Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.759
Filtrar
1.
Arch Virol ; 165(12): 3011-3015, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33025200

RESUMO

The hemagglutinin-esterase (HE) protein of betacoronavirus lineage A is a secondary receptor in the infection process and is involved in the emergence of new betacoronavirus genotypes with altered host specificity and tissue tropism. We previously reported a novel recombinant bovine coronavirus (BCoV) strain that was circulating in dairy cattle in China, but this virus was not successfully isolated, and the genetic characteristics of BCoV are still largely unknown. In this study, 20 diarrheic faecal samples were collected from a farm in Liaoning province that had an outbreak of calf diarrhea (≤ 3 months of age) in November 2018, and all of the samples tested positive for BCoV by RT-PCR. In addition, a BCoV strain with a recombinant HE (designated as SWUN/A1/2018) and another BCoV strain with a recombinant HE containing an insertion (designated as SWUN/A10/2018) were successfully isolated in cell culture (TCID50: 104.25/mL and 104.73/mL, respectively). Unexpectedly, we identified the emergence of a novel BCoV variant characterized by a 12-nt bovine gene insertion in the receptor-binding domain in a natural recombinant HE gene, suggesting a novel evolutionary pattern in BCoV.


Assuntos
Doenças dos Bovinos/epidemiologia , Infecções por Coronavirus/veterinária , Coronavirus Bovino/genética , Diarreia/veterinária , Hemaglutininas Virais/genética , RNA Viral/genética , Proteínas Virais de Fusão/genética , Animais , Bovinos , Doenças dos Bovinos/patologia , Doenças dos Bovinos/virologia , China/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Coronavirus Bovino/classificação , Coronavirus Bovino/isolamento & purificação , Diarreia/epidemiologia , Diarreia/patologia , Diarreia/virologia , Evolução Molecular , Fezes/virologia , Expressão Gênica , Genótipo , Modelos Moleculares , Mutagênese Insercional , Filogenia , Estrutura Secundária de Proteína , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA
2.
Proc Natl Acad Sci U S A ; 117(41): 25759-25770, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32994342

RESUMO

Human coronaviruses OC43 and HKU1 are respiratory pathogens of zoonotic origin that have gained worldwide distribution. OC43 apparently emerged from a bovine coronavirus (BCoV) spillover. All three viruses attach to 9-O-acetylated sialoglycans via spike protein S with hemagglutinin-esterase (HE) acting as a receptor-destroying enzyme. In BCoV, an HE lectin domain promotes esterase activity toward clustered substrates. OC43 and HKU1, however, lost HE lectin function as an adaptation to humans. Replaying OC43 evolution, we knocked out BCoV HE lectin function and performed forced evolution-population dynamics analysis. Loss of HE receptor binding selected for second-site mutations in S, decreasing S binding affinity by orders of magnitude. Irreversible HE mutations led to cooperativity in virus swarms with low-affinity S minority variants sustaining propagation of high-affinity majority phenotypes. Salvageable HE mutations induced successive second-site substitutions in both S and HE. Apparently, S and HE are functionally interdependent and coevolve to optimize the balance between attachment and release. This mechanism of glycan-based receptor usage, entailing a concerted, fine-tuned activity of two envelope protein species, is unique among CoVs, but reminiscent of that of influenza A viruses. Apparently, general principles fundamental to virion-sialoglycan interactions prompted convergent evolution of two important groups of human and animal pathogens.


Assuntos
Coronavirus/fisiologia , Hemaglutininas Virais/genética , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Virais de Fusão/genética , Vírion/metabolismo , Animais , Evolução Biológica , Linhagem Celular , Coronavirus/genética , Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Coronavirus Humano OC43/genética , Coronavirus Humano OC43/metabolismo , Coronavirus Humano OC43/fisiologia , Coronavirus Bovino/genética , Coronavirus Bovino/metabolismo , Coronavirus Bovino/fisiologia , Hemaglutininas Virais/química , Hemaglutininas Virais/metabolismo , Humanos , Lectinas/genética , Lectinas/metabolismo , Camundongos , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Virais/metabolismo , Seleção Genética , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/metabolismo , Vírion/genética , Ligação Viral , Liberação de Vírus
3.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611759

RESUMO

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.


