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1.
Microb Pathog ; 146: 104241, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32387389

RESUMO

The recent epidemic outbreak of a novel human coronavirus called SARS-CoV-2 and causing the respiratory tract disease COVID-19 has reached worldwide resonance and a global effort is being undertaken to characterize the molecular features and evolutionary origins of this virus. Therefore, rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. Additionally, general treatments, coronavirus-specific treatments, and antiviral treatments useful in fighting COVID-19 are addressed. This review sets out to shed light on the SARS-CoV-2 and host receptor recognition, a crucial factor for successful virus infection and taking immune-informatics approaches to identify B- and T-cell epitopes for surface glycoprotein of SARS-CoV-2. A variety of improved or new approaches also have been developed. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coronavirus infection. Moreover, the genomic sequence of the virus responsible for COVID-19, as well as the experimentally determined three-dimensional structure of the Main protease (Mpro) is available. The reported structure of the target Mpro was described in this review to identify potential drugs for COVID-19 using virtual high throughput screening.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/patologia , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/patologia , Receptores Virais/metabolismo , Antivirais/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/tratamento farmacológico , Cisteína Endopeptidases/metabolismo , Epitopos de Linfócito T/imunologia , Humanos , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/tratamento farmacológico , Conformação Proteica , Proteínas não Estruturais Virais/metabolismo
2.
Protein J ; 39(3): 198-216, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32447571

RESUMO

The devastating effects of the recent global pandemic (termed COVID-19 for "coronavirus disease 2019") caused by the severe acute respiratory syndrome coronavirus-2 (SARS CoV-2) are paramount with new cases and deaths growing at an exponential rate. In order to provide a better understanding of SARS CoV-2, this article will review the proteins found in the SARS CoV-2 that caused this global pandemic.


Assuntos
Betacoronavirus/química , Betacoronavirus/fisiologia , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Descoberta de Drogas/métodos , Genoma Viral , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Alinhamento de Sequência , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Proteínas da Matriz Viral/química , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
3.
Eur Rev Med Pharmacol Sci ; 24(4): 2006-2011, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32141569

RESUMO

The World Health Organization (WHO) has issued a warning that, although the 2019 novel coronavirus (COVID-19) from Wuhan City (China), is not pandemic, it should be contained to prevent the global spread. The COVID-19 virus was known earlier as 2019-nCoV. As of 12 February 2020, WHO reported 45,171 cases and 1115 deaths related to COVID-19. COVID-19 is similar to Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) virus in its pathogenicity, clinical spectrum, and epidemiology. Comparison of the genome sequences of COVID-19, SARS-CoV, and Middle East Respiratory Syndrome coronavirus (MERS-CoV) showed that COVID-19 has a better sequence identity with SARS-CoV compared to MERS CoV. However, the amino acid sequence of COVID-19 differs from other coronaviruses specifically in the regions of 1ab polyprotein and surface glycoprotein or S-protein. Although several animals have been speculated to be a reservoir for COVID-19, no animal reservoir has been already confirmed. COVID-19 causes COVID-19 disease that has similar symptoms as SARS-CoV. Studies suggest that the human receptor for COVID-19 may be angiotensin-converting enzyme 2 (ACE2) receptor similar to that of SARS-CoV. The nucleocapsid (N) protein of COVID-19 has nearly 90% amino acid sequence identity with SARS-CoV. The N protein antibodies of SARS-CoV may cross react with COVID-19 but may not provide cross-immunity. In a similar fashion to SARS-CoV, the N protein of COVID-19 may play an important role in suppressing the RNA interference (RNAi) to overcome the host defense. This mini-review aims at investigating the most recent trend of COVID-19.


