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1.
Nat Commun ; 10(1): 2098, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068585

RESUMO

Hepatitis D virus (HDV) doesn't encode envelope proteins for packaging of its ribonucleoprotein (RNP) and typically relies on the surface glycoproteins (GPs) from hepatitis B virus (HBV) for virion assembly, envelopment and cellular transmission. HDV RNA genome can efficiently replicate in different tissues and species, raising the possibility that it evolved, and/or is still able to transmit, independently of HBV. Here we show that alternative, HBV-unrelated viruses can act as helper viruses for HDV. In vitro, envelope GPs from several virus genera, including vesiculovirus, flavivirus and hepacivirus, can package HDV RNPs, allowing efficient egress of HDV particles in the extracellular milieu of co-infected cells and subsequent entry into cells expressing the relevant receptors. Furthermore, HCV can propagate HDV infection in the liver of co-infected humanized mice for several months. Further work is necessary to evaluate whether HDV is currently transmitted by HBV-unrelated viruses in humans.


Assuntos
Coinfecção/transmissão , Hepatite D/transmissão , Vírus Delta da Hepatite/fisiologia , Montagem de Vírus , Animais , Linhagem Celular Tumoral , Coinfecção/virologia , Flavivirus/metabolismo , Hepacivirus/metabolismo , Hepacivirus/patogenicidade , Hepatite D/virologia , Vírus Delta da Hepatite/isolamento & purificação , Vírus Delta da Hepatite/patogenicidade , Hepatócitos/transplante , Hepatócitos/virologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Cultura Primária de Células , RNA Viral/isolamento & purificação , Ribonucleoproteínas/metabolismo , Vesiculovirus/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo
2.
Nat Commun ; 10(1): 2265, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118422

RESUMO

Hepatitis delta virus (HDV) depends on the helper function of hepatitis B virus (HBV), which provides the envelope proteins for progeny virus secretion. Current infection-competent cell culture models do not support assembly and secretion of HDV. By stably transducing HepG2 cells with genes encoding the NTCP-receptor and the HBV envelope proteins we produce a cell line (HepNB2.7) that allows continuous secretion of infectious progeny HDV following primary infection. Evaluation of antiviral drugs shows that the entry inhibitor Myrcludex B (IC50: 1.4 nM) and interferon-α (IC50: 28 IU/ml, but max. 60-80% inhibition) interfere with primary infection. Lonafarnib inhibits virus secretion (IC50: 36 nM) but leads to a substantial intracellular accumulation of large hepatitis delta antigen and replicative intermediates, accompanied by the induction of innate immune responses. This work provides a cell line that supports the complete HDV replication cycle and presents a convenient tool for antiviral drug evaluation.


Assuntos
Antivirais/farmacologia , Vírus Delta da Hepatite/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Células Hep G2 , Vírus da Hepatite B/metabolismo , Vírus Delta da Hepatite/fisiologia , Antígenos da Hepatite delta/metabolismo , Humanos , Imunidade Inata/efeitos dos fármacos , Concentração Inibidora 50
3.
Soft Matter ; 15(22): 4525-4540, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31099376

RESUMO

The complex-type glycan shields of eukaryotic cells have a core layer of mannose residues buried under tiers of sugars that end with sialic acid (SA) residues. We investigate if the self-latching of mannose residues, earlier reported in pure monolayer studies, also manifests in the setting of a complex-type glycan shield. Would distal SA residues impede access to the mannose core? The interactions of mannobiose-, SA-, and lactose-coated probes with the complex-type VSV-G glycan shield on an HIV pseudovirus were studied with force-spectroscopy and gold-nanoparticle solutions. In force spectroscopy, the sugar probes can be forced to sample the depths of the glycan shield, whereas with sugar-coated nanoparticles, only interactions permitted by freely-diffusive contact occur. Deep-indentation mechanics was performed to verify the inferred structure of the engineered virus and to isolate the glycan shield layer for subsequent interaction studies. The adhesion between the sugar-probes and complex-type glycan shield was deconvoluted by comparing against the cross- and self- adhesions between the sugars in pure monolayers. Results from complementing systems were consistent with mannobiose-coated probes latching to the mannose core in the glycan shield, unhindered by the SA and distal sugars, with a short-range 'brittle' release of adhesion resulting in tightly coated viruses. SA-Coated probes, however, adhere to the terminal SA layer of a glycan shield with long-range and mechanically 'tough' adhesions resulting in large-scale virus aggregation. Lactose-coated probes exhibit ill-defined adherence to sialic residues. The selection and positioning of sugars within a glycan shield can influence how carbohydrate surfaces of different composition adhere.