Assuntos
Ebolavirus/efeitos dos fármacos , Proteína C1 de Niemann-Pick/antagonistas & inibidores , Receptores Virais/antagonistas & inibidores , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas Virais de Fusão/antagonistas & inibidores , Vírion/efeitos dos fármacos , Animais , Sítios de Ligação , Bioensaio , Chlorocebus aethiops , Clomifeno/química , Clomifeno/farmacologia , Ebolavirus/química , Ebolavirus/genética , Ebolavirus/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Temperatura Alta , Concentração de Íons de Hidrogênio , Simulação de Acoplamento Molecular , Proteína C1 de Niemann-Pick/química , Proteína C1 de Niemann-Pick/genética , Proteína C1 de Niemann-Pick/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Estrutura Terciária de Proteína , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/química , Tamoxifeno/farmacologia , Toremifeno/química , Toremifeno/farmacologia , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismo , Vírion/química , Vírion/genética , Vírion/metabolismo
4.
Int J Mol Sci ; 21(12)2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32604730

RESUMO

The recently emerged SARS-CoV-2 is the cause of the global health crisis of the coronavirus disease 2019 (COVID-19) pandemic. No evidence is yet available for CoV infection into hosts upon zoonotic disease outbreak, although the CoV epidemy resembles influenza viruses, which use sialic acid (SA). Currently, information on SARS-CoV-2 and its receptors is limited. O-acetylated SAs interact with the lectin-like spike glycoprotein of SARS CoV-2 for the initial attachment of viruses to enter into the host cells. SARS-CoV-2 hemagglutinin-esterase (HE) acts as the classical glycan-binding lectin and receptor-degrading enzyme. Most ß-CoVs recognize 9-O-acetyl-SAs but switched to recognizing the 4-O-acetyl-SA form during evolution of CoVs. Type I HE is specific for the 9-O-Ac-SAs and type II HE is specific for 4-O-Ac-SAs. The SA-binding shift proceeds through quasi-synchronous adaptations of the SA-recognition sites of the lectin and esterase domains. The molecular switching of HE acquisition of 4-O-acetyl binding from 9-O-acetyl SA binding is caused by protein-carbohydrate interaction (PCI) or lectin-carbohydrate interaction (LCI). The HE gene was transmitted to a ß-CoV lineage A progenitor by horizontal gene transfer from a 9-O-Ac-SA-specific HEF, as in influenza virus C/D. HE acquisition, and expansion takes place by cross-species transmission over HE evolution. This reflects viral evolutionary adaptation to host SA-containing glycans. Therefore, CoV HE receptor switching precedes virus evolution driven by the SA-glycan diversity of the hosts. The PCI or LCI stereochemistry potentiates the SA-ligand switch by a simple conformational shift of the lectin and esterase domains. Therefore, examination of new emerging viruses can lead to better understanding of virus evolution toward transitional host tropism. A clear example of HE gene transfer is found in the BCoV HE, which prefers 7,9-di-O-Ac-SAs, which is also known to be a target of the bovine torovirus HE. A more exciting case of such a switching event occurs in the murine CoVs, with the example of the ß-CoV lineage A type binding with two different subtypes of the typical 9-O-Ac-SA (type I) and the exclusive 4-O-Ac-SA (type II) attachment factors. The protein structure data for type II HE also imply the virus switching to binding 4-O acetyl SA from 9-O acetyl SA. Principles of the protein-glycan interaction and PCI stereochemistry potentiate the SA-ligand switch via simple conformational shifts of the lectin and esterase domains. Thus, our understanding of natural adaptation can be specified to how carbohydrate/glycan-recognizing proteins/molecules contribute to virus evolution toward host tropism. Under the current circumstances where reliable antiviral therapeutics or vaccination tools are lacking, several trials are underway to examine viral agents. As expected, structural and non-structural proteins of SARS-CoV-2 are currently being targeted for viral therapeutic designation and development. However, the modern global society needs SARS-CoV-2 preventive and therapeutic drugs for infected patients. In this review, the structure and sialobiology of SARS-CoV-2 are discussed in order to encourage and activate public research on glycan-specific interaction-based drug creation in the near future.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/virologia , Evolução Molecular , Interações entre Hospedeiro e Microrganismos/fisiologia , Pneumonia Viral/virologia , Receptores Virais/metabolismo , Internalização do Vírus , Acetilesterase/metabolismo , Animais , Betacoronavirus/genética , Sítios de Ligação , Linhagem Celular , Coronavirus/genética , Esterases , Transferência Genética Horizontal , Glicosaminoglicanos/metabolismo , Hemaglutininas Virais/genética , Humanos , Lectinas/metabolismo , Pandemias , Polissacarídeos , Receptores Virais/química , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/fisiologia , Torovirus , Proteínas Virais de Fusão/genética
5.
Arch Virol ; 165(9): 1959-1968, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32519007

