folding of disulfide bond containing proteins and modulating ZIKV secretion; aldo-keto reductase family 1 member C3 (AKR1C3), regulating capsid protein abundance and thus, ZIKV assembly; biliverdin reductase B (BLVRB) involved in ZIKV induced lipid peroxidation and increasing stability of viral transmembrane proteins; adenosylhomocysteinase (AHCY) indirectly promoting mA methylation of ZIKV RNA by decreasing the level of S- adenosyl homocysteine and thus, immune evasion. These results highlight the involvement of redox and methylation enzymes in the ZIKV life cycle and their accumulation at virally remodeled ER membranes.
Topics: Humans; Zika Virus; Zika Virus Infection; Methylation; Proviruses; Virus Replication; Viral Proteins; Oxidation-Reduction
PubMed: 38052817
DOI: 10.1038/s41467-023-43665-6 -
Journal of Materials Chemistry. B Jul 2023Protein cages and virus-like particles are often thought of as highly uniform structures that obey strict geometric rules for self-assembly. Yet, there is a growing... (Review)
Review
Protein cages and virus-like particles are often thought of as highly uniform structures that obey strict geometric rules for self-assembly. Yet, there is a growing number of examples where different architectures can emerge from the same native cage system through minor changes in experimental conditions or protein sequence. Access to diverse architectures can help tune the engineering of protein cages for biotechnology applications where shape and symmetry often affects function. In this review, we highlight the underappreciated diversity of polymorphic architectures that can be formed by protein cages and virus-like particles, categorising examples by their method of formation.
Topics: Proteins; Biotechnology
PubMed: 37345320
DOI: 10.1039/d3tb00991b -
Emerging Microbes & Infections Dec 2023Viral envelope glycoproteins are crucial for viral infections. In the process of enveloped viruses budding and release from the producer cells, viral envelope... (Review)
Review
Viral envelope glycoproteins are crucial for viral infections. In the process of enveloped viruses budding and release from the producer cells, viral envelope glycoproteins are presented on the viral membrane surface as spikes, promoting the virus's next-round infection of target cells. However, the host cells evolve counteracting mechanisms in the long-term virus-host co-evolutionary processes. For instance, the host cell antiviral factors could potently suppress viral replication by targeting their envelope glycoproteins through multiple channels, including their intracellular synthesis, glycosylation modification, assembly into virions, and binding to target cell receptors. Recently, a group of studies discovered that some host antiviral proteins specifically recognized host proprotein convertase (PC) furin and blocked its cleavage of viral envelope glycoproteins, thus impairing viral infectivity. Here, in this review, we briefly summarize several such host antiviral factors and analyze their roles in reducing furin cleavage of viral envelope glycoproteins, aiming at providing insights for future antiviral studies.
Topics: Humans; Furin; Viral Envelope Proteins; SARS-CoV-2; Ebolavirus; HIV-1; Antiviral Agents; Hemorrhagic Fever, Ebola; COVID-19; Glycoproteins; Virus Diseases
PubMed: 36591809
DOI: 10.1080/22221751.2022.2164742 -
International Journal of Molecular... Sep 2023Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion...
Porcine deltacoronavirus (PDCoV) is an emerging virus that poses a significant threat to the global swine industry. Its membrane (M) protein is crucial for virion assembly and virus-host interactions. We selected the hydrophilic region of M protein for prokaryotic expression, purification, and recombinant protein production. Utilizing hybridoma technology, we prepared the monoclonal antibody (mAb) 24-A6 against M protein. The mAb 24-A6 was shown to be suitable for use in immunofluorescence assays, western blotting, and immunoprecipitation, with specificity for PDCoV and no cross-reactivity with other five porcine viruses. The M protein was observed to be expressed as early as 3 h after PDCoV infection, increasing its expression over the duration of infection. Notably, the antigenic epitope of the M protein identified as SPESRL recognized by mAb 24-A6 was found within a conserved structural domain (SWWSFNPETNNL) of the coronavirus M protein, indicating a crucial overlap between a functionally important viral assembly region and a region recognized by the immune system. Our findings provide valuable insights into mAb 24-A6 targeting the antigenic epitope of M protein and may contribute to the development of diagnostic tools for PDCoV infection and fundamental research into the function of PDCoV M protein.
Topics: Animals; Swine; Membrane Proteins; Antibodies, Monoclonal; Deltacoronavirus; Epitopes
PubMed: 37762237
DOI: 10.3390/ijms241813934 -
Journal of Virology Feb 2024Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal...
