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2.
Cells ; 10(4)2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33917481

RESUMO

Coronavirus belongs to the family of Coronaviridae, comprising single-stranded, positive-sense RNA genome (+ ssRNA) of around 26 to 32 kilobases, and has been known to cause infection to a myriad of mammalian hosts, such as humans, cats, bats, civets, dogs, and camels with varied consequences in terms of death and debilitation. Strikingly, novel coronavirus (2019-nCoV), later renamed as severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), and found to be the causative agent of coronavirus disease-19 (COVID-19), shows 88% of sequence identity with bat-SL-CoVZC45 and bat-SL-CoVZXC21, 79% with SARS-CoV and 50% with MERS-CoV, respectively. Despite key amino acid residual variability, there is an incredible structural similarity between the receptor binding domain (RBD) of spike protein (S) of SARS-CoV-2 and SARS-CoV. During infection, spike protein of SARS-CoV-2 compared to SARS-CoV displays 10-20 times greater affinity for its cognate host cell receptor, angiotensin-converting enzyme 2 (ACE2), leading proteolytic cleavage of S protein by transmembrane protease serine 2 (TMPRSS2). Following cellular entry, the ORF-1a and ORF-1ab, located downstream to 5' end of + ssRNA genome, undergo translation, thereby forming two large polyproteins, pp1a and pp1ab. These polyproteins, following protease-induced cleavage and molecular assembly, form functional viral RNA polymerase, also referred to as replicase. Thereafter, uninterrupted orchestrated replication-transcription molecular events lead to the synthesis of multiple nested sets of subgenomic mRNAs (sgRNAs), which are finally translated to several structural and accessory proteins participating in structure formation and various molecular functions of virus, respectively. These multiple structural proteins assemble and encapsulate genomic RNA (gRNA), resulting in numerous viral progenies, which eventually exit the host cell, and spread infection to rest of the body. In this review, we primarily focus on genomic organization, structural and non-structural protein components, and potential prospective molecular targets for development of therapeutic drugs, convalescent plasm therapy, and a myriad of potential vaccines to tackle SARS-CoV-2 infection.


Assuntos
/terapia , Descoberta de Drogas , Proteínas não Estruturais Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Neutralizantes/uso terapêutico , /metabolismo , Desenho de Fármacos , Humanos , Imunização Passiva , Terapia de Alvo Molecular , /genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
Viruses ; 13(1)2021 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-33466596

RESUMO

Infectious Bursal Disease Virus (IBDV) has haunted the poultry industry with severe, prolonged immunosuppression of chickens when infected at an early age and can easily lead to other secondary infections. Understanding the pathogenic mechanisms could lead to effective prevention and control of Infectious Bursal Disease (IBD). Evidence suggests that the N-terminal domain of polymerase in segment B plays an important role, but it is not clear which part or residual is crucial for the pathogenicity. Using a reverse genetics technique, a molecular clone (rNN1172) of the parental vvIBDV strain NN1172 was generated, and its pathogenicity was found to be the same as the parental virus. Then, three recombinant chimeric viruses were rescued based on the rNN1172 and substituted with the counterparts in the N-terminal domain of the attenuated vaccine strain B87: the rNN1172-B87VP1a (substituting the full region of the 1-167 aa residuals), the rNN1172-B87VP1a∆4 (substituting the region of the 5-167 aa residuals), and the rNN1172-VP1∆4 (one single aa residual substitution V4I), to better explore the role of the N-terminal domain of polymerase on the viral pathogenicity. Interestingly, all these substitutions played different roles in the viral pathogenicity: the mortality of the rNN1172-B87VP1a-challenged chickens was significantly reduced from 30% to 0%. No obvious lesion was found in the histopathological examination, and the lowest viral genome copy number was also detected in the bursa when compared to the parental and two other recombinant viruses. The mortalities caused by rNN1172-B87VP1a∆4 and rNN1172-B87VP1∆4, respectively, were all reduced to 10% and had a delayed onset of death. Our results also revealed that the pathogenicity of the IBDV was consistent with the viral replication efficiency in vivo (bursae). This study demonstrated that the full region of the N-terminal of polymerase plays an important role in viral replication and pathogenicity, but the substitutions of its partial region or a single residual do not completely lead to the virus attenuation to Three-Yellow chickens, although that significantly reduces its pathogenicity.