RESUMO

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV) strains, has been one of the most problematic diseases affecting the poultry industry worldwide. Conventional vaccines provide effective protection for birds to survive ND outbreaks, but they may not completely suppress NDV shedding. NDV strains circulate on farms for a long time after the initial infection and cause potential risks. A new vaccine with fast clearance ability and low viral shedding is needed. In this study, we used interleukin-12 (IL-12) as an adjuvant and electroporation (EP) as an advanced delivery system to improve a DNA vaccine candidate. The fusion (F) protein gene from an NDV strain of the prevalent genotype VII.1.1 was cloned to prepare the vaccine. Chickens immunized with the F gene DNA vaccine co-delivered with an IL-12-expressing plasmid DNA showed higher neutralizing antibody levels and stronger concanavalin-A-induced lymphocyte proliferation than those treated with the F gene DNA vaccine alone. The co-delivered vaccine provided 100% protection, and less viral shedding and a shorter release time were observed in challenged chickens than when the F gene DNA vaccine was administered alone. The use of F gene DNA combined with IL-12 delivered by electroporation is a promising approach for vaccination against ND.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Interleucina-12/imunologia , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Galinhas , Eletroporação , Interleucina-12/administração & dosagem , Doença de Newcastle/imunologia , Doença de Newcastle/virologia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Vacinação , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Proteínas Virais de Fusão/administração & dosagem , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Eliminação de Partículas Virais
6.
PLoS One ; 15(6): e0234636, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32555720

RESUMO

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics.


Assuntos
Actinobacteria/virologia , Bacteriófagos/genética , Variação Genética , Genoma Viral , Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Composição de Bases , DNA Viral/genética , Genes Virais , Genômica , Filogenia , Proteínas Virais de Fusão/genética
7.
J Virol ; 94(15)2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32404526

RESUMO

We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance.IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.


Assuntos
Lipopeptídeos/farmacologia , Lipoproteínas/farmacologia , Síndrome de Imunodeficiência Adquirida dos Símios , Vírus da Imunodeficiência Símia/metabolismo , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus/efeitos dos fármacos , Substituição de Aminoácidos , Animais , Lipopeptídeos/química , Lipoproteínas/química , Macaca mulatta , Mutação de Sentido Incorreto , Síndrome de Imunodeficiência Adquirida dos Símios/tratamento farmacológico , Síndrome de Imunodeficiência Adquirida dos Símios/genética , Síndrome de Imunodeficiência Adquirida dos Símios/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/patologia , Vírus da Imunodeficiência Símia/genética , Proteínas Virais de Fusão/genética
8.
J Virol ; 94(12)2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32295904

RESUMO

Mumps virus (MuV), an enveloped RNA virus of the Paramyxoviridae family and the causative agent of mumps, affects the salivary glands and other glandular tissues as well as the central nervous system. The virus enters the cell by inducing the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by MuV envelope proteins: the hemagglutinin-neuraminidase and fusion (F) protein. Cleavage of the MuV F protein (MuV-F) into two subunits by the cellular protease furin is a prerequisite for fusion and virus infectivity. Here, we show that 293T (a derivative of HEK293) cells do not produce syncytia upon expression of MuV envelope proteins or MuV infection. This failure is caused by the inefficient MuV-F cleavage despite the presence of functional furin in 293T cells. An expression cloning strategy revealed that overexpression of lysosome-associated membrane proteins (LAMPs) confers on 293T cells the ability to produce syncytia upon expression of MuV envelope proteins. The LAMP family comprises the ubiquitously expressed LAMP1 and LAMP2, the interferon-stimulated gene product LAMP3, and the cell type-specific proteins. The expression level of the LAMP3 gene, but not of LAMP1 and LAMP2 genes, differed markedly between 293T and HEK293 cells. Overexpression of LAMP1, LAMP2, or LAMP3 allowed 293T cells to process MuV-F efficiently. Furthermore, these LAMPs were found to interact with both MuV-F and furin. Our results indicate that LAMPs support the furin-mediated cleavage of MuV-F and that, among them, LAMP3 may be critical for the process, at least in certain cells.IMPORTANCE The cellular protease furin mediates proteolytic cleavage of many host and pathogen proteins and plays an important role in viral envelope glycoprotein maturation. MuV, an enveloped RNA virus of the Paramyxoviridae family and an important human pathogen, enters the cell through the fusion of its envelope with the plasma membrane of the target cell. Membrane fusion is mediated by the viral attachment protein and the F protein. Cleavage of MuV-F into two subunits by furin is a prerequisite for fusion and virus infectivity. Here, we show that LAMPs support the furin-mediated cleavage of MuV-F. Expression levels of LAMPs affect the processing of MuV-F and MuV-mediated membrane fusion. Among LAMPs, the interferon-stimulated gene product LAMP3 is most critical in certain cells. Our study provides potential targets for anti-MuV therapeutics.