Assuntos
HIV-1/química , Manose/química , Glicoproteínas de Membrana/química , Ácido N-Acetilneuramínico/química , Proteínas do Envelope Viral/química , Células HEK293 , HIV-1/genética , HIV-1/metabolismo , Humanos , Manose/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Nanopartículas Metálicas/química , Ácido N-Acetilneuramínico/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
4.
Nat Commun ; 10(1): 1788, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996276

RESUMO

Three Ebolavirus genus viruses cause lethal disease and lack targeted therapeutics: Ebola virus, Sudan virus and Bundibugyo virus. Monoclonal antibody (mAb) cocktails against the surface glycoprotein (GP) present a potential therapeutic strategy. Here we report two crystal structures of the antibody BDBV223, alone and complexed with its GP2 stalk epitope, an interesting site for therapeutic/vaccine design due to its high sequence conservation among ebolaviruses. BDBV223, identified in a human survivor of Bundibugyo virus disease, neutralizes both Bundibugyo virus and Ebola virus, but not Sudan virus. Importantly, the structure suggests that BDBV223 binding interferes with both the trimeric bundle assembly of GP and the viral membrane by stabilizing a conformation in which the monomers are separated by GP lifting or bending. Targeted mutagenesis of BDBV223 to enhance SUDV GP recognition indicates that additional determinants of antibody binding likely lie outside the visualized interactions, and perhaps involve quaternary assembly or membrane-interacting regions.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Ebolavirus/efeitos dos fármacos , Doença pelo Vírus Ebola/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Monoclonais/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Anticorpos Neutralizantes/metabolismo , Anticorpos Antivirais/química , Anticorpos Antivirais/isolamento & purificação , Anticorpos Antivirais/metabolismo , Reações Cruzadas/imunologia , Cristalografia por Raios X , Ebolavirus/imunologia , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/sangue , Doença pelo Vírus Ebola/virologia , Humanos , Hibridomas , Mutagênese , Sobreviventes , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo
5.
Retrovirology ; 16(1): 9, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30940160

RESUMO

BACKGROUND: We previously showed that the gM of HSV-1 could restrict the release of infectious HIV-1 from cells. In this study, we analyzed if the four HSV-1 glycoproteins (gD, gB, and gH/gL), which are the minimum glycoproteins required for HSV-1 entry, restricted the release of infectious HIV-1. RESULTS: Of these four glycoproteins, gD and gH/gL restricted the production of infectious HIV-1 from cells transfected with an infectious molecular clone of HIV-1 (strain NL4-3) while gB had no significant effect. Pulse-chase analyses indicated that gD did not affect the biosynthesis and processing of gp160 into gp120/gp41, the transport of the gp120/gp41 to the cell surface, or the release of HIV-1 particles from the cell surface. Our analyses revealed that gD was incorporated into HIV-1 virus particles while gp120/gp41 was excluded from released virus particles. Truncated mutants of gD revealed that the cytoplasmic domain was dispensable but that a membrane bound gD was required for the restriction of release of infectious HIV-1. Finally, cell lines expressing gD also potently restricted the release of infectious virus. CONCLUSIONS: Due to its ability to exclude HIV-1 gp120/gp41 from maturing virus, gD may provide a useful tool in deciphering mechanisms of Env incorporation into maturing virus particles.


Assuntos
HIV-1/fisiologia , Herpesvirus Humano 1/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Linhagem Celular , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , Proteína gp160 do Envelope de HIV/genética , Proteína gp160 do Envelope de HIV/metabolismo , Herpesvirus Humano 1/genética , Humanos , Glicoproteínas de Membrana/genética , Proteínas do Envelope Viral/genética
6.
Arch Virol ; 164(5): 1323-1334, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30877450