Antibodies are frontline defenders against influenza virus infection, providing protection through multiple complementary mechanisms. Although a subset of monoclonal antibodies (mAbs) has been shown to restrict replication at the level of virus assembly and release, it remains unclear how potent and pervasive this mechanism of protection is, due in part to the challenge of separating this effect from other aspects of antibody function. To address this question, we developed imaging-based assays to determine how effectively a broad range of mAbs against the IAV surface proteins can specifically restrict viral egress. We find that classically neutralizing antibodies against hemagglutinin are broadly multifunctional, inhibiting virus assembly and release at concentrations 1-20-fold higher than the concentrations at which they inhibit viral entry. These antibodies are also capable of altering the morphological features of shed virions, reducing the proportion of filamentous particles. We find that antibodies against neuraminidase and M2 also restrict viral egress and that inhibition by anti-neuraminidase mAbs is only partly attributable to a loss in enzymatic activity. In all cases, antigen crosslinking-either on the surface of the infected cell, between the viral and cell membrane, or both-plays a critical role in inhibition, and we are able to distinguish between these modes experimentally and through a structure-based computational model. Together, these results provide a framework for dissecting antibody multifunctionality that could help guide the development of improved therapeutic antibodies or vaccines and that can be extended to other viral families and antibody isotypes.IMPORTANCEAntibodies against influenza A virus provide multifaceted protection against infection. Although sensitive and quantitative assays are widely used to measure inhibition of viral attachment and entry, the ability of diverse antibodies to inhibit viral egress is less clear. We address this challenge by developing an imaging-based approach to measure antibody inhibition of virus release across a panel of monoclonal antibodies targeting the influenza A virus surface proteins. Using this approach, we find that inhibition of viral egress is common and can have similar potency to the ability of an antibody to inhibit viral entry. Insights into this understudied aspect of antibody function may help guide the development of improved countermeasures.
Topics: Humans; Antibodies, Monoclonal; Antibodies, Neutralizing; Antibodies, Viral; Hemagglutinin Glycoproteins, Influenza Virus; Influenza A virus; Influenza Vaccines; Influenza, Human; Membrane Proteins; Neuraminidase; Virus Assembly
PubMed: 38179944
DOI: 10.1128/jvi.01398-23 -
Virology Aug 2023Senecavirus A (SVA) is an emerging virus, causing vesicular disease in swine. SVA is a single-stranded, positive-sense RNA virus, which is the only member of the genus... (Review)
Review
Senecavirus A (SVA) is an emerging virus, causing vesicular disease in swine. SVA is a single-stranded, positive-sense RNA virus, which is the only member of the genus Senecavirus in the family Picornaviridae. SVA genome encodes 12 proteins: L, VP4, VP2, VP3, VP1, 2A, 2B, 2C, 3A, 3B, 3C and 3D. The VP1 to VP4 are structural proteins, and the others are nonstructural proteins. The replication of SVA in host cells is a complex process coordinated by an elaborate interplay between the structural and nonstructural proteins. Structural proteins are primarily involved in the invasion and assembly of virions. Nonstructural proteins modulate viral RNA translation and replication, and also take part in antagonizing the antiviral host response and in disrupting some cellular processes to allow virus replication. Here, we systematically reviewed the molecular functions of SVA structural and nonstructural proteins by reference to literatures of SVA itself and other picornaviruses.
Topics: Animals; Swine; Picornaviridae; Viral Proteins; RNA, Viral
PubMed: 37348144
DOI: 10.1016/j.virol.2023.06.004 -
Methods in Molecular Biology (Clifton,... 2024Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW)...
Several mammarenaviruses cause hemorrhagic fever (HF) disease in humans and pose a significant public health problem in their endemic regions. The Old World (OW) mammarenavirus Lassa virus (LASV) is estimated to infect several hundred thousand people yearly in West Africa, resulting in high numbers of Lassa fever (LF) cases, a disease associated with high morbidity and mortality. No licensed vaccines are available to combat LASV infection, and anti-LASV drug therapy is limited to the off-label use of ribavirin whose efficacy remains controversial. The development of reverse genetics approaches has provided investigators with a powerful approach for the investigation of the molecular, cell biology and pathogenesis of mammarenaviruses. The use of cell-based minigenome systems has allowed examining the cis- and trans-acting factors involved in viral genome replication and gene transcription, assembly, and budding, which has facilitated the identification of several anti-mammarenavirus candidate drugs. Likewise, it is possible now to rescue infectious recombinant mammarenaviruses from cloned cDNAs containing predetermined mutations in their genomes to investigate virus-host interactions and mechanisms of viral pathogenesis. Reverse genetics have also allowed the generation of mammarenaviruses expressing foreign genes to facilitate virus detection, to identify antiviral drugs, and to generate live-attenuated vaccine (LAV) candidates. Likewise, reverse genetics techniques have allowed the generation of single-cycle infectious, reporter-expressing mammarenaviruses to study some aspects of the biology of HF-causing human mammarenavirus without the need of high security biocontainment laboratories. In this chapter, we describe the experimental procedures to generate recombinant (r)LASV using state-of-the-art plasmid-based reverse genetics.
