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1.
Emerg Microbes Infect ; 10(1): 1881-1889, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34490832

RESUMO

SARS-CoV-2 has been the causative pathogen of the pandemic of COVID-19, resulting in catastrophic health issues globally. It is important to develop human-like animal models for investigating the mechanisms that SARS-CoV-2 uses to infect humans and cause COVID-19. Several studies demonstrated that the non-human primate (NHP) is permissive for SARS-CoV-2 infection to cause typical clinical symptoms including fever, cough, breathing difficulty, and other diagnostic abnormalities such as immunopathogenesis and hyperplastic lesions in the lung. These NHP models have been used for investigating the potential infection route and host immune response to SARS-CoV-2, as well as testing vaccines and drugs. This review aims to summarize the benefits and caveats of NHP models currently available for SARS-CoV-2, and to discuss key topics including model optimization, extended application, and clinical translation.


Assuntos
COVID-19/virologia , Modelos Animais de Doenças , Primatas/virologia , SARS-CoV-2/fisiologia , Animais , Antivirais/administração & dosagem , COVID-19/tratamento farmacológico , COVID-19/imunologia , COVID-19/patologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/imunologia , Humanos , Primatas/imunologia , SARS-CoV-2/genética
5.
Signal Transduct Target Ther ; 6(1): 136, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790236

RESUMO

Epidemiological studies of the COVID-19 patients have suggested the male bias in outcomes of lung illness. To experimentally demonstrate the epidemiological results, we performed animal studies to infect male and female Syrian hamsters with SARS-CoV-2. Remarkably, high viral titer in nasal washings was detectable in male hamsters who presented symptoms of weight loss, weakness, piloerection, hunched back and abdominal respiration, as well as severe pneumonia, pulmonary edema, consolidation, and fibrosis. In contrast with the males, the female hamsters showed much lower shedding viral titers, moderate symptoms, and relatively mild lung pathogenesis. The obvious differences in the susceptibility to SARS-CoV-2 and severity of lung pathogenesis between male and female hamsters provided experimental evidence that SARS-CoV-2 infection and the severity of COVID-19 are associated with gender.


Assuntos
COVID-19 , SARS-CoV-2/metabolismo , Caracteres Sexuais , Animais , COVID-19/metabolismo , COVID-19/patologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Masculino , Mesocricetus
7.
Brief Bioinform ; 22(2): 1239-1253, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33006605

RESUMO

The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the binding to the permissive cells. The receptor-binding domain (RBD) of SARS-CoV-2 S protein directly interacts with the human angiotensin-converting enzyme 2 (ACE2) on the host cell membrane. In this study, we used computational saturation mutagenesis approaches, including structure-based energy calculations and sequence-based pathogenicity predictions, to quantify the systemic effects of missense mutations on SARS-CoV-2 S protein structure and function. A total of 18 354 mutations in S protein were analyzed, and we discovered that most of these mutations could destabilize the entire S protein and its RBD. Specifically, residues G431 and S514 in SARS-CoV-2 RBD are important for S protein stability. We analyzed 384 experimentally verified S missense variations and revealed that the dominant pandemic form, D614G, can stabilize the entire S protein. Moreover, many mutations in N-linked glycosylation sites can increase the stability of the S protein. In addition, we investigated 3705 mutations in SARS-CoV-2 RBD and 11 324 mutations in human ACE2 and found that SARS-CoV-2 neighbor residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. The findings comprehensively provide potential target sites in the development of drugs and vaccines against COVID-19.


Assuntos
Mutação de Sentido Incorreto , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/metabolismo , COVID-19/virologia , Humanos , Ligação Proteica , SARS-CoV-2/metabolismo , Termodinâmica
8.
bioRxiv ; 2020 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-33330860

RESUMO

Circular RNAs (circRNAs) encoded by DNA genomes have been identified across host and pathogen species as parts of the transcriptome. Accumulating evidences indicate that circRNAs play critical roles in autoimmune diseases and viral pathogenesis. Here we report that RNA viruses of the Betacoronavirus genus of Coronaviridae , SARS-CoV-2, SARS-CoV and MERS-CoV, encode a novel type of circRNAs. Through de novo circRNA analyses of publicly available coronavirus-infection related deep RNA-Sequencing data, we identified 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and characterized two major back-splice events shared by these viruses. Coronavirus-derived circRNAs are more abundant and longer compared to host genome-derived circRNAs. Using a systematic strategy to amplify and identify back-splice junction sequences, we experimentally identified over 100 viral circRNAs from SARS-CoV-2 infected Vero E6 cells. This collection of circRNAs provided the first line of evidence for the abundance and diversity of coronavirus-derived circRNAs and suggested possible mechanisms driving circRNA biogenesis from RNA genomes. Our findings highlight circRNAs as an important component of the coronavirus transcriptome. Summary: We report for the first time that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNAs that differ from circRNAs encoded by DNA genomes.

9.
Front Mol Biosci ; 7: 591873, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33363207

RESUMO

The ongoing outbreak of COVID-19 has been a serious threat to human health worldwide. The virus SARS-CoV-2 initiates its infection to the human body via the interaction of its spike (S) protein with the human Angiotensin-Converting Enzyme 2 (ACE2) of the host cells. Therefore, understanding the fundamental mechanisms of how SARS-CoV-2 S protein receptor binding domain (RBD) binds to ACE2 is highly demanded for developing treatments for COVID-19. Here we implemented multi-scale computational approaches to study the binding mechanisms of human ACE2 and S proteins of both SARS-CoV and SARS-CoV-2. Electrostatic features, including electrostatic potential, electric field lines, and electrostatic forces of SARS-CoV and SARS-CoV-2 were calculated and compared in detail. The results demonstrate that SARS-CoV and SARS-CoV-2 S proteins are both attractive to ACE2 by electrostatic forces even at different distances. However, the residues contributing to the electrostatic features are quite different due to the mutations between SARS-CoV S protein and SARS-CoV-2 S protein. Such differences are analyzed comprehensively. Compared to SARS-CoV, the SARS-CoV-2 binds with ACE2 using a more robust strategy: The electric field line related residues are distributed quite differently, which results in a more robust binding strategy of SARS-CoV-2. Also, SARS-CoV-2 has a higher electric field line density than that of SARS-CoV, which indicates stronger interaction between SARS-CoV-2 and ACE2, compared to that of SARS-CoV. Key residues involved in salt bridges and hydrogen bonds are identified in this study, which may help the future drug design against COVID-19.

11.
Autophagy ; 16(12): 2238-2251, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32924767

RESUMO

KPNA2/importin-alpha1 (karyopherin subunit alpha 2) is the primary nucleocytoplasmic transporter for some transcription factors to activate cellular proliferation and differentiation. Aberrant increase of KPNA2 level is identified as a prognostic marker in a variety of cancers. Yet, the turnover mechanism of KPNA2 remains unknown. Here, we demonstrate that KPNA2 is degraded via the chaperone-mediated autophagy (CMA) and that Zika virus (ZIKV) enhances the KPNA2 degradation. KPNA2 contains a CMA motif, which possesses an indispensable residue Gln109 for the CMA-mediated degradation. RNAi-mediated knockdown of LAMP2A, a vital component of the CMA pathway, led to a higher level of KPNA2. Moreover, ZIKV reduced KPNA2 via the viral NS2A protein, which contains an essential residue Thr100 for inducing the CMA-mediated KPNA2 degradation. Notably, mutant ZIKV with T100A alteration in NS2A replicates much weaker than the wild-type virus. Also, knockdown of KPNA2 led to a higher ZIKV viral yield, which indicates that KPNA2 mediates certain antiviral effects. These data provide insights into the KPNA2 turnover and the ZIKV-cell interactions.


Assuntos
Autofagia Mediada por Chaperonas , Proteólise , Proteínas não Estruturais Virais/metabolismo , Zika virus/metabolismo , alfa Carioferinas/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular Tumoral , Chlorocebus aethiops , Glutamina/genética , Células HEK293 , Meia-Vida , Humanos , Lisossomos/metabolismo , Mutação/genética , Relação Estrutura-Atividade , Treonina/metabolismo , Células Vero , Proteínas não Estruturais Virais/química , Replicação Viral , Zika virus/fisiologia , Infecção por Zika virus/virologia , alfa Carioferinas/química
12.
Viruses ; 12(9)2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32872150

RESUMO

The M112-113 gene is the first early gene of the murine cytomegalovirus (MCMV), and its expression is activated by the immediate-early 3 (IE3) protein during MCMV infection in permissive cells. At its 5' terminus, a 10-bp motif, upstream of the TATA box of the M112-113 gene, was identified to bind to IE3, and it is necessary for IE3 to activate M112-113 gene expression (Perez KJ et al. 2013 JVI). At the 3' terminus of the M112-113 gene, three poly(A) signals (PASs) are arranged closely, forming a PAS cluster. We asked whether it is necessary to have the PAS cluster for the M112-113 gene and wondered which PAS is required or important for M112-113 gene expression. In this study, we mutated one, two, or all three PASs in expressing plasmids. Then, we applied bacterial artificial chromosome (BAC) techniques to mutate PASs in viruses. Gene expression and viral replication were analyzed. We found that not all three PASs are needed for M112-113 gene expression. Moreover, we revealed that just one of the three poly(A)s is enough for MCMV replication. However, the deletion of all three PASs did not kill MCMV, although it significantly attenuated viral replication. Finally, an mRNA stability assay was performed and demonstrated that PASs are important to stabilize M112-113 mRNA. Therefore, we conclude that just one of the PASs of the M112-113 gene is sufficient and important for MCMV replication through the stabilization of M112-113 mRNA.


Assuntos
Infecções por Herpesviridae/veterinária , Muromegalovirus/genética , Poli A/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Doenças dos Roedores/virologia , Proteínas Virais/genética , Animais , Regulação Viral da Expressão Gênica , Infecções por Herpesviridae/virologia , Camundongos , Muromegalovirus/química , Muromegalovirus/fisiologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral
13.
Comput Struct Biotechnol J ; 18: 2100-2106, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32832038

RESUMO

ACE2 plays a critical role in SARS-CoV-2 infection to cause COVID-19 and SARS-CoV-2 spike protein binds to ACE2 and probably functionally inhibits ACE2 to aggravate the underlying diseases of COVID-19. The important factors that affect the severity and fatality of COVID-19 include patients' underlying diseases and ages. Therefore, particular care to the patients with underlying diseases is needed during the treatment of COVID-19 patients.

14.
Emerg Microbes Infect ; 9(1): 949-961, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32378471

RESUMO

The emergences of coronaviruses have caused a serious global public health problem because their infection in humans caused the severe acute respiratory disease and deaths. The outbreaks of lethal coronaviruses have taken place for three times within recent two decades (SARS-CoV in 2002, MERS-CoV in 2012 and SARS-CoV-2 in 2019). Much more serious than SARS-CoV in 2002, the current SARS-CoV-2 infection has been spreading to more than 213 countries, areas or territories and causing more than two million cases up to date (17 April 2020). Unfortunately, no vaccine and specific anti-coronavirus drugs are available at present time. Current clinical treatment at hand is inadequate to suppress viral replication and inflammation, and reverse organ failure. Intensive research efforts have focused on increasing our understanding of viral biology of SARS-CoV-2, improving antiviral therapy and vaccination strategies. The animal models are important for both the fundamental research and drug discovery of coronavirus. This review aims to summarize the animal models currently available for SARS-CoV and MERS-CoV, and their potential use for the study of SARS-CoV-2. We will discuss the benefits and caveats of these animal models and present critical findings that might guide the fundamental studies and urgent treatment of SARS-CoV-2-caused diseases.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronaviridae/patologia , Infecções por Coronaviridae/prevenção & controle , Infecções por Coronavirus/patologia , Infecções por Coronavirus/prevenção & controle , Modelos Animais de Doenças , Pandemias/prevenção & controle , Pneumonia Viral/patologia , Pneumonia Viral/prevenção & controle , Pesquisa/tendências , Animais , COVID-19 , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Vírus da SARS/fisiologia , SARS-CoV-2
16.
ACS Infect Dis ; 6(5): 811-819, 2020 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31840495

RESUMO

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been associated with neuropathology in fetuses and adults, imposing a serious health concern. Therefore, the development of a vaccine is a global health priority. Notably, neutralization tests have a significant value for vaccine development and virus diagnosis. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) is a common neutralization method for ZIKV. However, this method has some drawbacks, such as being time-consuming and labor-intensive and having low-throughput, which precludes its application in the detection of large numbers of specimens. To improve this problem, we developed a neutralization test based on an enzyme-linked immunospot assay (Nt-ELISPOT) for ZIKV and performed the assay in a 96-well format. A monoclonal antibody (mAb), 11C11, with high affinity and reactivity to ZIKV was used to detect ZIKV-infected cells. To optimize this method, the infectious dose of ZIKV was set at a multiplicity of infection (MOI) of 0.0625, and a detection experiment was performed after incubating for 24 h. As a result, under these conditions, the Nt-ELISPOT had good consistency with the traditional Nt-CPE to measure neutralizing titers of sera and neutralizing antibodies. Additionally, three neutralizing antibodies against ZIKV were screened by this method. Overall, we successfully developed an efficient neutralization test for ZIKV that is high-throughput and rapid. This Nt-ELISPOT can potentially be applied to detecting neutralizing titers of large numbers of specimens in vaccine evaluation and neutralizing antibody screening for ZIKV.


Assuntos
Anticorpos Antivirais , ELISPOT , Testes de Neutralização , Infecção por Zika virus , Zika virus , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Zika virus/imunologia , Infecção por Zika virus/diagnóstico
17.
J Biol Chem ; 294(49): 18742-18755, 2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31666336

RESUMO

The centrosome is a cytoplasmic nonenveloped organelle functioning as one of the microtubule-organizing centers and composing a centriole center surrounded by pericentriolar material (PCM) granules. PCM consists of many centrosomal proteins, including PCM1 and centrosomal protein 131 (CEP131), and helps maintain centrosome stability. Zika virus (ZIKV) is a flavivirus of the family Flaviviridae whose RNA and viral particles are replicated in the cytoplasm. However, how ZIKV interacts with host cell components during its productive infection stage is incompletely understood. Here, using several primate cell lines, we report that ZIKV infection disrupts and disperses the PCM granules. We demonstrate that PCM1- and CEP131-containing granules are dispersed in ZIKV-infected cells, whereas the centrioles remain intact. We found that ZIKV does not significantly alter cellular skeletal proteins, and, hence, these proteins may not be involved in the interaction between ZIKV and centrosomal proteins. Moreover, ZIKV infection decreased PCM1 and CEP131 protein, but not mRNA, levels. We further found that the protease inhibitor MG132 prevents the decrease in PCM1 and CEP131 levels and centriolar satellite dispersion. Therefore, we hypothesized that ZIKV infection induces proteasomal PCM1 and CEP131 degradation and thereby disrupts the PCM granules. Supporting this hypothesis, we show that ZIKV infection increases levels of mind bomb 1 (MIB1), previously demonstrated to be an E3 ubiquitin ligase for PCM1 and CEP131 and that ZIKV fails to degrade or disperse PCM in MIB1-ko cells. Our results imply that ZIKV infection activates MIB1-mediated ubiquitination that degrades PCM1 and CEP131, leading to PCM granule dispersion.


Assuntos
Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Infecção por Zika virus/metabolismo , Animais , Autoantígenos/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células HEK293 , Humanos , Immunoblotting , Ubiquitina-Proteína Ligases/genética , Células Vero , Zika virus , Infecção por Zika virus/genética
18.
Int J Mol Sci ; 20(18)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31510028

RESUMO

Vaccination has had great success in combating diseases, especially infectious diseases. However, traditional vaccination strategies are ineffective for several life-threatening diseases, including acquired immunodeficiency syndrome (AIDS), tuberculosis, malaria, and cancer. Viral vaccine vectors represent a promising strategy because they can efficiently deliver foreign genes and enhance antigen presentation in vivo. However, several limitations, including pre-existing immunity and packaging capacity, block the application of viral vectors. Cytomegalovirus (CMV) has been demonstrated as a new type of viral vector with additional advantages. CMV could systematically elicit and maintain high frequencies of effector memory T cells through the "memory inflation" mechanism. Studies have shown that CMV can be genetically modified to induce distinct patterns of CD8+ T-cell responses, while some unconventional CD8+ T-cell responses are rarely induced through conventional vaccine strategies. CMV has been used as a vaccine vector to deliver many disease-specific antigens, and the efficacy of these vaccines was tested in different animal models. Promising results demonstrated that the robust and unconventional T-cell responses elicited by the CMV-based vaccine vector are essential to control these diseases. These accumulated data and evidence strongly suggest that a CMV-based vaccine vector represents a promising approach to develop novel prophylactic and therapeutic vaccines against some epidemic pathogens and tumors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Citomegalovirus/imunologia , Vetores Genéticos/imunologia , Neoplasias/imunologia , Tuberculose/imunologia , Vacinas Virais/imunologia , Animais , Citomegalovirus/genética , Vetores Genéticos/genética , Humanos , Memória Imunológica/imunologia , Neoplasias/prevenção & controle , Tuberculose/prevenção & controle , Vacinação/métodos , Vacinas Virais/administração & dosagem
19.
Biomed Res Int ; 2019: 6870815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360721

RESUMO

[This corrects the article DOI: 10.1155/2011/124595.].

20.
Theranostics ; 9(7): 2115-2128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31037160

RESUMO

Rationale: Hepatocyte-like cells (HLCs) derived from human induced pluripotent stem cells (hiPSCs) have been developed to address the shortage of primary human hepatocytes (PHHs) for therapeutic applications. However, the in vivo repopulation capacity of HLCs remains limited. This study investigated the roles of agonist antibody activating the c-Met receptor in promoting the in vivo proliferation and repopulation of engrafted PHHs and/or HLCs in mice with liver injuries due to different causes. Methods: An agonist c-Met receptor antibody (5D5) was used to treat PHHs and hiPSC-HLCs in both cell culture and hepatocyte-engrafted immunodeficient mice mimicking various inherited and acquired liver diseases. The promoting roles and potential influence on the hepatic phenotype of the 5D5 regimen in cell transplantation-based therapeutic applications were systematically evaluated. Results: In hiPSC-HLC cell cultures, 5D5 treatment significantly stimulated c-Met receptor downstream signalling pathways and accelerated cell proliferation in dose-dependent and reversible manners. In contrast, only slight but nonsignificant promotion was observed in 5D5-treated PHHs. In vivo administration of 5D5 greatly promoted the expansion of implanted hiPSC-HLCs in fumarylacetoacetate hydrolase (Fah) deficient mice, resulting in significantly increased human albumin levels and high human liver chimerism (over 40%) in the transplanted mice at week 8 after transplantation. More importantly, transplantation of hiPSC-HLCs in combination with 5D5 significantly prolonged animal survival and ameliorated liver pathological changes in mice with acute and/or chronic liver injuries caused by Fas agonistic antibody treatment, carbon tetrachloride treatment and/or tyrosinemic stress. Conclusion: Our results demonstrated that the proliferation of hiPSC-HLCs can be enhanced by antibody-mediated modulation of c-Met signalling and facilitate hiPSC-HLC-based therapeutic applications for life-threatening liver diseases.


Assuntos
Anticorpos Monoclonais/farmacologia , Proliferação de Células/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Falência Hepática/tratamento farmacológico , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Transplante de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Modelos Animais de Doenças , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Falência Hepática/metabolismo , Camundongos
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