RESUMO

Porcine circovirus type 2 (PCV2) is the essential infectious agent causing porcine circovirus-associated disease (PCVD) in pigs and one of the important viruses that severely jeopardize the swine husbandry industry. PCV2 elicits the unfolded protein response (UPR) via activation of the PERK pathway, and its capsid protein (Cap) has also been found to induce UPR with subsequent activation of apoptosis. The open reading frame 5 (ORF5) protein is a recently discovered non-structural protein, and its function in PCV2 pathogenesis remains unknown. The aim of this study was to determine whether the PCV2 ORF5 protein could induce endoplasmic reticulum stress (ERS) and UPR in porcine alveolar macrophages (PAMs). pEGFP-tagged ORF5 protein was transiently overexpressed in PAMs. Transmission electron microscopy (TEM) was employed to examine changes in ER morphology, and quantitative real-time PCR and western blotting analysis were used to measure UPR-related cell signaling alterations. We found that the ORF5 protein triggers swelling and degranulation of the ER and upregulates the expression of ERS markers. Further experiments demonstrated that the PCV2 ORF5 protein induces ERS and UPR via the PERK (RNA-activated protein kinase-like endoplasmic reticulum kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol requiring enzyme 1) signaling pathways. Together with previous studies, we provide new information on the ERS-UPR induced by the PCV2 ORF5 protein.


Assuntos
Circovirus/genética , Estresse do Retículo Endoplasmático/genética , Retículo Endoplasmático/ultraestrutura , Macrófagos Alveolares/patologia , Resposta a Proteínas não Dobradas/genética , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Fator 6 Ativador da Transcrição/metabolismo , Animais , Linhagem Celular , Infecções por Circoviridae/patologia , Infecções por Circoviridae/veterinária , Retículo Endoplasmático/virologia , Endorribonucleases/metabolismo , Macrófagos Alveolares/virologia , Microscopia Eletrônica de Transmissão , Suínos , Doenças dos Suínos , Proteínas do Envelope Viral/metabolismo , eIF-2 Quinase/metabolismo
7.
J Biotechnol ; 295: 41-48, 2019 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-30826446

RESUMO

The recent Ebola virus disease (EVD) outbreaks make the development of efficacious and low cost vaccines against Ebola virus (EBOV) an urgent goal. Multiepitopic vaccines allow a rational design rendering vaccines able to induce proper immune responses in terms of polarization and potency. In addition, the pathogen variants can be easily covered by including epitopes conserved among relevant isolates. Other important aspects to consider in vaccination are the costs associated to production, distribution, and administration of the vaccine. Plants provide an advantageous platform for this purpose, since they yield biomass at very low costs and some species can be used to formulate purification-free oral vaccines. In the present study, a multiepitopic protein called Zerola, which carries epitopes from the EBOV glycoprotein (GP), was designed based on immunoinformatic approaches and current experimental evidence on B cell protective GP epitopes. Moreover, expression studies performed in nuclear-transformed tobacco lines confirmed the capacity of the plant cell to synthetize the Zerola antigenic protein. The generation of this plant-based candidate vaccine is a step forward in the development of highly efficient and low cost EBOV vaccines.


Assuntos
Vacinas contra Ebola , Ebolavirus/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes , Proteínas do Envelope Viral , Células Cultivadas , Vacinas contra Ebola/química , Vacinas contra Ebola/genética , Vacinas contra Ebola/metabolismo , Epitopos/química , Epitopos/genética , Epitopos/metabolismo , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Células Vegetais , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
8.
Int J Med Sci ; 16(3): 355-365, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911269

RESUMO

Dengue virus belongs to the Flaviviridae family which also includes viruses such as the Zika, West Nile and yellow fever virus. Dengue virus generally causes mild disease, however, more severe forms of the dengue virus infection, dengue haemorrhagic fever (DHF) and dengue haemorrhagic fever with shock syndrome (DSS) can also occur, resulting in multiple organ failure and even death, especially in children. The only dengue vaccine available in the market, CYD-TDV offers limited coverage for vaccinees from 9-45 years of age and is only recommended for individuals with prior dengue exposure. A number of mutations that were shown to attenuate virulence of dengue virus in vitro and/or in vivo have been identified in the literature. The mutations which fall within the conserved regions of all four dengue serotypes are discussed. This review hopes to provide information leading to the construction of a live attenuated dengue vaccine that is suitable for all ages, irrespective of the infecting dengue serotype and prior dengue exposure.