Assuntos
Betacoronavirus , Infecções por Coronavirus , Pneumonia Viral , Animais , Betacoronavirus/química , Betacoronavirus/genética , Betacoronavirus/metabolismo , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Genoma Viral , Humanos , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Ativação Viral , Organização Mundial da Saúde
4.
Vet Microbiol ; 239: 108498, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31767072

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) has caused huge economic losses to the pig industry worldwide over the last 30 years, yet the associated viral-host interactions remain poorly understood. S100A9 is a damage-associated molecular pattern of the S100 protein family. Here, we found that PRRSV infection stimulated S100A9 expression in porcine alveolar macrophages (PAMs) and Marc-145 cells. S100A9 inhibited PRRSV replication via cellular Ca2+ dependent manner. The viral nucleocapsid (N) protein co-localized with S100A9 in the cytoplasm, and directly interacted at amino acid 78 of S100A9 and amino acids 36-37 of N protein. Moreover, we also found that the mutant S100A9 (E78Q) protein exhibited decreased antiviral activity against PRRSV compared with the parent S100A9. Recombinant PRRSV rBB (36/37) with two mutations in amino acid 36-37 in the N protein exhibited greater replication than the parent PRRSV BB0907 in S100A9-overexpressed PAM and Marc-145 cells. Thus, S100A9 may restrict PRRSV proliferation by interacting with the viral N protein.


Assuntos
Calgranulina B/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica , Mutação , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Síndrome Respiratória e Reprodutiva Suína/fisiopatologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Proteínas Recombinantes/metabolismo , Suínos , Replicação Viral/fisiologia
5.
Emerg Microbes Infect ; 8(1): 1347-1360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516086

RESUMO

The polymerase complex of Ebola virus (EBOV) is the functional unit for transcription and replication of viral genome. Nucleoprotein (NP) is a multifunctional protein with high RNA binding affinity and recruits other viral proteins to form functional polymerase complex. In our study, we investigated host proteins associated with EBOV polymerase complex using NP as bait in a transcription and replication competent minigenome system by mass spectrometry analysis and identified SET and MYND domain-containing protein 3 (SMYD3) as a novel host protein which was required for the replication of EBOV. SMYD3 specifically interacted with NP and was recruited to EBOV inclusion bodies through NP. The depletion of SMYD3 dramatically suppressed EBOV mRNA production. A mimic of non-phosphorylated VP30, which is a transcription activator, could partially rescue the viral mRNA production downregulated by the depletion of SMYD3. In addition, SMYD3 promoted NP-VP30 interaction in a dose-dependent manner. These results revealed that SMYD3 was a novel host factor recruited by NP to supporting EBOV mRNA transcription through increasing the binding of VP30 to NP. Thus, our study provided a new understanding of mechanism underlying the transcription of EBOV genome, and a novel anti-EBOV drug design strategy by targeting SMYD3.


Assuntos
Ebolavirus/fisiologia , Histona-Lisina N-Metiltransferase/metabolismo , Interações Hospedeiro-Patógeno , Proteínas do Nucleocapsídeo/metabolismo , RNA Mensageiro/biossíntese , RNA Viral/biossíntese , Transcrição Genética , Células HEK293 , Humanos , Espectrometria de Massas , Ligação Proteica , Mapeamento de Interação de Proteínas
6.
Virus Genes ; 55(5): 660-672, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31375995

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) infection causes one of the most economically important swine diseases worldwide. Tripartite motif-containing 22 (TRIM22), a TRIM family protein, has been identified as a crucial restriction factor that inhibits a group of human viruses. Currently, the role of cellular TRIM22 in PRRSV infection remains unclear. In the present study, we analyzed the effect of TRIM22 on PRRSV replication in vitro and explored the underlying mechanism. Ectopic expression of TRIM22 impaired the viral replication, while TRIM22-RNAi favored the replication of PRRSV in MARC-145 cells. Additionally, we observed that TRIM22 deletion SPRY domain or Nuclear localization signal (NLS) losses the ability to inhibit PRRSV replication. Finally, Co-IP analysis identified that TRIM22 interacts with PRRSV nucleocapsid (N) protein through the SPRY domain, while the NLS2 motif of N protein is involved in interaction with TRIM22. Although the concentration of PRRSV N protein was not altered in the presence of TRIM22, the abundance of N proteins from simian hemorrhagic fever virus (SHFV), equine arteritis virus (EAV), and murine lactate dehydrogenase-elevating virus (LDV) diminished considerably with increasing TRIM22 expression. Together, our findings uncover a previously unrecognized role for TRIM22 and extend the antiviral effects of TRIM22 to arteriviruses.