Assuntos
Infecções por Birnaviridae/veterinária , RNA Polimerases Dirigidas por DNA/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Doenças das Aves Domésticas/virologia , Domínios e Motivos de Interação entre Proteínas , Replicação Viral , Substituição de Aminoácidos , Animais , Células Cultivadas , Embrião de Galinha , Clonagem Molecular , RNA Polimerases Dirigidas por DNA/química , Fibroblastos , Genoma Viral , Vírus da Doença Infecciosa da Bursa/patogenicidade , Mutação , Ligação Proteica , Genética Reversa , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismo , Virulência , Replicação Viral/genética
4.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408172

RESUMO

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.IMPORTANCE hPIV-2 V protein interferes with interaction between FTH1 and NCOA4 and inhibits NCOA4-mediated ferritin degradation, leading to the inhibition of iron release to the cytoplasm. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2.


Assuntos
Homeostase , Ferro/metabolismo , Vírus da Parainfluenza 2 Humana/fisiologia , Proteínas Estruturais Virais/metabolismo , Apoptose , Linhagem Celular , Ferritinas/genética , Ferritinas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Coativadores de Receptor Nuclear/genética , Coativadores de Receptor Nuclear/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Vírus da Parainfluenza 2 Humana/crescimento & desenvolvimento , Vírus da Parainfluenza 2 Humana/metabolismo , Ligação Proteica
5.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33361427

RESUMO

Infectious bursal disease virus (IBDV) is the archetypal member of the family Birnaviridae and the etiological agent of Gumboro disease, a highly contagious immunosuppressive infection of concern to the global poultry sector for its adverse health effects in chicks. Unlike most double-stranded RNA (dsRNA) viruses, which enclose their genomes within specialized cores throughout their viral replication cycle, birnaviruses organize their bisegmented dsRNA genome in ribonucleoprotein (RNP) structures. Recently, we demonstrated that IBDV exploits endosomal membranes for replication. The establishment of IBDV replication machinery on the cytosolic leaflet of endosomal compartments is mediated by the viral protein VP3 and its intrinsic ability to target endosomes. In this study, we identified the early endosomal phosphatidylinositol 3-phosphate [PtdIns(3)P] as a key host factor of VP3 association with endosomal membranes and consequent establishment of IBDV replication complexes in early endosomes. Indeed, our data reveal a crucial role for PtdIns(3)P in IBDV replication. Overall, our findings provide new insights into the replicative strategy of birnaviruses and strongly suggest that it resembles those of positive-strand RNA (+ssRNA) viruses, which replicate in association with host membranes. Furthermore, our findings support the role of birnaviruses as evolutionary intermediaries between +ssRNA and dsRNA viruses and, importantly, demonstrate a novel role for PtdIns(3)P in the replication of a dsRNA virus.IMPORTANCE Infectious bursal disease virus (IBDV) infects chicks and is the causative agent of Gumboro disease. During IBDV outbreaks in recent decades, the emergence of very virulent variants and the lack of effective prevention/treatment strategies to fight this disease have had devastating consequences for the poultry industry. IBDV belongs to the peculiar family Birnaviridae Unlike most dsRNA viruses, birnaviruses organize their genomes in ribonucleoprotein complexes and replicate in a core-independent manner. We recently demonstrated that IBDV exploits host cell endosomes as platforms for viral replication, a process that depends on the VP3 viral protein. In this study, we delved deeper into the molecular characterization of IBDV-endosome association and investigated the role of host cell phosphatidylinositide lipids in VP3 protein localization and IBDV infection. Together, our findings demonstrate that PtdIns(3)P serves as a scaffold for the association of VP3 to endosomes and reveal its essential role for IBDV replication.