Assuntos
Vacinas contra Dengue/farmacologia , Vírus da Dengue/genética , Vírus da Dengue/patogenicidade , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Capsídeo/química , Ensaios Clínicos Fase III como Assunto , Dengue/prevenção & controle , Vacinas contra Dengue/imunologia , Genoma Viral , Humanos , Mutação , Vacinas Atenuadas/farmacologia , Vacinas Sintéticas/farmacologia , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Virulência , Fatores de Virulência/química , Fatores de Virulência/genética
9.
Oncol Rep ; 41(5): 2927-2936, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896862

RESUMO

Glioblastoma (GBM) is the most aggressive form of brain tumor in adults, with a devastating outcome. Emerging evidence shows that human cytomegalovirus (HCMV) proteins and nucleic acids are present in GBM tissues. DNA methylation is important for the initiation and progression of cancer and is an established host response against invading nucleic acids. The expression and localization of DNA methyltransferase 1 (DNMT­1) was assessed, and the effects of DNA methylation inhibitor 5­azacytidine (5AZA) were analyzed in the context of the viral replication, proliferation and invasion capacities of HCMV­infected GBM U343MG cells. In addition, the expression of various HCMV proteins and DNMT­1 was examined in GBM tissue specimens obtained from five patients. DNMT­1 was localized in the nucleus of cells expressing HCMV­immediate early, whereas in cells expressing HCMV­glycoprotein gB (gB), extranuclear/cytoplasmic localization was observed. This was also observed in vitro in U343MG cells. In addition, DNMT­1 was localized to the extranuclear/cytoplasmic space of cells lining blood vessel walls within the GBM tumors. Treatment of infected U343MG cells with 5AZA did not affect viral replication, but reduced cell invasion and proliferation (P=0.05 and P<0.0001, respectively). However, 5AZA treatment of uninfected cells did not affect cell invasion (P=0.09), but proliferation was significantly reduced (P<0.0001). These findings may be of importance in further investigations aimed at using DNA methylation and viral inhibitors in GBM therapy.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Glioblastoma/tratamento farmacológico , Adulto , Idoso , Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Encéfalo/patologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/virologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/patogenicidade , Citomegalovirus/fisiologia , Citoplasma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Progressão da Doença , Feminino , Glioblastoma/patologia , Glioblastoma/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Resultado do Tratamento , Proteínas do Envelope Viral/metabolismo , Replicação Viral/efeitos dos fármacos
10.
Nat Microbiol ; 4(5): 876-887, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30886357

RESUMO

Arboviruses cycle between, and replicate in, both invertebrate and vertebrate hosts, which for Zika virus (ZIKV) involves Aedes mosquitoes and primates1. The viral determinants required for replication in such obligate hosts are under strong purifying selection during natural virus evolution, making it challenging to resolve which determinants are optimal for viral fitness in each host. Herein we describe a deep mutational scanning (DMS) strategy2-5 whereby a viral cDNA library was constructed containing all codon substitutions in the C-terminal 204 amino acids of ZIKV envelope protein (E). The cDNA library was transfected into C6/36 (Aedes) and Vero (primate) cells, with subsequent deep sequencing and computational analyses of recovered viruses showing that substitutions K316Q and S461G, or Q350L and T397S, conferred substantial replicative advantages in mosquito and primate cells, respectively. A 316Q/461G virus was constructed and shown to be replication-defective in mammalian cells due to severely compromised virus particle formation and secretion. The 316Q/461G virus was also highly attenuated in human brain organoids, and illustrated utility as a vaccine in mice. This approach can thus imitate evolutionary selection in a matter of days and identify amino acids key to the regulation of virus replication in specific host environments.


Assuntos
Análise Mutacional de DNA/métodos , Tropismo Viral , Infecção por Zika virus/virologia , Zika virus/fisiologia , Aedes/virologia , Animais , Evolução Biológica , Cercopithecus aethiops , Feminino , Especificidade de Hospedeiro , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mosquitos Vetores/virologia , Mutação , Seleção Genética , Células Vero , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Zika virus/química , Zika virus/genética
11.
Nat Commun ; 10(1): 1121, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850661

RESUMO

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.