Assuntos
Interações Hospedeiro-Patógeno , Sinais de Localização Nuclear , Vírus da Síndrome Respiratória e Reprodutiva Suína/crescimento & desenvolvimento , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Replicação Viral , Animais , Linhagem Celular , Chlorocebus aethiops , Inativação Gênica , Proteínas do Nucleocapsídeo/metabolismo , Mapeamento de Interação de Proteínas , Deleção de Sequência
7.
Virol Sin ; 34(6): 712-721, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31292829

RESUMO

Our previous study has shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) p48 (ac103) gene is essential for the nuclear egress of nucleocapsids and the formation of occlusion-derived virions (ODVs). However, the exact role of p48 in the morphogenesis of ODVs remains unknown. In this study, we demonstrated that p48 was required for the efficient formation of intranuclear microvesicles. To further understand its functional role in intranuclear microvesicle formation, we characterized the distribution of the P48 protein, which was found to be associated with the nucleocapsid and envelope fractions of both budded virions and ODVs. In AcMNPV-infected cells, P48 was predominantly localized to nucleocapsids in the virogenic stroma and the nucleocapsids enveloped in ODVs, with a limited but discernible distribution in the plasma membrane, nuclear envelope, intranuclear microvesicles, and ODV envelope. Furthermore, coimmunoprecipitation assays showed that among the viral proteins required for intranuclear microvesicle formation, P48 associated with Ac93 in the absence of viral infection.


Assuntos
Núcleo Celular/virologia , Genes Virais , Proteínas do Nucleocapsídeo/metabolismo , Nucleopoliedrovírus/fisiologia , Animais , Membrana Celular/metabolismo , Núcleo Celular/ultraestrutura , Técnicas de Inativação de Genes , Larva/virologia , Mariposas/virologia , Membrana Nuclear/metabolismo , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/ultraestrutura , Corpos de Oclusão Virais/metabolismo , Ligação Proteica , Células Sf9 , Vírion/metabolismo
8.
J Gen Virol ; 100(8): 1208-1221, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31268416

RESUMO

The family Hantaviridae mostly comprises rodent-borne segmented negative-sense RNA viruses, many of which are capable of causing devastating disease in humans. In contrast, hantavirus infection of rodent hosts results in a persistent and inapparent infection through their ability to evade immune detection and inhibit apoptosis. In this study, we used Tula hantavirus (TULV) to investigate the interplay between viral and host apoptotic responses during early, peak and persistent phases of virus infection in cell culture. Examination of early-phase TULV infection revealed that infected cells were refractory to apoptosis, as evidenced by the complete lack of cleaved caspase-3 (casp-3C) staining, whereas in non-infected bystander cells casp-3C was highly abundant. Interestingly, at later time points, casp-3C was abundant in infected cells, but the cells remained viable and able to continue shedding infectious virus, and together these observations were suggestive of a TULV-associated apoptotic block. To investigate this block, we viewed TULV-infected cells using laser scanning confocal and wide-field deconvolution microscopy, which revealed that TULV nucleocapsid protein (NP) colocalized with, and sequestered, casp-3C within cytoplasmic ultrastructures. Consistent with casp-3C colocalization, we showed for the first time that TULV NP was cleaved in cells and that TULV NP and casp-3C could be co-immunoprecipitated, suggesting that this interaction was stable and thus unlikely to be solely confined to NP binding as a substrate to the casp-3C active site. To account for these findings, we propose a novel mechanism by which TULV NP inhibits apoptosis by spatially sequestering casp-3C from its downstream apoptotic targets within the cytosol.