Assuntos
Endossomos/metabolismo , Vírus da Doença Infecciosa da Bursa/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , /metabolismo , Animais , Linhagem Celular , Endossomos/virologia , Membranas Intracelulares/metabolismo , Codorniz , Proteínas Estruturais Virais/metabolismo , Replicação Viral
6.
Viruses ; 12(12)2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33339336

RESUMO

Using molecular techniques and bioinformatics tools, we studied the vector-host interactions and the molecular epidemiology of West Nile virus (WNV) in western Iran. Mosquitoes were collected during 2017 and 2018. DNA typing assays were used to study vector-host interactions. Mosquitoes were screened by RT-PCR for the genomes of five virus families. WNV-positive samples were fully sequenced and evolutionary tree and molecular architecture were constructed by Geneious software and SWISS-MODEL workspace, respectively. A total of 5028 mosquito specimens were collected and identified. The most prevalent species was Culex (Cx.) pipiens complex (57.3%). Analysis of the blood-feeding preferences of blood-fed mosquitoes revealed six mammalian and one bird species as hosts. One mosquito pool containing non-blood-fed Cx. theileri and one blood-fed Culex pipiens pipiens (Cpp.) biotype pipiens were positive for WNV. A phylogram indicated that the obtained WNV sequences belonged to lineage 2, subclade 2 g. Several amino acid substitutions suspected as virulence markers were observed in the Iranian WNV strains. The three-dimensional structural homology model of the E-protein identified hot spot domains known to facilitate virus invasion and neurotropism. The recent detection of WNV lineage 2 in mosquitoes from several regions of Iran in consecutive years suggests that the virus is established in the country.


Assuntos
Vetores de Doenças , Interações Hospedeiro-Patógeno , Febre do Nilo Ocidental/transmissão , Febre do Nilo Ocidental/virologia , Vírus do Nilo Ocidental/fisiologia , Sequência de Aminoácidos , Animais , Evolução Molecular , Genoma Viral , Genômica/métodos , Geografia Médica , Humanos , Irã (Geográfico)/epidemiologia , Mosquitos Vetores/virologia , Filogenia , Dinâmica Populacional , Prevalência , Conformação Proteica , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/metabolismo , Virulência , Fatores de Virulência , Sequenciamento Completo do Genoma
7.
Viruses ; 13(1)2020 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-33374251

RESUMO

African swine fever virus (ASFV) is a highly pathogenic large DNA virus that causes African swine fever (ASF) in domestic pigs and wild boars. The p17 protein, encoded by the D117L gene, is a major transmembrane protein of the capsid and the inner lipid envelope. The aim of this study was to investigate the effects of p17 on cell proliferation and the underlying mechanisms of action. The effects of p17 on cell proliferation, cell cycle, apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress have been examined in 293T, PK15, and PAM cells, respectively. The results showed that p17 reduced cell proliferation by causing cell cycle arrest at G2/M phase. Further, p17-induced oxidative stress and increased the level of intracellular reactive oxygen species (ROS). Decreasing the level of ROS partially reversed the cell cycle arrest and prevented the decrease of cell proliferation induced by p17 protein. In addition, p17-induced ER stress, and alleviating ER stress decreased the production of ROS and prevented the decrease of cell proliferation induced by p17. Taken together, this study suggests that p17 can inhibit cell proliferation through ER stress and ROS-mediated cell cycle arrest, which might implicate the involvement of p17 in ASF pathogenesis.