Assuntos
Furina/genética , Interações Hospedeiro-Patógeno/genética , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Lisossomos/virologia , Vírus da Caxumba/genética , Proteínas de Neoplasias/genética , Proteínas Virais de Fusão/genética , Células A549 , Animais , Membrana Celular/metabolismo , Membrana Celular/virologia , Chlorocebus aethiops , Furina/metabolismo , Regulação da Expressão Gênica , Células Gigantes/química , Células Gigantes/metabolismo , Células HEK293 , Proteína HN/genética , Proteína HN/metabolismo , Células HeLa , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Lisossomos/metabolismo , Vírus da Caxumba/metabolismo , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Proteólise , Transdução de Sinais , Células Vero , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus
9.
J Virol ; 94(11)2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32213613

RESUMO

Interferon alpha (IFN-α) and IFN-ß are type I IFNs that are induced by virus infection and are important in the host's innate antiviral response. EBV infection activates multiple cell signaling pathways, resulting in the production of type I IFN which inhibits EBV infection and virus-induced B-cell transformation. We reported previously that EBV tegument protein BGLF2 activates p38 and enhances EBV reactivation. To further understand the role of BGLF2 in EBV infection, we used mass spectrometry to identify cellular proteins that interact with BGLF2. We found that BGLF2 binds to Tyk2 and confirmed this interaction by coimmunoprecipitation. BGLF2 blocked type I IFN-induced Tyk2, STAT1, and STAT3 phosphorylation and the expression of IFN-stimulated genes (ISGs) IRF1, IRF7, and MxA. In contrast, BGLF2 did not inhibit STAT1 phosphorylation induced by IFN-γ. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of the protein to repress type I IFN signaling. Treatment of gastric carcinoma and Raji cells with IFN-α blocked BZLF1 expression and EBV reactivation; however, expression of BGLF2 reduced the ability of IFN-α to inhibit BZLF1 expression and enhanced EBV reactivation. In summary, EBV BGLF2 interacts with Tyk2, inhibiting Tyk2, STAT1, and STAT3 phosphorylation and impairs type I IFN signaling; BGLF2 also counteracts the ability of IFN-α to suppress EBV reactivation.IMPORTANCE Type I interferons are important for controlling virus infection. We have found that the Epstein-Barr virus (EBV) BGLF2 tegument protein binds to a protein in the type I interferon signaling pathway Tyk2 and inhibits the expression of genes induced by type I interferons. Treatment of EBV-infected cells with type I interferon inhibits reactivation of the virus, while expression of EBV BGLF2 reduces the ability of type I interferon to inhibit virus reactivation. Thus, a tegument protein delivered to cells during virus infection inhibits the host's antiviral response and promotes virus reactivation of latently infected cells. Therefore, EBV BGLF2 might protect virus-infected cells from the type I interferon response in cells undergoing lytic virus replication.


Assuntos
Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/fisiologia , Interferon Tipo I/imunologia , Transdução de Sinais/imunologia , Proteínas Virais de Fusão/imunologia , Ativação Viral/imunologia , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/patologia , Células HEK293 , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , Interferon Tipo I/genética , Interferon gama/genética , Interferon gama/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/genética , TYK2 Quinase/genética , TYK2 Quinase/imunologia , Proteínas Virais de Fusão/genética , Ativação Viral/genética
10.
J Virol ; 94(10)2020 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132237

RESUMO

For cell entry, vaccinia virus requires fusion with the host membrane via a viral fusion complex of 11 proteins, but the mechanism remains unclear. It was shown previously that the viral proteins A56 and K2 are expressed on infected cells to prevent superinfection by extracellular vaccinia virus through binding to two components of the viral fusion complex (G9 and A16), thereby inhibiting membrane fusion. To investigate how the A56/K2 complex inhibits membrane fusion, we performed experimental evolutionary analyses by repeatedly passaging vaccinia virus in HeLa cells overexpressing the A56 and K2 proteins to isolate adaptive mutant viruses. Genome sequencing of adaptive mutants revealed that they had accumulated a unique G9R open reading frame (ORF) mutation, resulting in a single His44Tyr amino acid change. We engineered a recombinant vaccinia virus to express the G9H44Y mutant protein, and it readily infected HeLa-A56/K2 cells. Moreover, similar to the ΔA56 virus, the G9H44Y mutant virus on HeLa cells had a cell fusion phenotype, indicating that G9H44Y-mediated membrane fusion was less prone to inhibition by A56/K2. Coimmunoprecipitation experiments demonstrated that the G9H44Y protein bound to A56/K2 at neutral pH, suggesting that the H44Y mutation did not eliminate the binding of G9 to A56/K2. Interestingly, upon acid treatment to inactivate A56/K2-mediated fusion inhibition, the G9H44Y mutant virus induced robust cell-cell fusion at pH 6, unlike the pH 4.7 required for control and revertant vaccinia viruses. Thus, A56/K2 fusion suppression mainly targets the G9 protein. Moreover, the G9H44Y mutant protein escapes A56/K2-mediated membrane fusion inhibition most likely because it mimics an acid-induced intermediate conformation more prone to membrane fusion.IMPORTANCE It remains unclear how the multiprotein entry fusion complex of vaccinia virus mediates membrane fusion. Moreover, vaccinia virus contains fusion suppressor proteins to prevent the aberrant activation of this multiprotein complex. Here, we used experimental evolution to identify adaptive mutant viruses that overcome membrane fusion inhibition mediated by the A56/K2 protein complex. We show that the H44Y mutation of the G9 protein is sufficient to overcome A56/K2-mediated membrane fusion inhibition. Treatment of virus-infected cells at different pHs indicated that the H44Y mutation lowers the threshold of fusion inhibition by A56/K2. Our study provides evidence that A56/K2 inhibits the viral fusion complex via the latter's G9 subcomponent. Although the G9H44Y mutant protein still binds to A56/K2 at neutral pH, it is less dependent on low pH for fusion activation, implying that it may adopt a subtle conformational change that mimics a structural intermediate induced by low pH.