Assuntos
Apoptose , Caspase 3/metabolismo , Infecções por Hantavirus/enzimologia , Hantavirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Animais , Caspase 3/genética , Citosol/enzimologia , Citosol/virologia , Hantavirus/genética , Infecções por Hantavirus/genética , Infecções por Hantavirus/fisiopatologia , Infecções por Hantavirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Proteínas do Nucleocapsídeo/genética , Ligação Proteica
9.
Virol Sin ; 34(6): 701-711, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31317397

RESUMO

Baculoviridae is a family of large DNA viruses that specifically infect insects. It contains four genera, Alpha-, Beta-, Gamma-, and Deltabaculovirus. Alphabaculovirus is further divided into Group I and II, and Group I appears to be emerged most recently among all baculoviruses. Interestingly, there are 12 Group I specific genes that are only found in this lineage. Studying these genes is helpful to understand how baculoviruses evolved. Here, we reported the functional analyzing results of ac73, a function unknown Group I specific gene of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) which is the type species of baculovirus. The AC73 protein encoded by ac73 was found to be expressed during the late stage of infection and incorporated into the nucleocapsids of budded virus (BV) and occlusion-derived virus (ODV). In infected cells, AC73 resided mainly in the ring zone region of the nucleus, and appeared to be assembled into occlusion bodies (OBs). The ac73 knockout and repaired viruses were constructed and studied by in vitro and in vivo infection. Although ac73 was not essential for BV and ODV or OB formation, the BV titer and viral infectivity in insect larvae of ac73 knockout AcMNPV decreased by about 5-8 and 3-4 fold compared to those of wild type virus, respectively, suggesting ac73 contributed to infectious BV production and viral infectivity in vivo. This research provides new insight into the function of this Group I specific gene.


Assuntos
Genes Virais , Proteínas do Nucleocapsídeo/metabolismo , Nucleopoliedrovírus/fisiologia , Animais , Núcleo Celular/metabolismo , Técnicas de Inativação de Genes , Larva/virologia , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/patogenicidade , Nucleopoliedrovírus/ultraestrutura , Corpos de Oclusão Virais/metabolismo , Células Sf9 , Spodoptera/virologia , Transcrição Genética , Replicação Viral
10.
Nat Commun ; 10(1): 2453, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31165735

RESUMO

RNA chaperones are proteins that aid in the folding of nucleic acids, but remarkably, many of these proteins are intrinsically disordered. How can these proteins function without a well-defined three-dimensional structure? Here, we address this question by studying the hepatitis C virus core protein, a chaperone that promotes viral genome dimerization. Using single-molecule fluorescence spectroscopy, we find that this positively charged disordered protein facilitates the formation of compact nucleic acid conformations by acting as a flexible macromolecular counterion that locally screens repulsive electrostatic interactions with an efficiency equivalent to molar salt concentrations. The resulting compaction can bias unfolded nucleic acids towards folding, resulting in faster folding kinetics. This potentially widespread mechanism is supported by molecular simulations that rationalize the experimental findings by describing the chaperone as an unstructured polyelectrolyte.


Assuntos
Hepacivirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Dobramento de RNA , RNA Viral/metabolismo , Proteínas do Core Viral/metabolismo , Dimerização , Genoma Viral , Chaperonas Moleculares/metabolismo , Ácidos Nucleicos/metabolismo , Imagem Individual de Molécula , Espectrometria de Fluorescência , Eletricidade Estática
11.
Arch Biochem Biophys ; 671: 77-86, 2019 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-31229488