Assuntos
Vírus da Febre Suína Africana/fisiologia , Febre Suína Africana/metabolismo , Febre Suína Africana/virologia , Pontos de Checagem do Ciclo Celular , Estresse do Retículo Endoplasmático , Espécies Reativas de Oxigênio/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Apoptose , Linhagem Celular , Proliferação de Células , Retículo Endoplasmático/metabolismo , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , L-Lactato Desidrogenase/metabolismo , Suínos
8.
PLoS Pathog ; 16(9): e1008850, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956404

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Proteínas Estruturais Virais , Montagem de Vírus , Linhagem Celular Tumoral , Deleção de Genes , Humanos , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
9.
Sheng Wu Gong Cheng Xue Bao ; 36(8): 1536-1545, 2020 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-32924352

RESUMO

Moschus chrysogaster (sifanicus) viral hemorrhagic disease (McVHD) is an acute and highly lethal infectious disease caused by Moschus chrysogaster hemorrhagic disease virus (McHDV) whose genome sequence is highly homologous with rabbit hemorrhagic disease virus. To screen the protective antigen of McHDV and set the basis for study of McVHD vaccine, the antigen epitope of major structural protein VP60 of McHDV was analyzed, and the specific primers were designed to obtain three amplified DNA sequences encoding the main antigen epitope of VP60 from McHDV by using RT-PCR. Then the three DNA fragments were sequenced and cloned to prokaryotic expression vector with pET-28a(+) by using overlap extension PCR, and finally the prokaryotic expression plasmid pET-truncated-VP60 was constructed. Subsequently, the pET-truncated-VP60 was transformed into Escherichia coli BL21(DE3), and the recombinant proteins were expressed by IPTG induction. Finally, the expressed protein was purified and applied to immunize that without immunizing with RHD vaccine, then the antiserum titers were evaluated by the hemagglutination inhibition test, and the immune-protective efficacy of the recombinant proteins was observed and analyzed through animal challenge test. The results showed that the multi-epitope DNA fragments of VP60 of McHDV was successfully expressed in the form of inclusion bodies in E. coli, and the relative molecular weight of recombinant proteins is about 45 kDa. After immunized with the recombinant proteins, 100% of New Zealand white rabbits were resistant to attack of McHDV, which indicates efficient immune-protective efficacy of chosen epitope recombinant protein. The study laid a foundation for the development of the new subunit vaccines of McVHD.


Assuntos
Infecções por Caliciviridae , Expressão Gênica , Vírus da Doença Hemorrágica de Coelhos , Proteínas Estruturais Virais , Animais , Infecções por Caliciviridae/imunologia , Infecções por Caliciviridae/virologia , Epitopos/genética , Escherichia coli/genética , Coelhos , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
10.
J Virol ; 94(18)2020 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-32611749