Assuntos
Fusão de Membrana , Mutação , Vírus Vaccinia/genética , Vírus Vaccinia/isolamento & purificação , Proteínas Virais/genética , Fusão Celular , Membrana Celular , Evolução Molecular , Regulação Viral da Expressão Gênica , Genes Virais/genética , Genoma Viral , Células HeLa , Humanos , Proteínas Recombinantes , Vírus Vaccinia/crescimento & desenvolvimento , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/isolamento & purificação , Proteínas Virais/isolamento & purificação , Internalização do Vírus
11.
Arch Virol ; 165(5): 1057-1067, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144542

RESUMO

Human respiratory syncytial virus (hRSV) is the primary cause of severe respiratory tract disease in children and infants as well as in elderly and immunocompromised adults. The fusion protein (F) of hRSV is the major antigen eliciting a neutralizing antibody response and protective immunity in the host, especially those recognizing the prefusion F protein (pre-F). In this study, we made genetic constructs for expression of a recombinant prefusion F protein in Pichia pastoris GS115, called RGF. Using Escherichia coli BL21, we expressed the pre-F and postfusion F protein (Post-F), called RBF and Post-RBF, respectively. RGF and RBF showed high affinity for 5C4, a highly potent monoclonal antibody specific for pre-F. We studied the immunogenicity of RGF and RBF in mice. Compared to mice immunized with formalin-inactivated RSV (FI-RSV), mice immunized with RGF or RBF exhibited superior protective immunity, which was confirmed by serum neutralizing activity and viral clearance after challenge. As judged from the IgG1/IgG2a ratios and numbers of IFN-γ- and IL-4-secreting cells, RGF or RBF with alum adjuvant induced a balanced Th1-biased immune response and produced no signs of enhanced respiratory disease (ERD) upon hRSV challenge. In addition, the immunogenicity and protective efficacy of RGF were superior to those of RBF in mice. Therefore, RGF represents a potential vaccine candidate for the prevention of human infection with hRSV.


Assuntos
Proteínas Recombinantes/imunologia , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Vacinas contra Vírus Sincicial Respiratório/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Antígenos Virais/imunologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Infecções por Vírus Respiratório Sincicial/patologia , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/genética , Vacinas contra Vírus Sincicial Respiratório/isolamento & purificação , Células Th1/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/isolamento & purificação , Proteínas Virais de Fusão/genética , Viremia/imunologia
12.
Arch Virol ; 165(5): 1069-1077, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32144544

RESUMO

We investigated the molecular epidemiology of respiratory syncytial virus (RSV) isolated from children during 28 consecutive seasons (1990-2018) and the genetic variability of the duplication region of RSV genotypes ON1 and BA in South Korea. RSV was identified using culture-based methods in Hep-2 cells and was grouped as RSV-A or RSV-B by an immunofluorescence assay. The second hypervariable region of the G gene was sequenced for genotyping. The nucleotide and deduced amino acid sequences of the duplication region of RSV ON1 and BA were analyzed. A total of 670 RSV-A and 233 RSV-B isolates were obtained. For RSV-A, the NA1 genotype predominated during the 2004/2005-2011/2012 seasons. The ON1 genotype was first detected in 2011 and has since replaced all other genotypes. For RSV-B, the GB3 genotype predominated during the 1999/2000-2005/2006 seasons, but the BA genotype also replaced all other genotypes of RSV-B after the first season in which it was isolated (2005/2006). In ON1 and BA genotype RSV strains, novel sequence types of the duplication region of the G gene were identified in 50-95% and 33-80% of the isolates, respectively, in each season. The ON1 and BA9 genotypes are responsible for the current epidemics of RSV infection in South Korea. The sequences in the duplication region of the G gene have evolved continuously and might be sufficient for the identification of specific strains of the RSV-A ON1 and RSV-B BA genotypes.


