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1.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33397718

RESUMO

Macrodomains are proteins that recognize and hydrolyze ADP ribose (ADPR) modifications of intracellular proteins. Macrodomains are implicated in viral genome replication and interference with host cell immune responses. They are important to the infectious cycle of Coronaviridae and Togaviridae viruses. We describe crystal structures of the conserved macrodomain from the bat coronavirus (CoV) HKU4 in complex with ligands. The structures reveal a binding cavity that accommodates ADPR and analogs via local structural changes within the pocket. Using a radioactive assay, we present evidence of mono-ADPR (MAR) hydrolase activity. In silico analysis presents further evidence on recognition of the ADPR modification for hydrolysis. Mutational analysis of residues within the binding pocket resulted in diminished enzymatic activity and binding affinity. We conclude that the common structural features observed in the macrodomain in a bat CoV contribute to a conserved function that can be extended to other known macrodomains.


Assuntos
Adenosina Difosfato Ribose/química , Coronavirus/enzimologia , Pirofosfatases/química , Proteínas não Estruturais Virais/química , Animais , Sítios de Ligação , Quirópteros , Coronavirus/genética , Cristalografia por Raios X , Hidrólise , Pirofosfatases/genética , Proteínas não Estruturais Virais/genética
2.
Sci Rep ; 10(1): 22387, 2020 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-33372181

RESUMO

In the genome of SARS-CoV-2, the 5'-terminus encodes a polyprotein, which is further cleaved into 15 non-structural proteins whereas the 3' terminus encodes four structural proteins and eight accessory proteins. Among these 27 proteins, the present study aimed to discover likely antigenic proteins and epitopes to be used for the development of a vaccine or serodiagnostic assay using an in silico approach. For this purpose, after the full genome analysis of SARS-CoV-2 Wuhan isolate and variant proteins that are detected frequently, surface proteins including spike, envelope, and membrane proteins as well as proteins with signal peptide were determined as probable vaccine candidates whereas the remaining were considered as possible antigens to be used during the development of serodiagnostic assays. According to results obtained, among 27 proteins, 26 of them were predicted as probable antigen. In 26 proteins, spike protein was selected as the best vaccine candidate because of having a signal peptide, negative GRAVY value, one transmembrane helix, moderate aliphatic index, a big molecular weight, a long-estimated half-life, beta wrap motifs as well as having stable, soluble and non-allergic features. In addition, orf7a, orf8, and nsp-10 proteins with signal peptide were considered as potential vaccine candidates. Nucleocapsid protein and a highly antigenic GGDGKMKD epitope were identified as ideal antigens to be used in the development of serodiagnostic assays. Moreover, considering MHC-I alleles, highly antigenic KLNDLCFTNV and ITLCFTLKRK epitopes can be used to develop an epitope-based peptide vaccine.


Assuntos
/imunologia , /genética , Glicoproteína da Espícula de Coronavírus/imunologia , /diagnóstico , Simulação por Computador , /imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Genoma Viral/genética , Humanos , Simulação de Acoplamento Molecular , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
3.
ACS Infect Dis ; 6(12): 3174-3189, 2020 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-33263384

RESUMO

Human coronaviruses (hCoVs) have become a threat to global health and society, as evident from the SARS outbreak in 2002 caused by SARS-CoV-1 and the most recent COVID-19 pandemic caused by SARS-CoV-2. Despite a high sequence similarity between SARS-CoV-1 and -2, each strain has a distinctive virulence. A better understanding of the basic molecular mechanisms mediating changes in virulence is needed. Here, we profile the virus-host protein-protein interactions of two hCoV nonstructural proteins (nsps) that are critical for virus replication. We use tandem mass tag-multiplexed quantitative proteomics to sensitively compare and contrast the interactomes of nsp2 and nsp4 from three betacoronavirus strains: SARS-CoV-1, SARS-CoV-2, and hCoV-OC43-an endemic strain associated with the common cold. This approach enables the identification of both unique and shared host cell protein binding partners and the ability to further compare the enrichment of common interactions across homologues from related strains. We identify common nsp2 interactors involved in endoplasmic reticulum (ER) Ca2+ signaling and mitochondria biogenesis. We also identify nsp4 interactors unique to each strain, such as E3 ubiquitin ligase complexes for SARS-CoV-1 and ER homeostasis factors for SARS-CoV-2. Common nsp4 interactors include N-linked glycosylation machinery, unfolded protein response associated proteins, and antiviral innate immune signaling factors. Both nsp2 and nsp4 interactors are strongly enriched in proteins localized at mitochondria-associated ER membranes suggesting a new functional role for modulating host processes, such as calcium homeostasis, at these organelle contact sites. Our results shed light on the role these hCoV proteins play in the infection cycle, as well as host factors that may mediate the divergent pathogenesis of OC43 from SARS strains. Our mass spectrometry workflow enables rapid and robust comparisons of multiple bait proteins, which can be applied to additional viral proteins. Furthermore, the identified common interactions may present new targets for exploration by host-directed antiviral therapeutics.


Assuntos
/metabolismo , Interações Hospedeiro-Patógeno/genética , Proteínas não Estruturais Virais/metabolismo , /virologia , Coronavirus Humano OC43/patogenicidade , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas/genética , Vírus da SARS/patogenicidade , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Transfecção , Proteínas não Estruturais Virais/genética , Virulência/genética , Replicação Viral/genética
4.
Med Hypotheses ; 144: 110296, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33254487

RESUMO

The factors that may contribute to a COVID-19 patient remaining in the asymptomatic stage, or to the infection evolving into the more serious stages are examined. In particular, we refer to the TMPRSS2 expression profile, balance of androgen and estrogen, blood group-A and/or B, nonsynonymous mutations in ORF3, and proteins NS7b and NS8 in SARS-CoV-2. Also, we review other factors related to the susceptibility and pathogenicity of SARS-CoV-2.


Assuntos
Infecções Assintomáticas , Predisposição Genética para Doença , Serina Endopeptidases/genética , Alelos , Androgênios/metabolismo , /virologia , Exoma , Feminino , Perfilação da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Masculino , Modelos Teóricos , Mutação , Fases de Leitura Aberta , Polimorfismo de Nucleotídeo Único , Proteínas não Estruturais Virais/genética , Vitamina D/análogos & derivados , Vitamina D/metabolismo
5.
Mol Cell ; 80(6): 1055-1066.e6, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33188728

RESUMO

The causative virus of the COVID-19 pandemic, SARS-CoV-2, uses its nonstructural protein 1 (Nsp1) to suppress cellular, but not viral, protein synthesis through yet unknown mechanisms. We show here that among all viral proteins, Nsp1 has the largest impact on host viability in the cells of human lung origin. Differential expression analysis of mRNA-seq data revealed that Nsp1 broadly alters the cellular transcriptome. Our cryo-EM structure of the Nsp1-40S ribosome complex shows that Nsp1 inhibits translation by plugging the mRNA entry channel of the 40S. We also determined the structure of the 48S preinitiation complex formed by Nsp1, 40S, and the cricket paralysis virus internal ribosome entry site (IRES) RNA, which shows that it is nonfunctional because of the incorrect position of the mRNA 3' region. Our results elucidate the mechanism of host translation inhibition by SARS-CoV-2 and advance understanding of the impacts from a major pathogenicity factor of SARS-CoV-2.


Assuntos
/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , /patogenicidade , Proteínas não Estruturais Virais/metabolismo , Animais , /patologia , Chlorocebus aethiops , Microscopia Crioeletrônica , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Subunidades Ribossômicas Menores de Eucariotos/virologia , /ultraestrutura , Células Vero , Proteínas não Estruturais Virais/genética
6.
Open Biol ; 10(11): 200237, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33202171

RESUMO

Viral macrodomains possess the ability to counteract host ADP-ribosylation, a post-translational modification implicated in the creation of an antiviral environment via immune response regulation. This brought them into focus as promising therapeutic targets, albeit the close homology to some of the human macrodomains raised concerns regarding potential cross-reactivity and adverse effects for the host. Here, we evaluate the structure and function of the macrodomain of SARS-CoV-2, the causative agent of COVID-19. We show that it can antagonize ADP-ribosylation by PARP14, a cellular (ADP-ribosyl)transferase necessary for the restriction of coronaviral infections. Furthermore, our structural studies together with ligand modelling revealed the structural basis for poly(ADP-ribose) binding and hydrolysis, an emerging new aspect of viral macrodomain biology. These new insights were used in an extensive evolutionary analysis aimed at evaluating the druggability of viral macrodomains not only from the Coronaviridae but also Togaviridae and Iridoviridae genera (causing diseases such as Chikungunya and infectious spleen and kidney necrosis virus disease, respectively). We found that they contain conserved features, distinct from their human counterparts, which may be exploited during drug design.


Assuntos
ADP-Ribosilação , Simulação de Acoplamento Molecular , Poli(ADP-Ribose) Polimerases/química , Proteínas não Estruturais Virais/química , Adenosina Difosfato Ribose/química , Adenosina Difosfato Ribose/metabolismo , Sítios de Ligação , Evolução Molecular , Humanos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica , Domínios Proteicos , /metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
7.
Sheng Wu Gong Cheng Xue Bao ; 36(10): 2206-2215, 2020 Oct 25.
Artigo em Chinês | MEDLINE | ID: mdl-33169584

RESUMO

Dengue virus (DENV) is the most widely transmitted arbovirus in the world. Due to the lack of diagnostic technology to quickly identify the virus serotypes in patients, severe dengue hemorrhagic fever cases caused by repeated infections remain high. To realize the rapid differential diagnosis of different serotypes of DENV infection by immunological methods, in this study, four DENV serotype NS1 proteins were expressed and purified in mammalian cells. Monoclonal antibodies (MAbs) against NS1 protein were obtained by hybridoma technology after immunizing BALB/c mice. Enzyme-linked immunosorbent assay, indirect immunofluorescence assay, dot blotting, and Western blotting were used to confirm the reactivity of MAbs to viral native NS1 and recombinant NS1 protein. These MAbs include not only the universal antibodies that recognize all DENV 1-4 serotype NS1, but also serotype-specific antibodies against DENV-1, DENV-2 and DENV-4. Double antibody sandwich ELISA was established based on these antibodies, which can be used to achieve rapid differential diagnosis of serotypes of DENV infection. Preparation of DENV serotype-specific MAbs and establishment of an ELISA technology for identifying DENV serotypes has laid the foundation for the rapid diagnosis of DENV clinical infection.


Assuntos
Anticorpos Antivirais , Vírus da Dengue , Dengue , Animais , Anticorpos Monoclonais , Anticorpos Antivirais/classificação , Anticorpos Antivirais/genética , Anticorpos Antivirais/metabolismo , Dengue/diagnóstico , Vírus da Dengue/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Sorogrupo , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia
8.
PLoS One ; 15(11): e0241592, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180795

RESUMO

Superinfection exclusion (SIE) is a process by which a virally infected cell is protected from subsequent infection by the same or a closely related virus. By preventing cell coinfection, SIE favors preservation of genome integrity of a viral strain and limits its recombination potential with other viral genomes, thereby impacting viral evolution. Although described in virtually all viral families, the precise step(s) impacted by SIE during the viral life cycle have not been systematically explored. Here, we describe for the first time SIE triggered by chikungunya virus (CHIKV), an alphavirus of public health importance. Using single-cell technologies, we demonstrate that CHIKV excludes subsequent infection with: CHIKV; Sindbis virus, a related alphavirus; and influenza A, an unrelated RNA virus. We further demonstrate that SIE does not depend on the action of type I interferon, nor does it rely on host cell transcription. Moreover, exclusion is not mediated by the action of a single CHIKV protein; in particular, we observed no role for non-structural protein 2 (nsP2), making CHIKV unique among characterized alphaviruses. By stepping through the viral life cycle, we show that CHIKV exclusion occurs at the level of replication, but does not directly influence virus binding, nor viral structural protein translation. In sum, we characterized co-infection during CHIKV replication, which likely influences the rate of viral diversification and evolution.


Assuntos
Febre de Chikungunya/virologia , Vírus Chikungunya/fisiologia , Superinfecção/virologia , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Células Cultivadas , Vírus Chikungunya/genética , Vírus Chikungunya/patogenicidade , Chlorocebus aethiops , Genoma Viral , Vírus da Influenza A/patogenicidade , Camundongos , Vírus Sindbis/patogenicidade , Células Vero , Proteínas não Estruturais Virais/genética
9.
Proc Natl Acad Sci U S A ; 117(49): 31519-31526, 2020 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-33203681

RESUMO

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.


Assuntos
Epistasia Genética/genética , Genes Virais/genética , /genética , /patologia , /genética , Exorribonucleases/genética , Genoma Viral/genética , Humanos , Metiltransferases/genética , RNA Helicases/genética , Seleção Genética/genética , Proteínas não Estruturais Virais/genética , Proteínas Virais/genética , /genética
10.
Nat Commun ; 11(1): 5877, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208735

RESUMO

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen that causes the disease COVID-19, produces replicase polyproteins 1a and 1ab that contain, respectively, 11 or 16 nonstructural proteins (nsp). Nsp5 is the main protease (Mpro) responsible for cleavage at eleven positions along these polyproteins, including at its own N- and C-terminal boundaries, representing essential processing events for subsequent viral assembly and maturation. We have determined X-ray crystallographic structures of this cysteine protease in its wild-type free active site state at 1.8 Å resolution, in its acyl-enzyme intermediate state with the native C-terminal autocleavage sequence at 1.95 Å resolution and in its product bound state at 2.0 Å resolution by employing an active site mutation (C145A). We characterize the stereochemical features of the acyl-enzyme intermediate including critical hydrogen bonding distances underlying catalysis in the Cys/His dyad and oxyanion hole. We also identify a highly ordered water molecule in a position compatible for a role as the deacylating nucleophile in the catalytic mechanism and characterize the binding groove conformational changes and dimerization interface that occur upon formation of the acyl-enzyme. Collectively, these crystallographic snapshots provide valuable mechanistic and structural insights for future antiviral therapeutic development including revised molecular docking strategies based on Mpro inhibition.


Assuntos
Betacoronavirus/enzimologia , Cisteína Endopeptidases/química , Proteínas não Estruturais Virais/química , Betacoronavirus/química , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Dimerização , Humanos , Modelos Moleculares , Mutação , Inibidores de Proteases/metabolismo , Conformação Proteica , Especificidade por Substrato , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
11.
Nat Commun ; 11(1): 5874, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33208736

RESUMO

Non-structural proteins (nsp) constitute the SARS-CoV-2 replication and transcription complex (RTC) to play a pivotal role in the virus life cycle. Here we determine the atomic structure of a SARS-CoV-2 mini RTC, assembled by viral RNA-dependent RNA polymerase (RdRp, nsp12) with a template-primer RNA, nsp7 and nsp8, and two helicase molecules (nsp13-1 and nsp13-2), by cryo-electron microscopy. Two groups of mini RTCs with different conformations of nsp13-1 are identified. In both of them, nsp13-1 stabilizes overall architecture of the mini RTC by contacting with nsp13-2, which anchors the 5'-extension of RNA template, as well as interacting with nsp7-nsp8-nsp12-RNA. Orientation shifts of nsp13-1 results in its variable interactions with other components in two forms of mini RTC. The mutations on nsp13-1:nsp12 and nsp13-1:nsp13-2 interfaces prohibit the enhancement of helicase activity achieved by mini RTCs. These results provide an insight into how helicase couples with polymerase to facilitate its function in virus replication and transcription.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Replicação Viral , Betacoronavirus/genética , Betacoronavirus/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Metiltransferases/química , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , RNA Helicases/química , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Viral/metabolismo , Relação Estrutura-Atividade , Transcrição Genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
12.
Sheng Wu Gong Cheng Xue Bao ; 36(11): 2357-2366, 2020 Nov 25.
Artigo em Chinês | MEDLINE | ID: mdl-33244930

RESUMO

Antigenic purity is important for quality control of the foot-and-mouth (FMD) whole virus inactivated vaccine. The recommended method for evaluation the antigenic purity of FMD vaccine is to check the serum conversion to non-structural protein (NSP) 3AB antibody after 2 to 3 times inoculation of animals with inactivated vaccine. In this study, we developed a quantitative ELISA to detect the amount of residual 3AB in vaccine antigen, to provide a reference to evaluate the antigenic purity of FMD vaccine. Monoclonal antibody (Mab) of NSP 3A and HRP-conjugated Mab of NSP 3B were used to establish a sandwich ELISA to quantify the NSP 3AB in vaccine antigen of FMD. Purified NSP 3AB expressed in Escherichia coli was serially diluted and detected to draw the standard curve. The detectable limit was determined to be the lowest concentration of standard where the ratio of its OD value to OD blank well was not less than 2.0. Results: The OD value was linearly corelated with the concentration of 3AB protein within the range between 4.7 and 600 ng/mL. The correlation coefficient R² is greater than 0.99, and the lowest detectable limit is 4.7 ng/mL. The amount of 3AB protein in non-purified inactivated virus antigen was detected between 9.3 and 200 ng/mL depending on the 12 different virus strains, whereas the amount of 3AB in purified virus antigen was below the lowest detectable limit. The amount of 3AB in 9 batches of commercial FMD vaccine antigens was between 9.0 and 74 ng/mL, whereas it was below the detectable limit in other 24 batches of commercial vaccine antigens. Conclusion: the sandwich ELISA established in this study is specific and sensitive to detect the content of 3AB protein in vaccine antigen of FMD, which will be a useful method for evaluation of the antigenic purity and quality control of FMD inactivated vaccine.


Assuntos
Vírus da Febre Aftosa , Febre Aftosa , Vacinas Virais , Animais , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Febre Aftosa/prevenção & controle , Proteínas não Estruturais Virais/genética
13.
Indian J Med Microbiol ; 38(3 & 4): 451-456, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33154262

RESUMO

In the current COVID-19 crisis, many national healthcare systems are confronted with a huge demand for mass testing and an acute shortage of diagnostic resources. Considering group testing as a viable solution, this pilot study was carried out to find the maximum number of samples that can be pooled together to accurately detect one positive sample carrying the severe acute respiratory syndrome-coronavirus 2 viral RNA from different pools. We made different pool sizes ranging from 5 to 30 samples. Three positive samples, covering the common range of polymerase chain reaction (PCR) threshold cycle values (an indirect indicator of viral load) observed in our patients, were selected, and different pools were made with known negative samples. The pools underwent real-time qualitative PCR for the determination of effective maximum pool size. It was observed that up to 20-sample pools of all positive samples could accurately be detected in terms of both E gene and RdRp gene, leading to considerable conservation of resources, time and workforce. However, while deciding the optimal pool size, the infection level in that particular geographical area and sensitivity of the test assay used (limit of detection) have to be taken into account.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Programas de Rastreamento/métodos , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Testes Diagnósticos de Rotina/métodos , Humanos , Índia , Pandemias , RNA Viral/genética , Manejo de Espécimes , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
14.
Vaccine ; 38(48): 7612-7628, 2020 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-33082015

RESUMO

SARS-CoV-2 causes a severe respiratory disease called COVID-19. Currently, global health is facing its devastating outbreak. However, there is no vaccine available against this virus up to now. In this study, a novel multi-epitope vaccine against SARS-CoV-2 was designed to provoke both innate and adaptive immune responses. The immunodominant regions of six non-structural proteins (nsp7, nsp8, nsp9, nsp10, nsp12 and nsp14) of SARS-CoV-2 were selected by multiple immunoinformatic tools to provoke T cell immune response. Also, immunodominant fragment of the functional region of SARS-CoV-2 spike (400-510 residues) protein was selected for inducing neutralizing antibodies production. The selected regions' sequences were connected to each other by furin-sensitive linker (RVRR). Moreover, the functional region of ß-defensin as a well-known agonist for the TLR-4/MD complex was added at the N-terminus of the vaccine using (EAAAK)3 linker. Also, a CD4 + T-helper epitope, PADRE, was used at the C-terminal of the vaccine by GPGPG and A(EAAAK)2A linkers to form the final vaccine construct. The physicochemical properties, allergenicity, antigenicity, functionality and population coverage of the final vaccine construct were analyzed. The final vaccine construct was an immunogenic, non-allergen and unfunctional protein which contained multiple CD8 + and CD4 + overlapping epitopes, IFN-γ inducing epitopes, linear and conformational B cell epitopes. It could form stable and significant interactions with TLR-4/MD according to molecular docking and dynamics simulations. Global population coverage of the vaccine for HLA-I and II were estimated 96.2% and 97.1%, respectively. At last, the final vaccine construct was reverse translated to design the DNA vaccine. Although the designed vaccine exhibited high efficacy in silico, further experimental validation is necessary.


Assuntos
Anticorpos Antivirais/biossíntese , Betacoronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Proteínas não Estruturais Virais/imunologia , Vacinas Virais/biossíntese , Sequência de Aminoácidos , Betacoronavirus/patogenicidade , Biologia Computacional , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Epitopos de Linfócito B/química , Epitopos de Linfócito B/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Inata/efeitos dos fármacos , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Pneumonia Viral/epidemiologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Ligação Proteica , Estrutura Secundária de Proteína , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Vacinas Atenuadas , Vacinas de DNA , Vacinas de Subunidades , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Vacinas Virais/genética , Vacinas Virais/metabolismo
15.
Proc Natl Acad Sci U S A ; 117(44): 27598-27607, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33060297

RESUMO

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.


Assuntos
Enterovirus/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Proteínas de Membrana/metabolismo , Replicação Viral/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Resfriado Comum/imunologia , Resfriado Comum/virologia , Enterovirus/genética , Enterovirus/imunologia , Enterovirus/metabolismo , Células HeLa , Humanos , Imunidade Inata , Fator Regulador 3 de Interferon/metabolismo , Lipoilação , Proteínas de Membrana/agonistas , Mutação , Domínios Proteicos/genética , Transdução de Sinais , Especificidade da Espécie , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo
16.
PLoS Pathog ; 16(10): e1008951, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33052957

RESUMO

Both mosquito species-specific differences and virus strain -specific differences impact vector competence. Previous results in our laboratory with individual populations of N. American mosquitoes support studies suggesting Aedes aegypti are more competent than Ae. albopictus for American Zika virus (ZIKV) strains and demonstrate that U.S. Ae. albopictus have higher competence for an ancestral Asian ZIKV strain. A982V, an amino acid substitution in the NS1 gene acquired prior to the American outbreak, has been shown to increase competence in Ae. aegypti. We hypothesized that variability in the NS1 could therefore contribute to species-specific differences and developed a reverse genetics system based on a 2016 ZIKV isolate from Honduras (ZIKV-WTic) to evaluate the phenotypic correlates of individual amino acid substitutions. In addition to A982V, we evaluated G894A, which was acquired during circulation in the Americas. Reversion of 982 and 894 to ancestral residues increased infectivity, transmissibility and viral loads in Ae. albopictus but had no effect on competence or replication in Ae. aegypti. In addition, while host cell-specific differences in NS1 secretion were measured, with significantly higher secretion in mammalian cells relative to mosquito cells, strain-specific differences in secretion were not detected, despite previous reports. These results demonstrate that individual mutations in NS1 can influence competence in a species-specific manner independent of differences in NS1 secretion and further indicate that ancestral NS1 residues confer increased competence in Ae. albopictus. Lastly, experimental infections of Ifnar1-/- mice demonstrated that these NS1 substitutions can influence viral replication in the host and, specifically, that G894A could represent a compensatory change following a fitness loss from A982V with some viral genetic backgrounds. Together these data suggest a possible role for epistatic interactions in ZIKV fitness in invertebrate and vertebrate hosts and demonstrate that strains with increased transmission potential in U.S. Ae. albopictus could emerge.


Assuntos
Aedes/virologia , Interações Hospedeiro-Patógeno , Mosquitos Vetores/virologia , Carga Viral , Proteínas não Estruturais Virais/genética , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia , Animais , Chlorocebus aethiops , Feminino , Camundongos , Camundongos Knockout , Mutação , Receptor de Interferon alfa e beta/fisiologia , Células Vero , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Zika virus/classificação , Zika virus/genética
17.
Vet Microbiol ; 251: 108878, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33069035

RESUMO

Canine chaphamaparvovirus (CaChPV) is a newly recognised parvovirus discovered by metagenomic analysis during an outbreak of diarrhoea in dogs in Colorado, USA, in 2017 and more recently detected in diarrhoeic dogs in China. Whether the virus plays a role as canine pathogen and whether it is distributed elsewhere, in other geographical areas, is not known. We performed a case-control study to investigate the possible association of CaChPV with enteritis in dogs. CaChPV DNA was detected both in the stools of diarrhoeic dogs (1.9 %, 3/155) and of healthy animals (1.6 %, 2/120). All the CaChPV-infected dogs with diarrhea were mixed infected with other enteric viruses such as canine parvovirus (formerly CPV-2), canine bufavirus (CBuV) and canine coronavirus (CCoV), whilst none of the asymptomatic CaChPV positive animals resulted co-infected. The nearly full-length genome and the partial capsid protein (VP) gene of three canine strains, Te/36OVUD/19/ITA, Te/37OVUD/19/ITA and Te/70OVUD/19/ITA, were reconstructed. Upon phylogenetic analyses based on the NS1 and VP aa sequences, the Italian CaChPV strains tightly clustered with the American reference viruses. Distinctive residues could be mapped to the deduced variable regions of the VP of canine and feline chaphamaparvoviruses, considered as important markers of host range and pathogenicity for parvoviruses.


Assuntos
Diarreia/veterinária , Doenças do Cão/virologia , Genoma Viral , Infecções por Parvoviridae/veterinária , Parvovirus Canino/classificação , Animais , Proteínas do Capsídeo/genética , Estudos de Casos e Controles , Diarreia/virologia , Cães/virologia , Fezes/virologia , Especificidade de Hospedeiro , Itália , Infecções por Parvoviridae/diagnóstico , Infecções por Parvoviridae/virologia , Parvovirus Canino/isolamento & purificação , Animais de Estimação/virologia , Filogenia , Proteínas não Estruturais Virais/genética
18.
PLoS One ; 15(10): e0227239, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33064724

RESUMO

Species of the genus Flavivirus are widespread in Brazil and are a major public health concern. The country's largest city, São Paulo, is in a highly urbanized area with a few forest fragments which are commonly used for recreation. These can be considered to present a potential risk of flavivirus transmission to humans as they are home simultaneously to vertebrate hosts and mosquitoes that are potential flavivirus vectors. The aim of this study was to conduct flavivirus surveillance in field-collected mosquitoes in the Capivari-Monos Environmental Protection Area (EPA) and identify the flavivirus species by sequence analysis in flavivirus IFA-positive pools. Monthly mosquito collections were carried out from March 2016 to April 2017 with CO2-baited CDC light traps. Specimens were identified morphologically and grouped in pools of up to 10 individuals according to their taxonomic category. A total of 260 pools of non-engorged females were inoculated into C6/36 cell culture, and the cell suspensions were analyzed by indirect immunofluorescence assay (IFA) after the incubation period. IFA-positive pools were tested by qRT-PCR with genus-specific primers targeting the flavivirus NS5 gene to confirm IFA-positive results and sequenced to identify the species. Anopheles cruzii (19.5%) and Wyeomyia confusa (15.3%) were the most frequent vector species collected. IFA was positive for flaviviruses in 2.3% (6/260) of the sample pools. This was confirmed by qRT-PCR in five pools (83.3%). All five flavivirus-positive pools were successfully sequenced and the species identified. DENV serotype 2 (DENV-2) was detected in Culex spp. and Culex vaxus pools, while ZIKV was identified in An. cruzii, Limatus durhamii and Wy. confusa pools. To the best of our knowledge, detection of flavivirus species of medical importance has never previously been reported in these species of wild-caught mosquitoes. The finding of DENV-2 and ZIKV circulating in wild mosquitoes suggests the existence of an enzootic cycle in the area. In-depth studies of DENV-2 and ZIKV, including investigation of mosquito infection, vector competence and infection in sylvatic hosts, are needed to shed light on the transmission dynamics of these important viruses and the potential risk of future outbreaks of DENV-2 and ZIKV infections in the region.


Assuntos
Vírus da Dengue/isolamento & purificação , Mosquitos Vetores/virologia , Proteínas não Estruturais Virais/genética , Zika virus/isolamento & purificação , Animais , Anopheles/virologia , Brasil/epidemiologia , Culex/virologia , Vírus da Dengue/genética , Feminino , Vigilância da População , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Meio Selvagem , Zika virus/genética
19.
Nat Commun ; 11(1): 5278, 2020 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-33077712

RESUMO

There are no licensed therapeutics or vaccines available against Zika virus (ZIKV) to counteract its potential for congenital disease. Antibody-based countermeasures targeting the ZIKV envelope protein have been hampered by concerns for cross-reactive responses that induce antibody-dependent enhancement (ADE) of heterologous flavivirus infection. Nonstructural protein 1 (NS1) is a membrane-associated and secreted glycoprotein that functions in flavivirus replication and immune evasion but is absent from the virion. Although some studies suggest that antibodies against ZIKV NS1 are protective, their activity during congenital infection is unknown. Here we develop mouse and human anti-NS1 monoclonal antibodies that protect against ZIKV in both non-pregnant and pregnant mice. Avidity of antibody binding to cell-surface NS1 along with Fc effector functions engagement correlate with protection in vivo. Protective mAbs map to exposed epitopes in the wing domain and loop face of the ß-platform. Anti-NS1 antibodies provide an alternative strategy for protection against congenital ZIKV infection without causing ADE.


Assuntos
Anticorpos Antivirais/administração & dosagem , Complicações Infecciosas na Gravidez/prevenção & controle , Proteínas não Estruturais Virais/imunologia , Infecção por Zika virus/prevenção & controle , Zika virus/imunologia , Animais , Anticorpos Antivirais/imunologia , Afinidade de Anticorpos , Anticorpos Facilitadores , Reações Cruzadas , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/virologia , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Zika virus/química , Zika virus/genética , Infecção por Zika virus/congênito , Infecção por Zika virus/imunologia , Infecção por Zika virus/virologia
20.
Signal Transduct Target Ther ; 5(1): 221, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33024073
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