RESUMO

Us3 proteins of herpes simplex virus 1 (HSV-1) and HSV-2 are multifunctional serine-threonine protein kinases. Here, we identified an HSV-2 tegument protein, UL7, as a novel physiological substrate of HSV-2 Us3. Mutations in HSV-2 UL7, which precluded Us3 phosphorylation of the viral protein, significantly reduced mortality, viral replication in the vagina, and development of vaginal disease in mice following vaginal infection. These results indicated that Us3 phosphorylation of UL7 in HSV-2 was required for efficient viral replication and pathogenicity in vivo Of note, this phosphorylation was conserved in UL7 of chimpanzee herpesvirus (ChHV), which phylogenetically forms a monophyletic group with HSV-2 and the resurrected last common ancestral UL7 for HSV-2 and ChHV. In contrast, the phosphorylation was not conserved in UL7s of HSV-1, which belongs to a sister clade of the monophyletic group, the resurrected last common ancestor for HSV-1, HSV-2, and ChHV, and other members of the genus Simplexvirus that are phylogenetically close to these viruses. Thus, evolution of Us3 phosphorylation of UL7 coincided with the phylogeny of simplex viruses. Furthermore, artificially induced Us3 phosphorylation of UL7 in HSV-1, in contrast to phosphorylation in HSV-2, had no effect on viral replication and pathogenicity in mice. Our results suggest that HSV-2 and ChHV have acquired and maintained Us3 phosphoregulation of UL7 during their evolution because the phosphoregulation had an impact on viral fitness in vivo, whereas most other simplex viruses have not because the phosphorylation was not necessary for efficient fitness of the viruses in vivo IMPORTANCE It has been hypothesized that the evolution of protein phosphoregulation drives phenotypic diversity across species of organisms, which impacts fitness during their evolution. However, there is a lack of information regarding linkage between the evolution of viral phosphoregulation and the phylogeny of virus species. In this study, we clarified the novel HSV-2 Us3 phosphoregulation of UL7 in infected cells, which is important for viral replication and pathogenicity in vivo We also showed that the evolution of Us3 phosphoregulation of UL7 was linked to the phylogeny of viruses that are phylogenetically close to HSV-2 and to the phosphorylation requirements for the efficient in vivo viral fitness of HSV-2 and HSV-1, which are representative of viruses that have and have not evolved phosphoregulation, respectively. This study reports the first evidence showing that evolution of viral phosphoregulation coincides with phylogeny of virus species and supports the hypothesis regarding the evolution of viral phosphoregulation during viral evolution.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Genital/virologia , Herpesvirus Humano 2/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas da Matriz Viral/genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Modelos Animais de Doenças , Evolução Molecular , Feminino , Aptidão Genética , Células HEK293 , Herpes Genital/mortalidade , Herpesvirus Humano 1/classificação , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Herpesvirus Humano 1/patogenicidade , Herpesvirus Humano 2/classificação , Herpesvirus Humano 2/metabolismo , Herpesvirus Humano 2/patogenicidade , Humanos , Camundongos , Fosforilação , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Vagina/virologia , Células Vero , Proteínas da Matriz Viral/metabolismo , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Virulência , Replicação Viral
12.
Sci Rep ; 10(1): 5658, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32221415

RESUMO

Duck plague virus (DPV), a member of the alphaherpesviruses subfamily, causes massive ducks death and results in a devastating hit to duck industries in China. It is of great significance for us to analyze the functions of DPV genes for controlling the outbreak of duck plague. Thus, glycoproteins E (gE) of DPV, which requires viral cell-to-cell spreading and the final envelopment in herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), was chosen herein. The gE mutant virus BAC-CHv-ΔgE was constructed by using a markerless two-step Red recombination system implemented on the DPV genome cloned into a bacterial artificial chromosome (BAC). Viral plaques on duck embryo fibroblast (DEF) cells of BAC-CHv-ΔgE were on average approximately 60% smaller than those produced by BAC-CHv virus. Viral replication kinetics showed that BAC-CHv-ΔgE grew to lower titers than BAC-CHv virus did in DEF cells. Electron microscopy confirmed that deleting of DPV gE resulted in a large number of capsids accumulating around vesicles and very few of them could bud into vesicles. The drastic inhibition of virion formation in the absence of the DPV gE indicated that it played an important role in virion morphogenesis before the final envelopment of intracytoplasmic nucleocapsids.


Assuntos
Alphaherpesvirinae/metabolismo , Capsídeo/metabolismo , Citoplasma/metabolismo , Vesículas Citoplasmáticas/metabolismo , Patos/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion/metabolismo , Animais , Linhagem Celular , Cromossomos Artificiais Bacterianos/metabolismo , Citoplasma/virologia , Vesículas Citoplasmáticas/virologia , Patos/virologia , Glicoproteínas/metabolismo , Herpesvirus Humano 1/metabolismo , Herpesvirus Suídeo 1/metabolismo , Mardivirus/metabolismo , Montagem de Vírus/fisiologia , Replicação Viral/fisiologia
13.
Sci Rep ; 10(1): 1589, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-32005959

RESUMO

Mumps virus is one of the main cause of respiratory illnesses in humans, especially children. Among the viral surface glycoproteins, the hemagglutinin - neuraminidase, MuV-HN, plays key roles in virus entry into host cells and infectivity, thus representing an ideal target for the design of novel inhibitors. Here we report the detailed analysis of the molecular recognition of host cell surface sialylated glycans by the viral glycoprotein MuV-HN. By a combined use of NMR, docking, molecular modelling and CORCEMA-ST, the structural features of sialoglycans/MuV-HN complexes were revealed. Evidence for a different enzyme activity toward longer and complex substrates compared to unbranched ligands was also examined by an accurate NMR kinetic analysis. Our results provide the basis for the structure-based design of effective drugs against mumps-induced diseases.