Topics: Humans; Lassa virus; Reverse Genetics; Arenaviridae; Lassa Fever; Hemorrhagic Fevers, Viral; Plasmids
PubMed: 38064030
DOI: 10.1007/978-1-0716-3533-9_8 -
Nature Communications Sep 2023Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template...
Human Respiratory Syncytial Virus (HRSV) is a prevalent cause of severe respiratory infections in children and the elderly. The helical HRSV nucleocapsid is a template for the viral RNA synthesis and a scaffold for the virion assembly. This cryo-electron microscopy analysis reveals the non-canonical arrangement of the HRSV nucleocapsid helix, composed of 16 nucleoproteins per asymmetric unit, and the resulting systematic variations in the RNA accessibility. We demonstrate that this unique helical symmetry originates from longitudinal interactions by the C-terminal arm of the HRSV nucleoprotein. We explore the polymorphism of the nucleocapsid-like assemblies, report five structures of the full-length particles and two alternative arrangements formed by a C-terminally truncated nucleoprotein mutant, and demonstrate the functional importance of the identified longitudinal interfaces. We put all these findings in the context of the HRSV RNA synthesis machinery and delineate the structural basis for its further investigation.
Topics: Child; Aged; Humans; Respiratory Syncytial Virus, Human; Cryoelectron Microscopy; Nucleocapsid; RNA, Viral; Nucleoproteins
PubMed: 37714861
DOI: 10.1038/s41467-023-41439-8 -
International Journal of Biological... Dec 2023Porcine Epidemic diarrhea virus (PEDV), which can result in severe vomiting, diarrhea, dehydration and death in newborn piglets, poses a great threat to the pig industry...
Porcine Epidemic diarrhea virus (PEDV), which can result in severe vomiting, diarrhea, dehydration and death in newborn piglets, poses a great threat to the pig industry around the world. The S1 subunit of S protein is crucial for triggering neutralizing antibodies binding to the receptor. Based on the advantages of high immunogenicity and precise assembly of nanoparticles, the mi3 nanoparticles and truncated S1 protein were assembled by the SpyTag/SpyCatcher system and then expressed in HEK293F cells, whereafter high-efficiency monoclonal antibodies (mAbs) were produced and identified. The obtained five mAbs can bind to various genotypes of PEDV, including a mAb (12G) which can neutralize G1 and G2 genotypes of PEDV in vitro. By further identification of monoclonal antibody target sequences, FNDHSF and LFYNVTNSYG were first identified as B-cell linear epitopes, in which LFYNVTNSYG was a neutralizing epitope. Alanine scans identified the key amino acid sites of two epitopes. Moreover, the results of multiple sequence alignment analysis showed that these two epitopes were highly conserved in various subtype variants. In brief, these findings can serve as a basis for additional research of PEDV and prospective resources for the creation of later detection and diagnostic techniques.
Topics: Animals; Swine; Antibodies, Monoclonal; Antibodies, Viral; Porcine epidemic diarrhea virus; Prospective Studies; Antibodies, Neutralizing; Epitopes, B-Lymphocyte
PubMed: 37804887
DOI: 10.1016/j.ijbiomac.2023.127276 -
Nature Communications Apr 2024Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of...
Controlled assembly of a protein shell around a viral genome is a key step in the life cycle of many viruses. Here we report a strategy for regulating the co-assembly of nonviral proteins and nucleic acids into highly ordered nucleocapsids in vitro. By fusing maltose binding protein to the subunits of NC-4, an engineered protein cage that encapsulates its own encoding mRNA, we successfully blocked spontaneous capsid assembly, allowing isolation of the individual monomers in soluble form. To initiate RNA-templated nucleocapsid formation, the steric block can be simply removed by selective proteolysis. Analyses by transmission and cryo-electron microscopy confirmed that the resulting assemblies are structurally identical to their RNA-containing counterparts produced in vivo. Enzymatically triggered cage formation broadens the range of RNA molecules that can be encapsulated by NC-4, provides unique opportunities to study the co-assembly of capsid and cargo, and could be useful for studying other nonviral and viral assemblies.
Topics: Nucleocapsid; Cryoelectron Microscopy; Maltose-Binding Proteins; Virus Assembly; Capsid; RNA, Viral; Capsid Proteins; RNA, Messenger
PubMed: 38678040
DOI: 10.1038/s41467-024-47808-1