Assuntos
Variação Genética , Genótipo , Infecções por Vírus Respiratório Sincicial/epidemiologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/classificação , Proteínas Virais de Fusão/genética , Duplicação Gênica , Humanos , Epidemiologia Molecular , República da Coreia/epidemiologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/isolamento & purificação , Análise de Sequência de DNA
13.
J Gen Virol ; 101(3): 309-321, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32003710

RESUMO

Our previous study showed that the Bombyx mori nucleopolyhedrovirus (BmNPV) F-like protein Bm14 is intrinsically related to the production of occlusion bodies, occlusion-derived virus (ODV) embedding and virulence in infected larvae. However, the exact mechanism by which Bm14 affects primary infection remains unknown. In this report, we characterized the detailed distribution and topology of Bm14 in occlusion bodies (OBs) and ODVs, and then further investigated the functional role of Bm14 in primary infection. A combination of Western blot and immunoelectron microscopy showed that Bm14 is mainly present on the surface of ODVs within OBs, but rarely in the OB matrix. Further phase separation and topology analysis of Bm14 by selective permeabilization revealed that Bm14 is a type I integral membrane protein with an N-terminus hidden in the endoplasmic reticulum (ER) lumen and a C-terminus exposed to the cytosol. In vivo assays demonstrated that the disruption of bm14 impaired the interactions of ODV with midgut epithelia, resulting in delayed spread in larval tissues. As the essential trigger of primary infection, some per os infectivity factors (PIFs) were verified to interact with Bm14 via a series of coimmunoprecipitation analyses. Further partially denaturing SDS-PAGE and BN-PAGE assays clearly showed that the deletion of bm14 did not affect the formation and presence of the PIF complex. In conclusion, Bm14 functions as a type I integral membrane protein to regulate ODV attachment to the midgut epithelial cells.


Assuntos
Bombyx/virologia , Células Epiteliais/metabolismo , Mucosa Intestinal/citologia , Proteínas de Membrana/metabolismo , Nucleopoliedrovírus/metabolismo , Corpos de Oclusão Virais/metabolismo , Proteínas Virais de Fusão/metabolismo , Ligação Viral , Animais , Bombyx/citologia , Linhagem Celular , Retículo Endoplasmático/metabolismo , Técnicas de Inativação de Genes , Larva/virologia , Proteínas de Membrana/genética , Nucleopoliedrovírus/química , Transfecção , Proteínas Virais de Fusão/genética , Vírion/metabolismo
14.
PLoS One ; 15(2): e0228572, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045432

RESUMO

Human respiratory syncytial virus (RSV) and parainfluenza virus type 3 (HPIV3) are among the most common viral causes of childhood bronchiolitis and pneumonia worldwide, and lack effective antiviral drugs or vaccines. Recombinant (r) HPIV3 was modified to express the RSV fusion (F) glycoprotein, the major RSV neutralization and protective antigen, providing a live intranasal bivalent HPIV3/RSV vaccine candidate. This extends previous studies using a chimeric bovine-human PIV3 vector (rB/HPIV3). One advantage is that rHPIV3 expresses all of the HPIV3 antigens compared to only two for rB/HPIV3. In addition, the use of rHPIV3 as vector should avoid excessive attenuation following addition of the modified RSV F gene, which may occur with rB/HPIV3. To enhance its immunogenicity, RSV F was modified (i) to increase the stability of the prefusion (pre-F) conformation and (ii) by replacement of its transmembrane (TM) and cytoplasmic tail (CT) domains with those of HPIV3 F (H3TMCT) to increase incorporation in the vector virion. RSV F (+/- H3TMCT) was expressed from the first (F/preN) or the second (F/N-P) gene position of rHPIV3. The H3TMCT modification dramatically increased packaging of RSV F into the vector virion and, in hamsters, resulted in significant increases in the titer of high-quality serum RSV-neutralizing antibodies, in addition to the increase conferred by pre-F stabilization. Only F-H3TMCT/preN replication was significantly attenuated in the nasal turbinates by the RSV F insert. F-H3TMCT/preN, F/N-P, and F-H3TMCT/N-P provided complete protection against wt RSV challenge. F-H3TMCT/N-P exhibited the most stable and highest expression of RSV F, providing impetus for its further development.


Assuntos
Vacinas contra Parainfluenza/genética , Vírus da Parainfluenza 3 Humana/imunologia , Vacinas contra Vírus Sincicial Respiratório/genética , Proteínas Virais de Fusão/genética , Montagem de Vírus , Administração Intranasal , Animais , Chlorocebus aethiops , Cricetinae , Feminino , Humanos , Imunogenicidade da Vacina , Macaca mulatta , Mesocricetus , Vacinas contra Parainfluenza/administração & dosagem , Vacinas contra Parainfluenza/imunologia , Vírus da Parainfluenza 3 Humana/genética , Vírus da Parainfluenza 3 Humana/fisiologia , Estabilidade Proteica , Vacinas contra Vírus Sincicial Respiratório/administração & dosagem , Vacinas contra Vírus Sincicial Respiratório/imunologia , Células Vero , Proteínas Virais de Fusão/metabolismo
15.
J Gen Virol ; 101(5): 467-472, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32100701