Assuntos
Hemaglutininas/metabolismo , Vírus da Caxumba/metabolismo , Neuraminidase/metabolismo , Polissacarídeos/metabolismo , Proteínas Estruturais Virais/metabolismo , Sítios de Ligação , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Conformação Proteica
14.
J Gen Virol ; 101(4): 440-452, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32003709

RESUMO

We report the isolation of Australian strains of Bustos virus and Ngewotan virus, two insect-specific viruses in the newly identified taxon Negevirus, originally isolated from Southeast Asian mosquitoes. Consistent with the expected insect-specific tropism of negeviruses, these isolates of Ngewotan and Bustos viruses, alongside the Australian negevirus Castlerea virus, replicated exclusively in mosquito cells but not in vertebrate cells, even when their temperature was reduced to 34 °C. Our data confirmed the existence of two structural proteins, putatively one membrane protein forming the majority of the virus particle, and one glycoprotein forming a projection on the apex of the virions. We generated and characterized 71 monoclonal antibodies to both structural proteins of the two viruses, most of which were neutralizing. Overall, these data increase our knowledge of negevirus mechanisms of infection and replication in vitro.


Assuntos
Anticorpos Monoclonais/imunologia , Culicidae/virologia , Vírus de Insetos/fisiologia , Proteínas Estruturais Virais/imunologia , Vírion/metabolismo , Replicação Viral/genética , Animais , Austrália , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Genoma Viral , Glicoproteínas/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Especificidade de Hospedeiro/fisiologia , Hibridomas/imunologia , Vírus de Insetos/genética , Vírus de Insetos/imunologia , Vírus de Insetos/isolamento & purificação , Proteínas de Membrana/imunologia , Microscopia Eletrônica , Filogenia , Células Vero , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo , Vírion/ultraestrutura
15.
Proc Natl Acad Sci U S A ; 117(4): 2099-2107, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31953264

RESUMO

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Chaperonas Moleculares/química , Vírus da Raiva/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/química , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA/genética , Regulação Viral da Expressão Gênica , Humanos , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Vírus da Raiva/química , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Transcrição Genética , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
16.
Virology ; 539: 80-91, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31706163

RESUMO

To identify potential pathogens responsible for a disease outbreak of cultured peafowls in China in 2013, metagenomic sequencing was conducted. The genomes of two closely related parvoviruses, namely peafowl parvovirus 1 (PePV1) and PePV2, were identified with size of 4428 bp and 4348 bp, respectively. Phylogenetic analysis revealed that both viruses are novel parvoviruses, belonging to the proposed genus Chapparvovirus of Parvoviridae. The transcriptional profile of PePV1 was analyzed by transfecting a nearly complete PePV1 genome into HEK-293T cells. Results revealed that PePV1 employs one promoter and two polyadenylation sites to start and terminate its transcriptions, with one donor site and two acceptor sites for pre-mRNA splicing. PePV1 DNA and structural protein were detected in several tissues of a dead peafowl, which appeared to have suffered enteritis, pneumonia and viremia. These results provide novel information of chapparvoviruses, and call for attention to the potential pathogens.