Assuntos
Antígenos CD/química , Apoferritinas/química , Proteínas de Protozoários/química , Receptores da Transferrina/química , Receptores Virais/química , Transferrina/química , Proteínas do Envelope Viral/química , Antígenos CD/genética , Antígenos CD/metabolismo , Apoferritinas/genética , Apoferritinas/metabolismo , Arenavirus do Novo Mundo/genética , Arenavirus do Novo Mundo/metabolismo , Sítios de Ligação , Clonagem Molecular , Microscopia Crioeletrônica , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HeLa , Proteína da Hemocromatose/química , Proteína da Hemocromatose/genética , Proteína da Hemocromatose/metabolismo , Humanos , Plasmodium vivax/genética , Plasmodium vivax/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores da Transferrina/genética , Receptores da Transferrina/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transferrina/genética , Transferrina/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo
12.
Methods Mol Biol ; 1937: 125-134, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30706393

RESUMO

Lentiviral vectors (LVs) are widely used in gene transfer protocols due to many advantages that include stable gene expression, higher transgene payloads, and, importantly, the ability to pseudotype the vectors with a diverse number of heterologous viral envelopes with broad or restricted cell tropism depending on the need. The pseudotyping process also allows for incorporation of specific antibodies/ligands to engineer LVs. These features greatly facilitate customization of lentiviral vectors for cell/tissue specific gene delivery. The VSV-G protein containing envelope remains the most widely used among the viral glycoproteins used for LV pseudotyping due to its versatile host range and stability. However, many other viral envelopes are being identified for special applications of LVs. Here we describe the methodology to generate pseudotyped LVs using a four-plasmid transient transfection system focusing on aspects to generate high-titer vector stocks.


Assuntos
Lentivirus/fisiologia , Plasmídeos/genética , Proteínas do Envelope Viral/metabolismo , Cultura de Vírus/métodos , Técnicas de Transferência de Genes , Vetores Genéticos , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas do Envelope Viral/genética , Carga Viral , Tropismo Viral , Montagem de Vírus
13.
Virus Res ; 264: 32-39, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30797826

RESUMO

The lifecycle of avian leukosis virus subgroup J (ALV-J), a typical tumorigenic retrovirus, is highly dependent upon host cellular proteins. However, there have been few studies directed at uncovering the host proteins responsible for ALV-J replication, which could provide insights into new strategies for ALV-J prevention and control. Here, we used proteomics to identify the association of differential levels of collagen triple helix-repeat-containing 1 (CTHRC1) and with viral replication. Our results revealed that CTHRC1 was significantly upregulated in ALV-J-infected cells in vitro, and these findings were confirmed in vivo. Additionally, CTHRC1 overexpression facilitated ALV-J replication, whereas CTHRC1 knockdown suppressed this activity. Moreover, we found that ALV-J drove CTHRC1 translocation from the nucleus to the cytosol through interactions with the ALV-J envelope glycoprotein. These results revealed CTHRC1 as a shutting protein is recruited by ALV-J to facilitate viral replication.


Assuntos
Vírus da Leucose Aviária/fisiologia , Proteínas da Matriz Extracelular/genética , Interações entre Hospedeiro e Microrganismos , Proteínas do Envelope Viral/genética , Replicação Viral , Animais , Linhagem Celular , Embrião de Galinha , Galinhas/virologia , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/virologia , Proteômica , Proteínas do Envelope Viral/metabolismo
14.
PLoS Pathog ; 15(2): e1007163, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30730994

RESUMO

Hepatitis C virus (HCV) assembly and envelopment are coordinated by a complex protein interaction network that includes most of the viral structural and nonstructural proteins. While the nonstructural protein 4A (NS4A) is known to be important for viral particle production, the specific function of NS4A in this process is not well understood. We performed mutagenesis of the C-terminal acidic domain of NS4A and found that mutation of several of these amino acids prevented the formation of the viral envelope, and therefore the production of infectious virions, without affecting viral RNA replication. In an overexpression system, we found that NS4A interacted with several viral proteins known to coordinate envelopment, including the viral E1 glycoprotein. One of the NS4A C-terminal mutations, Y45F, disrupted the interaction of NS4A with E1. Specifically, NS4A interacted with the first hydrophobic region of E1, a region previously described as regulating viral particle production. Indeed, we found that an E1 mutation in this region, D72A, also disrupted the interaction of NS4A with E1. Supernatants from HCV NS4A Y45F transfected cells had significantly reduced levels of HCV RNA, however they contained equivalent levels of Core protein. Interestingly, the Core protein secreted from these cells formed high order oligomers with a density matching the infectious virus secreted from wild-type cells. These results suggest that this Y45F mutation in NS4A causes secretion of low-density Core particles lacking genomic HCV RNA. These results corroborate previous findings showing that the E1 D72A mutation also causes secretion of Core complexes lacking genomic HCV RNA, and therefore suggest that the interaction between NS4A and E1 is involved in the incorporation of viral RNA into infectious HCV particles. Our findings define a new role for NS4A in the HCV lifecycle and help elucidate the protein interactions necessary for production of infectious virus.


Assuntos
Proteínas de Transporte/metabolismo , Hepacivirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Linhagem Celular , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C Crônica/virologia , Humanos , Mutação , Domínios Proteicos , RNA Viral , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética , Vírion/metabolismo , Vírion/fisiologia , Montagem de Vírus , Replicação Viral
15.
Nat Commun ; 10(1): 846, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30783086

RESUMO

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.


Assuntos
Vírus de Archaea/química , Proteínas de Fusão de Membrana/química , Proteínas do Envelope Viral/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Tomografia com Microscopia Eletrônica , Halorubrum/virologia , Fusão de Membrana , Proteínas de Fusão de Membrana/genética , Proteínas de Fusão de Membrana/metabolismo , Domínios Proteicos , Dobramento de Proteína , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Vírion/química
16.
Nat Commun ; 10(1): 879, 2019 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-30787296

RESUMO

Orthobunyaviruses (OBVs) form a distinct genus of arthropod-borne bunyaviruses that can cause severe disease upon zoonotic transmission to humans. Antigenic drift or genome segment re-assortment have in the past resulted in new pathogenic OBVs, making them potential candidates for causing emerging zoonoses in the future. Low-resolution electron cryo-tomography studies have shown that OBV particles feature prominent trimeric spikes, but their molecular organization remained unknown. Here we report X-ray crystallography studies of four different OBVs showing that the spikes are formed by an N-terminal extension of the fusion glycoprotein Gc. Using Schmallenberg virus, a recently emerged OBV, we also show that the projecting spike is the major target of the neutralizing antibody response, and provide X-ray structures in complex with two protecting antibodies. We further show that immunization of mice with the spike domains elicits virtually sterilizing immunity, providing fundamental knowledge essential in the preparation for potential newly emerging OBV zoonoses.


Assuntos
Anticorpos Neutralizantes/imunologia , Orthobunyavirus/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismo , Estruturas Virais/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Cercopithecus aethiops , Cricetinae , Cristalografia por Raios X , Feminino , Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Estrutura Terciária de Proteína , Ruminantes/virologia , Células Vero
17.
J Biol Chem ; 294(9): 3249-3260, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30617182

RESUMO

N 6-methyladenosine (m6A) modification of HIV-1 RNA regulates viral replication and protein expression. The m6A modification is regulated by two groups of cellular proteins named writers and erasers that add or remove m6A, respectively. HIV-1 infection of CD4+ T-cells increases m6A levels of cellular mRNA, but the underlying mechanism is unknown. Here, we show that HIV-1 infection of CD4+ primary T-cells or Jurkat cells significantly increases m6A levels of cellular RNA independently of viral replication. Compared with HIV-1-infected CD4+ T-cells, similar m6A up-regulation was detected in total RNA from HIV-1-infected cells treated with a reverse-transcriptase inhibitor or with heat-inactivated HIV-1. Compared with mock controls, significantly increased m6A levels were detected in total RNA from Jurkat cells infected by single-cycle HIV-1 pseudotyped with an HIV-1 envelope (Env) glycoprotein, but not with vesicular stomatitis virus glycoprotein G (VSV-G). Overexpression of HIV-1 Env in HEK293T cells did not affect m6A levels of cellular RNA, suggesting that de novo synthesis of Env is not required for m6A up-regulation. Interestingly, treatment of Jurkat cells with recombinant gp120 of HIV-1 Env significantly increased m6A levels of cellular RNA, which was reduced by a gp120-neutralizing antibody. Preincubation of Jurkat cells with a CD4 receptor-neutralizing antibody blocked HIV-1-induced up-regulation of m6A levels in cellular RNA. Moreover, HIV-1 infection or gp120 treatment did not alter the protein expression of m6A writers and erasers in cells. Our findings suggest that HIV-1 gp120 binding to the CD4 receptor is required for m6A up-regulation in cells.


Assuntos
Adenosina/análogos & derivados , HIV-1/fisiologia , RNA/metabolismo , Regulação para Cima , Proteínas do Envelope Viral/metabolismo , Replicação Viral , Adenosina/metabolismo , Linfócitos T CD4-Positivos/virologia , Células HEK293 , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Células Jurkat
18.
Phytomedicine ; 53: 62-69, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30668413

RESUMO

BACKGROUND: Despite the advent of direct-acting antivirals (DAAs), HCV remains an important public health problem globally. There is at present no effective vaccine against the virus, and the DAAs in current use cannot prevent de novo infection, including in liver transplant setting wherein donor livers inevitably become re-infected. Developing inhibitors to HCV entry using nature-derived small molecules may help to expand/complement the current treatment options. PURPOSE: In this study, we explored the effect of the plant alkaloid berberine (BBR) on HCV early viral entry. METHODS: Cell culture-derived HCV (HCVcc), viral pseudoparticles bearing HCV glycoproteins (HCVpp), and entry-related assays were employed to assess BBR's bioactivity. Molecular docking was used to predict BBR-HCV glycoproteins interaction, and the compound's antiviral activity was confirmed against HCVcc infection of primary human hepatocytes (PHHs). RESULTS: BBR specifically impeded HCVcc attachment and entry/fusion steps without inactivating the free virus particles or affecting the expression of host cell entry factors and post-entry viral replication. BBR also effectively inhibited infection by viral pseudoparticles expressing HCV E1/E2 glycoproteins and molecular docking analysis pointed at potential interaction with HCV E2. Finally, BBR could suppress HCVcc infection of PHHs. CONCLUSIONS: We identified BBR as a potent HCV entry inhibitor, which merits further evaluation particularly for use in transplant setting against graft re-infection by HCV.


Assuntos
Antivirais/farmacologia , Berberina/farmacologia , Hepacivirus/efeitos dos fármacos , Proteínas do Envelope Viral/metabolismo , Antivirais/química , Berberina/química , Células Cultivadas , Hepacivirus/patogenicidade , Hepacivirus/fisiologia , Hepatite C/tratamento farmacológico , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Simulação de Acoplamento Molecular , Terapia de Alvo Molecular , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
DNA Cell Biol ; 38(2): 115-120, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30615471

RESUMO

Ebola virus (EBOV) is an enveloped filamentous virus that causes severe hemorrhagic fever in humans and nonhuman primates with up to 90% fatality. Accumulating evidence indicates that various viruses, including EBOV, exploit the host apoptotic clearance machinery to enhance their entry into host cells by externalizing phosphatidylserine (PS) in the viral envelope. PS is typically distributed in the inner layer of the plasma membrane (PM) in normal cells. Progeny EBOV virions bud from the PM of infected cells, suggesting that PS is likely flipped to the outer leaflet of the envelope of Ebola virions. Currently, the intracellular dynamics of PS during EBOV infection are poorly understood. This review summarizes recent progress in determining the molecular mechanism of externalization of PS in the envelope of EBOV particles. We also discuss future directions and how viral apoptotic mimicry could be targeted for therapeutics.


Assuntos
Ebolavirus/metabolismo , Fosfatidilserinas/metabolismo , Proteínas do Envelope Viral/metabolismo , Animais , Membrana Celular/virologia , Doença pelo Vírus Ebola/virologia , Humanos , Vírion/metabolismo
20.
Methods Mol Biol ; 1911: 33-45, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30593616

RESUMO

For a long time, the study of the HCV infectious cycle has been a major challenge for researchers because of the difficulties in generating an efficient cell culture system leading to a productive viral infection. The development of HCVpp and later on HCVcc model allowing for functional studies of HCV in cell culture completely revolutionized HCV research. The aim of this review is to provide the reader with a brief overview of the development of these two models. We describe the advantages of each model as well as their limitations in the study of the HCV life cycle, with a particular emphasis on virus entry. A comparison between these two models is presented in terms of virion composition and their use as tools for the characterization of entry factors, envelope glycoprotein functions, and antibody neutralization. We also compare the production and biosafety level of these two types of viral particles. Globally, this review provides a general description of the most adequate applications for HCVpp and HCVcc in HCV research.


Assuntos
Técnicas de Cultura de Células/métodos , Hepacivirus/fisiologia , Hepatite C/metabolismo , Proteínas do Envelope Viral/metabolismo , Vírion/fisiologia , Internalização do Vírus , Animais , Anticorpos Neutralizantes/metabolismo , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Hepatite C/virologia , Humanos , Vírion/crescimento & desenvolvimento , Vírion/metabolismo
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