RESUMO

Virus from the Mononegavirales order share common features ranging from virion structure arrangement to mechanisms of replication and transcription. One of them is the way the nucleoprotein (N) wraps and protects the RNA genome from degradation by forming a highly ordered helical nucleocapsid. However, crystal structures from numerous Mononegavirales reveal that binding to the nucleoprotein results in occluded nucleotides that hinder base pairing necessary for transcription and replication. This hints at the existence of alternative conformations of the N protein that would impact on the protein-RNA interface, allowing for transient exposure of the nucleotides without complete RNA release. Moreover, the regulation between the alternative conformations should be finely tuned. Recombinant expression of N from the respiratory syncytial virus form regular N/RNA common among all Mononegavirales, and these constitute an ideal minimal unit for investigating the mechanisms through which these structures protect RNA so efficiently while allowing for partial accessibility during transcription and replication. Neither pH nor high ionic strength could dissociate the RNA but led to irreversible aggregation of the nucleoprotein. Low concentrations of guanidine chloride dissociated the RNA moiety but leading to irreversible aggregation of the protein moiety. On the other hand, high concentrations of urea and long incubation periods were required to remove bound RNA. Both denaturants eventually led to unfolding but converged in the formation of an RNA-free ß-enriched intermediate species that remained decameric even at high denaturant concentrations. Although the N-RNA rings interact with the phosphoprotein P, the scaffold of the RNA polymerase complex, this interaction did not lead to RNA dissociation from the rings in vitro. Thus, we have uncovered complex equilibria involving changes in secondary structure of N and RNA loosening, processes that must take place in the context of RNA transcription and replication, whose detailed mechanisms and cellular and viral participants need to be established.


Assuntos
Proteínas do Nucleocapsídeo/metabolismo , RNA Viral/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Proteínas do Nucleocapsídeo/química , Concentração Osmolar , Ligação Proteica , Estrutura Secundária de Proteína , Estabilidade de RNA , RNA Viral/química , Vírus Sincicial Respiratório Humano/química , Temperatura , Termodinâmica
12.
Vet Microbiol ; 233: 140-146, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31176400

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is caused by PRRS virus (PRRSV), and is characterized by respiratory diseases in piglet and reproductive disorders in sow. Identification of sustainable and effective measures to mitigate PRRSV transmission is a pressing problem. The nucleocapsid (N) protein of PRRSV plays a crucial role in inhibiting host innate immunity during PRRSV infection. In the current study, a new host-restricted factor, tripartite motif protein 25 (TRIM25), was identified as an inhibitor of PRRSV replication. Co-immunoprecipitation assay indicated that the PRRSV N protein interferes with TRIM25-RIG-I interactions by competitively interacting with TRIM25. Furthermore, N protein inhibits the expression of TRIM25 and TRIM25-mediated RIG-I ubiquitination to suppress interferon ß production. Furthermore, with increasing TRIM25 expression, the inhibitory effect of N protein on the ubiquitination of RIG-I diminished. These results indicate for the first time that TRIM25 inhibits PRRSV replication and that the N protein antagonizes the antiviral activity by interfering with TRIM25-mediated RIG-I ubiquitination. This not only provides a theoretical basis for the development of drugs to control PRRSV replication, but also better explains the mechanism through which the PRRSV N protein inhibits innate immune responses of the host.


Assuntos
Proteína DEAD-box 58/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Proteínas com Motivo Tripartido/antagonistas & inibidores , Proteínas com Motivo Tripartido/genética , Ubiquitinação , Motivos de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Proteínas do Nucleocapsídeo/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Ligação Proteica , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Suínos , Transfecção , Replicação Viral
13.
Vet Res Commun ; 43(3): 187-195, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104196

RESUMO

The emergence of virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV), causing atypical and severe outbreaks, has been notified worldwide. This study assesses the expression, distribution and kinetics of PRRSV N-protein, CD163 and CD107a in the lung and tonsil from experimentally-infected piglets with three different PRRSV-1 strains: a virulent PRRSV-1 subtype 3 strain (SU1-bel) and two low-virulent subtype 1 strains, Lelystad virus (LV) and 215-06. SU1-bel replicated more efficiently in the lungs and tonsils. The number of CD163+ cells decreased in both tissues from all infected groups at 7 dpi, followed by an increase at the end of the study, highlighting a negative correlation with the number of N-protein+-infected cells. A significant increase in CD107a was observed in all infected groups at 35 dpi but no differences were observed among them. Whereas the initial decrease of CD163+ cells appears to be associated to virus replication and cell death, the later recovery of the CD163+ population may be due to either the induction of CD163 in immature cells, the recruitment of CD163+ cells in the area of infection, or both. These results highlight the ability of macrophage subpopulations in infected animals to recover and restore their potential biological functions at one-month post-infection, with the greatest improvement observed in SU1-bel-infected animals.