Assuntos
Doenças das Aves/virologia , Galliformes/virologia , Perfilação da Expressão Gênica , Genoma Viral/genética , Infecções por Parvoviridae/veterinária , Parvovirinae/genética , Processamento Alternativo/genética , Animais , Doenças das Aves/epidemiologia , China/epidemiologia , DNA Viral/genética , DNA Viral/metabolismo , Células HEK293 , Humanos , Metagenômica , Infecções por Parvoviridae/epidemiologia , Infecções por Parvoviridae/virologia , Parvovirinae/classificação , Filogenia , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/metabolismo
17.
Uirusu ; 70(1): 29-36, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33967109

RESUMO

Coronaviruses are pathogens that infect many of animals, resulting in respiratory or enteric diseases. Coronaviruses constitute Nidovirales together with Arteriviridae. Most of human coronaviruses are known to cause mild illness and common cold. However, an epidemic of severe acute respiratory syndrome (SARS) occurred in 2002, ten years after SARS epidemic Middle East respiratory syndrome (MERS) emerged in 2012. Now, we face on a novel coronavirus which emerges in end of 2019. This novel coronavirus is named as SARS-CoV-2. SARS-CoV-2 is spread to worldwide within one to two months and causes coronavirus disease 2019 (COVID-19), respiratory illness. Coronaviruses are enveloped viruses possessing a positive-sense and large single stranded RNA genomes. The 5' two-thirds of the CoV genome consists of two overlapping open reading frames (ORFs 1a and 1b) that encode non-structural proteins (nsps). The other one-third of the genome consists of ORFs encoding structural proteins, including spike (S), membrane (M), envelope (E) and nucleocapsid (N) proteins, and accessory proteins. Upon infection of CoV into host cells, the translation of two precursor polyproteins, pp1a and pp1ab, occurs and these polyproteins are cleaved into 16 nsps by viral proteases. Structural proteins assemble to the vesicles located from ER to Golgi (ER Golgiintermediate compartment) and virions bud into the vesicles. Virions are released from infectedcells via exocytosis.


Assuntos
COVID-19/virologia , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , Animais , Retículo Endoplasmático/metabolismo , Genoma Viral/genética , Complexo de Golgi/metabolismo , Humanos , Fases de Leitura Aberta , Poliproteínas/metabolismo , RNA Viral/genética , Proteases Virais , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Vírion
18.
Sci Rep ; 9(1): 16710, 2019 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-31723221

RESUMO

Nipah virus (NiV) is a pathogenic paramyxovirus and zoononis with very high human fatality rates. Previous protein over-expression studies have shown that various mutations to the common N-terminal STAT1-binding motif of the NiV P, V, and W proteins affected the STAT1-binding ability of these proteins thus interfering with he JAK/STAT pathway and reducing their ability to inhibit type-I IFN signaling, but due to differing techniques it was unclear which amino acids were most important in this interaction or what impact this had on pathogenesis in vivo. We compared all previously described mutations in parallel and found the amino acid mutation Y116E demonstrated the greatest reduction in binding to STAT1 and the greatest reduction in interferon antagonism. A similar reduction in binding and activity was seen for a deletion of twenty amino acids constituting the described STAT1-binding domain. To investigate the contribution of this STAT1-binding motif in NiV-mediated disease, we produced rNiVs with complete deletion of the STAT1-binding motif or the Y116E mutation for ferret challenge studies (rNiVM-STAT1blind). Despite the reduced IFN inhibitory function, ferrets challenged with these rNiVM-STAT1blind mutants had a lethal, albeit altered, NiV-mediated disease course. These data, together with our previously published data, suggest that the major role of NiV P, V, and W in NiV-mediated disease in the ferret model are likely to be in the inhibition of viral recognition/innate immune signaling induction with a minor role for inhibition of IFN signaling.


Assuntos
Infecções por Henipavirus/patologia , Infecções por Henipavirus/virologia , Vírus Nipah/fisiologia , Fosfoproteínas/metabolismo , Fator de Transcrição STAT1/antagonistas & inibidores , Proteínas Virais/metabolismo , Proteínas Estruturais Virais/metabolismo , Animais , Anticorpos Neutralizantes/imunologia , Sítios de Ligação , Modelos Animais de Doenças , Progressão da Doença , Feminino , Furões , Infecções por Henipavirus/metabolismo , Fosfoproteínas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Proteínas Virais/genética , Proteínas Estruturais Virais/genética
19.
Biophys J ; 117(8): 1387-1392, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31585705

RESUMO

Scaffolding proteins (SPs) are required for the capsid shell assembly of many tailed double-stranded DNA bacteriophages, some archaeal viruses, herpesviruses, and adenoviruses. Despite their importance, only one high-resolution structure is available for SPs within procapsids. Here, we use the inherent size limit of NMR to identify mobile segments of the 303-residue phage P22 SP free in solution and when incorporated into a ∼23 MDa procapsid complex. Free SP gives NMR signals from its acidic N-terminus (residues 1-40) and basic C-terminus (residues 264-303), whereas NMR signals from the middle segment (residues 41-263) are missing because of intermediate conformational exchange on the NMR chemical shift timescale. When SP is incorporated into P22 procapsids, NMR signals from the C-terminal helix-turn-helix domain disappear because of binding to the procapsid interior. Signals from the N-terminal domain persist, indicating that this segment retains flexibility when bound to procapsids. The unstructured character of the N-terminus, coupled with its high content of negative charges, is likely important for dissociation and release of SP during the double-stranded DNA genome packaging step accompanying phage maturation.


Assuntos
Bacteriófago P22/química , Capsídeo/química , Dobramento de Proteína , Proteínas Estruturais Virais/química , Bacteriófago P22/metabolismo , Capsídeo/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Domínios Proteicos , Proteínas Estruturais Virais/metabolismo
20.
PLoS Pathog ; 15(10): e1007956, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589653

RESUMO

We report the analysis of a complex enveloped human virus, herpes simplex virus (HSV), assembled after in vivo incorporation of bio-orthogonal methionine analogues homopropargylglycine (HPG) or azidohomoalanine (AHA). We optimised protocols for the production of virions incorporating AHA (termed HSVAHA), identifying conditions which resulted in normal yields of HSV and normal particle/pfu ratios. Moreover we show that essentially every single HSVAHA capsid-containing particle was detectable at the individual particle level by chemical ligation of azide-linked fluorochromes to AHA-containing structural proteins. This was a completely specific chemical ligation, with no capsids assembled under normal methionine-containing conditions detected in parallel. We demonstrate by quantitative mass spectrometric analysis that HSVAHA virions exhibit no qualitative or quantitative differences in the repertoires of structural proteins compared to virions assembled under normal conditions. Individual proteins and AHA incorporation sites were identified in capsid, tegument and envelope compartments, including major essential structural proteins. Finally we reveal novel aspects of entry pathways using HSVAHA and chemical fluorochrome ligation that were not apparent from conventional immunofluorescence. Since ligation targets total AHA-containing protein and peptides, our results demonstrate the presence of abundant AHA-labelled products in cytoplasmic macrodomains and tubules which no longer contain intact particles detectable by immunofluorescence. Although these do not co-localise with lysosomal markers, we propose they may represent sites of proteolytic virion processing. Analysis of HSVAHA also enabled the discrimination from primary entering from secondary assembling virions, demonstrating assembly and second round infection within 6 hrs of initial infection and dual infections of primary and secondary virus in spatially restricted cytoplasmic areas of the same cell. Together with other demonstrated applications e.g., in genome biology, lipid and protein trafficking, this work further exemplifies the utility and potential of bio-orthogonal chemistry for studies in many aspects of virus-host interactions.


Assuntos
Aminoácidos/metabolismo , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Epitélio Pigmentado da Retina/virologia , Proteínas Estruturais Virais/metabolismo , Montagem de Vírus , Internalização do Vírus , Proliferação de Células , Células Cultivadas , Herpes Simples/metabolismo , Humanos , Epitélio Pigmentado da Retina/metabolismo
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