RESUMO

The paramyxoviruses Hendra virus (HeV) and parainfluenza virus 5 (PIV5) require the fusion (F) protein to efficiently infect cells. For fusion to occur, F undergoes dramatic, essentially irreversible conformational changes to merge the viral and cell membranes into a continuous bilayer. Recently, a transmembrane (TM) domain leucine/isoleucine (L/I) zipper was shown to be critical in maintaining the expression, stability and pre-fusion conformation of HeV F, allowing for fine-tuned timing of membrane fusion. To analyse the effect of the TM domain L/I zipper in another paramyxovirus, we created alanine mutations to the TM domain of PIV5 F, a paramyxovirus model system. Our data show that while the PIV5 F TM L/I zipper does not significantly affect total expression and only modestly affects surface expression and pre-fusion stability, it is critical for fusogenic activity. These results suggest that the roles of TM L/I zipper motifs differ among members of the family Paramyxoviridae.


Assuntos
Membrana Celular/genética , Isoleucina/genética , Leucina/genética , Mutação/genética , Vírus da Parainfluenza 5/genética , Domínios Proteicos/genética , Proteínas Virais de Fusão/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Fusão de Membrana/genética , Paramyxovirinae/genética , Células Vero
16.
Mater Sci Eng C Mater Biol Appl ; 108: 110426, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31923928

RESUMO

A DNA fragment amplification/expression technology for the production of new generation biomaterials for scientific, industrial and biomedical applications is described. The technology enables the formation of artificial Open Reading Frames (ORFs) encoding concatemeric RNAs and proteins. It recruits the Type IIS SapI restriction endonuclease (REase) for an assembling of DNA fragments in an ordered head-to-tail-orientation. The technology employs a vector-enzymatic system, dedicated to the expression of newly formed, concatemeric ORFs from strong promoters. Four vector series were constructed to suit specialised needs. As a proof of concept, a model amplification of a 7-amino acid (aa) epitope from the S protein of HBV virus was performed, resulting in 500 copies of the epitope-coding DNA segment, consecutively linked and expressed in Escherichia coli (E. coli). Furthermore, a peptide with potential pro-regenerative properties (derived from an angiopoietin-related growth factor) was designed. Its aa sequence was back-translated, codon usage optimized and synthesized as a continuous ORF 10-mer. The 10-mer was cloned into the amplification vector, enabling the N-terminal fusion and multiplication of the encoded protein with MalE signal sequence. The obtained genes were expressed, and the proteins were purified. Conclusively, we show that the proteins are neither cytotoxic nor immunogenic and they have a very low allergic potential.


Assuntos
Materiais Biocompatíveis , DNA Concatenado , Escherichia coli , Expressão Gênica , Técnicas de Amplificação de Ácido Nucleico , Fases de Leitura Aberta , DNA Concatenado/genética , DNA Concatenado/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Vírus da Hepatite B/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Virais de Fusão/biossíntese , Proteínas Virais de Fusão/genética
17.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31852779

RESUMO

Respiratory syncytial virus (RSV) is a top cause of severe lower respiratory tract disease and mortality in infants and the elderly. Currently, no vaccine or effective treatment exists for RSV. The RSV G glycoprotein mediates viral attachment to cells and contributes to pathogenesis by modulating host immunity through interactions with the human chemokine receptor CX3CR1. Antibodies targeting the RSV G central conserved domain are protective in both prophylactic and postinfection animal models. Here, we describe the crystal structure of the broadly neutralizing human monoclonal antibody 3G12 bound to the RSV G central conserved domain. Antibody 3G12 binds to a conformational epitope composed of highly conserved residues, explaining its broad neutralization activity. Surprisingly, RSV G complexed with 3G12 adopts a distinct conformation not observed in previously described RSV G-antibody structures. Comparison to other structures reveals that the RSV G central conserved domain is flexible and can adopt multiple conformations in the regions flanking the cysteine noose. We also show that restriction of RSV G flexibility with a proline mutation abolishes binding to antibody 3G12 but not antibody 3D3, which recognizes a different conformation of RSV G. Our studies provide new insights for rational vaccine design, indicating the importance of preserving both the global structural integrity of antigens and local conformational flexibility at antigenic sites, which may elicit a more diverse antibody response and broader protection against infection and disease.IMPORTANCE Respiratory syncytial virus (RSV) causes severe respiratory infections in infants, young children, and the elderly, and currently, no licensed vaccine exists. In this study, we describe the crystal structure of the RSV surface glycoprotein G in complex with a broadly neutralizing human monoclonal antibody. The antibody binds to RSV G at a highly conserved region stabilized by two disulfide bonds, but it captures RSV G in a conformation not previously observed, revealing that this region is both structured and flexible. Importantly, our findings provide insight for the design of vaccines that elicit diverse antibodies, which may provide broad protection from infection and disease.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Epitopos/química , Epitopos/imunologia , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais de Fusão/química , Proteínas Virais de Fusão/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Receptor 1 de Quimiocina CX3C/metabolismo , Cristalografia por Raios X , Epitopos/genética , Humanos , Modelos Moleculares , Mutação , Conformação Proteica , Vírus Sincicial Respiratório Humano/genética , Proteínas Virais de Fusão/genética
18.
Vet Microbiol ; 238: 108425, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31648723