Assuntos
Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/genética , Regulação da Expressão Gênica/imunologia , Pulmão/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/genética , Tonsila Palatina/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Receptores de Superfície Celular/genética , Animais , Antígenos CD/imunologia , Antígenos de Diferenciação Mielomonocítica/imunologia , Proteína 1 de Membrana Associada ao Lisossomo/imunologia , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Receptores de Superfície Celular/imunologia , Suínos , Virulência/imunologia
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 97-102, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975272

RESUMO

Objective To construct the plasmid expressing the fusion protein of Hantaan virus nucleocapsid protein (HTNV NP) with affinity tag, and isolate the host factors interacting with NP using the affinity purification. Methods The synthetic streptavidin-FLAG (SF) gene and HTNV NP gene were cloned into the mammalian eukaryotic expression vector to obtain the recombinant expression plasmid (pCAGGS-SF-NP). The plasmid pCAGGS-SF-NP was transfected into HEK293T cells, and the expression of SF-NP was detected by Western blotting. Next, cell lysates were mixed with StrepTrapTM HP agar beads. After incubating overnight at 4DegreesCelsius, the agar beads were transferred into affinity chromatography column and washed with elution buffer. Finally, the binding proteins that interacted with SF-NP were collected by competitive elution buffer with desthiobiotin, and then were subjected to SDS-PAGE. Results The recombinant SF-NP proteins were highly expressed in eukaryotic cells. The host factors interacting with SF-NP were successfully enriched by affinity purification, and confirmed by SDS-PAGE. Conclusion The host factors interacting with HTNV NP can be isolated by affinity purification.


Assuntos
Vírus Hantaan , Fatores Celulares Derivados do Hospedeiro/isolamento & purificação , Proteínas do Nucleocapsídeo/metabolismo , Animais , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Células HEK293 , Vírus Hantaan/metabolismo , Humanos , Proteínas do Nucleocapsídeo/genética , Plasmídeos/genética , Proteínas Recombinantes/metabolismo
15.
J Virol ; 93(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30867297

RESUMO

Andes virus (ANDV) causes hantavirus pulmonary syndrome (HPS) and is the only hantavirus shown to spread person to person and cause a highly lethal HPS-like disease in Syrian hamsters. The unique ability of ANDV N protein to inhibit beta interferon (IFNß) induction may contribute to its virulence and spread. Here we analyzed IFNß regulation by ANDV N protein substituted with divergent residues from the nearly identical Maporal virus (MAPV) N protein. We found that MAPV N fails to inhibit IFNß signaling and that replacing ANDV residues 252 to 296 with a hypervariable domain (HVD) from MAPV N prevents IFNß regulation. In addition, changing ANDV residue S386 to the histidine present in MAPV N or the alanine present in other hantaviruses prevented ANDV N from regulating IFNß induction. In contrast, replacing serine with phosphoserine-mimetic aspartic acid (S386D) in ANDV N robustly inhibited interferon regulatory factor 3 (IRF3) phosphorylation and IFNß induction. Additionally, the MAPV N protein gained the ability to inhibit IRF3 phosphorylation and IFNß induction when ANDV HVD and H386D replaced MAPV residues. Mass spectroscopy analysis of N protein from ANDV-infected cells revealed that S386 is phosphorylated, newly classifying ANDV N as a phosphoprotein and phosphorylated S386 as a unique determinant of IFN regulation. In this context, the finding that the ANDV HVD is required for IFN regulation by S386 but dispensable for IFN regulation by D386 suggests a role for HVD in kinase recruitment and S386 phosphorylation. These findings delineate elements within the ANDV N protein that can be targeted to attenuate ANDV and suggest targeting cellular kinases as potential ANDV therapeutics.IMPORTANCE ANDV contains virulence determinants that uniquely permit it to spread person to person and cause highly lethal HPS in immunocompetent hamsters. We discovered that ANDV S386 and an ANDV-specific hypervariable domain permit ANDV N to inhibit IFN induction and that IFN regulation is directed by phosphomimetic S386D substitutions in ANDV N. In addition, MAPV N proteins containing D386 and ANDV HVD gained the ability to inhibit IFN induction. Validating these findings, mass spectroscopy analysis revealed that S386 of ANDV N protein is uniquely phosphorylated during ANDV infection. Collectively, these findings reveal new paradigms for ANDV N protein as a phosphoprotein and IFN pathway regulator and suggest new mechanisms for hantavirus regulation of cellular kinases and signaling pathways. Our findings define novel IFN-regulating virulence determinants of ANDV, identify residues that can be modified to attenuate ANDV for vaccine development, and suggest the potential for kinase inhibitors to therapeutically restrict ANDV replication.


Assuntos
Hantavirus/metabolismo , Interferon beta/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Animais , Chlorocebus aethiops , Células Endoteliais/virologia , Células HEK293 , Hantavirus/patogenicidade , Infecções por Hantavirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Interferon beta/fisiologia , Interferons/metabolismo , Interferons/fisiologia , Proteínas do Nucleocapsídeo/fisiologia , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismo , Transdução de Sinais , Células Vero , Virulência , Fatores de Virulência/metabolismo , Replicação Viral
16.
J Gen Virol ; 100(3): 392-402, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30720418

RESUMO

The Nairoviridae family within the Bunyavirales order comprise tick-borne segmented negative-sense RNA viruses that cause serious disease in a broad range of mammals, yet cause a latent and lifelong infection in tick hosts. An important member of this family is Crimean-Congo haemorrhagic fever virus (CCHFV), which is responsible for serious human disease that results in case fatality rates of up to 30 %, and which exhibits the most geographically broad distribution of any tick-borne virus. Here, we explored differences in the cellular response of both mammalian and tick cells to nairovirus infection using Hazara virus (HAZV), which is a close relative of CCHFV within the CCHFV serogroup. We show that HAZV infection of human-derived SW13 cells led to induction of apoptosis, evidenced by activation of cellular caspases 3, 7 and 9. This was followed by cleavage of the classical apoptosis marker poly ADP-ribose polymerase, as well as cellular genome fragmentation. In addition, we show that the HAZV nucleocapsid (N) protein was abundantly cleaved by caspase 3 in these mammalian cells at a conserved DQVD motif exposed at the tip of its arm domain, and that cleaved HAZV-N was subsequently packaged into nascent virions. However, in stark contrast, we show for the first time that nairovirus infection of cells of the tick vector failed to induce apoptosis, as evidenced by undetectable levels of cleaved caspases and lack of cleaved HAZV-N. Our findings reveal that nairoviruses elicit diametrically opposed cellular responses in mammalian and tick cells, which may influence the infection outcome in the respective hosts.


Assuntos
Apoptose , Infecções por Bunyaviridae/fisiopatologia , Nairovirus/metabolismo , Proteínas do Nucleocapsídeo/metabolismo , Carrapatos/virologia , Motivos de Aminoácidos , Animais , Infecções por Bunyaviridae/enzimologia , Infecções por Bunyaviridae/genética , Infecções por Bunyaviridae/virologia , Caspase 3/genética , Caspase 3/metabolismo , Caspase 7/genética , Caspase 7/metabolismo , Linhagem Celular , Interações Hospedeiro-Patógeno , Humanos , Nairovirus/química , Nairovirus/genética , Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/genética , Processamento de Proteína Pós-Traducional
17.
J Biol Chem ; 294(13): 5023-5037, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30723154

RESUMO

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.


Assuntos
Vírus da Febre Hemorrágica da Crimeia-Congo/fisiologia , Febre Hemorrágica da Crimeia/virologia , Proteínas do Nucleocapsídeo/metabolismo , RNA/metabolismo , Sítios de Ligação , Vírus da Febre Hemorrágica da Crimeia-Congo/química , Febre Hemorrágica da Crimeia/metabolismo , Humanos , Modelos Moleculares , Nairovirus/química , Nairovirus/fisiologia , Proteínas do Nucleocapsídeo/química , Domínios Proteicos , Replicação Viral
18.
Biochem Biophys Res Commun ; 510(3): 472-478, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30737028

RESUMO

Retroviral nucleocapsid (NC) proteins are multifunctional nucleic acid binding proteins, playing critical roles in essentially every step of the viral replication cycle. As a small, basic protein, NC contains one or two highly conserved zinc-finger domains, each having an invariant CCHC motif, flanked by basic residues. In this study, we report for the first time, to our knowledge, the thermostable property of equine infectious anemia virus (EIAV) NCp11. About 43% of purified NCp11 remained soluble after incubation at 100 °C for 60 min, and heat-treated NCp11 maintained its abilities to bind to the E. coli RNA and the EIAV packaging signal sequence. At a very high degree of sequence occupancy, NCp11 inhibited first-strand cDNA synthesis catalyzed by either a commercial or the purified EIAV reverse transcriptase, and heat-treated NCp11 still inhibited the first-strand cDNA synthesis. We also found that protein concentrations, at a range from 0.1 to 0.9 µg/µl, have not affected the NCp11 thermostability significantly. However, NCp11 at acidic pH was more thermostable. Our findings highlight a new feature of the NC protein. Detailed understanding of NC's properties and functions will facilitate the development of effective and rational therapeutic strategies against retroviruses.


Assuntos
Proteínas do Nucleocapsídeo/química , Proteínas do Nucleocapsídeo/metabolismo , DNA Complementar/biossíntese , Ácido Edético , Temperatura Alta , Concentração de Íons de Hidrogênio , Estabilidade Proteica , RNA/metabolismo
19.
Viruses ; 11(1)2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30609802

RESUMO

The filoviruses Ebolavirus and Marburgvirus are among the deadliest viral pathogens known to infect humans, causing emerging diseases with fatality rates of up to 90% during some outbreaks. The replication cycles of these viruses are comprised of numerous complex molecular processes and interactions with their human host, with one key feature being the means by which nascent virions exit host cells to spread to new cells and ultimately to a new host. This review focuses on our current knowledge of filovirus egress and the viral and host factors and processes that are involved. Within the virus, these factors consist of the major matrix protein, viral protein 40 (VP40), which is necessary and sufficient for viral particle release, and nucleocapsid and glycoprotein that interact with VP40 to promote egress. In the host cell, some proteins are hijacked by filoviruses in order to enhance virion budding capacity that include members of the family of E3 ubiquitin ligase and the endosomal sorting complexes required for transport (ESCRT) pathway, while others such as tetherin inhibit viral egress. An understanding of these molecular interactions that modulate viral particle egress provides an important opportunity to identify new targets for the development of antivirals to prevent and treat filovirus infections.


Assuntos
Ebolavirus/fisiologia , Interações entre Hospedeiro e Microrganismos , Marburgvirus/fisiologia , Liberação de Vírus , Animais , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HEK293 , Humanos , Camundongos , Nucleocapsídeo/genética , Nucleocapsídeo/metabolismo , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo
20.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541836

RESUMO

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.


Assuntos
Hantavirus/genética , Proteínas do Nucleocapsídeo/metabolismo , Capuzes de RNA/genética , RNA Replicase/genética , RNA Mensageiro/metabolismo , RNA Viral/biossíntese , Linhagem Celular , Células Endoteliais , Genoma Viral/genética , Infecções por Hantavirus/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , RNA/genética , RNA Mensageiro/genética , RNA Viral/genética , Proteínas de Ligação a RNA/metabolismo , Transcrição Genética/genética , Replicação Viral/genética
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