RESUMO

Duck Tembusu virus (DTMUV) has caused enormous economic losses to the poultry industry in China. In the current study, we generated chimeric virus-like particles (VLPs) containing E protein of the DTMUV and HA2 protein of the H3N2 avian influenza virus (AIV). The chimeric VLPs could induce specific antibody responses in both mice (n = 5/group) and ducks (n = 10/group). After immunizing ducklings with the chimeric VLPs, all immunized ducks (n = 10/group) were 100% (10/10) protected against homologous DTMUV strain and virus shedding was not detected on day 5 post-challenge, whereas 60% (6/10) of the ducklings immunized with PBS presented typical symptoms with a virus shedding rate of 90% (9/10). Furthermore, viral loads were significantly decreased in the birds of the chimeric VLPs immunized group, comparing to that of the PBS immunized group. Our data demonstrated that the chimeric VLPs used in the current study could be applied as a potential vaccine candidate to control DTMUV infections in young ducks.


Assuntos
Antígenos Virais/imunologia , Flavivirus/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais de Fusão/imunologia , Animais , Anticorpos Antivirais/sangue , Patos , Flavivirus/genética , Infecções por Flavivirus/imunologia , Imunogenicidade da Vacina/imunologia , Camundongos , Proteínas Virais de Fusão/genética , Carga Viral
19.
Genes (Basel) ; 10(9)2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31470607

RESUMO

Enteroviruses affect millions of people worldwide and are of significant clinical importance. The standard method for enterovirus identification and genotyping still relies on Sanger sequencing of short diagnostic amplicons. In this study, we assessed the feasibility of nanopore sequencing using the new flow cell "Flongle" for fast, cost-effective, and accurate genotyping of human enteroviruses from clinical samples. PCR amplification of partial VP1 gene was performed from multiple patient samples, which were multiplexed together after barcoding PCR and sequenced multiple times on Flongle flow cells. The nanopore consensus sequences obtained from mapping reads to a reference database were compared to their Sanger sequence counterparts. Using clinical specimens sampled over different years, we were able to correctly identify enterovirus species and genotypes for all tested samples, even when doubling the number of barcoded samples on one flow cell. Average sequence identity across sequencing runs was >99.7%. Phylogenetic analysis showed that the consensus sequences achieved with Flongle delivered accurate genotyping. We conclude that the new Flongle-based assay with its fast turnover time, low cost investment, and low cost per sample represents an accurate, reproducible, and cost-effective platform for enterovirus identification and genotyping.


Assuntos
Enterovirus/genética , Técnicas de Genotipagem/métodos , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de RNA/métodos , Sequência Consenso , Técnicas de Genotipagem/economia , Técnicas de Genotipagem/instrumentação , Técnicas de Diagnóstico Molecular/economia , Técnicas de Diagnóstico Molecular/instrumentação , Nanoporos , Análise de Sequência de RNA/economia , Análise de Sequência de RNA/instrumentação , Proteínas Virais de Fusão/genética
20.
Virology ; 535: 102-110, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31299486

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease with a high fatality rate, caused by SFTS virus (SFTSV). Because little is known about the nature of SFTSV, basic studies are required for the developments of vaccines and effective therapies. In the present study, we identified the amino acid residue important for membrane fusion induced by the SFTSV glycoprotein (GP). Syncytium formations were observed in cells expressing the GPs of SFTSV Japanese strain (YG-1 and SPL030). In contrast, no or only weak syncytium formations were induced in cells expressing GP of SFTSV Chinese strain (HB29). The replacement of arginine at amino acid residue 962 with serine in HB29 GP (R962S) induced membrane fusion, while the replacement of serine at residue 962 with arginine in YG1 GP (S962R) did not. These data indicate that serine at residue 962 in the SFTSV-GP is critical for inducing membrane fusion and viral infection.


Assuntos
Phlebovirus/fisiologia , Proteínas Virais de Fusão/metabolismo , Internalização do Vírus , Substituição de Aminoácidos , Fusão Celular , Células Gigantes/citologia , Células Gigantes/virologia , Mutagênese Sítio-Dirigida , Phlebovirus/genética , Proteínas Virais de Fusão/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA