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1.
Cells ; 10(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200500

RESUMO

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.


Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/virologia , SARS-CoV-2/fisiologia , Fenômenos Fisiológicos Virais , Replicação Viral/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Internalização do Vírus , Vírus/patogenicidade
2.
Cells ; 10(6)2021 06 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1259432

RESUMO

The host nucleocytoplasmic trafficking system is often hijacked by viruses to accomplish their replication and to suppress the host immune response. Viruses encode many factors that interact with the host nuclear transport receptors (NTRs) and the nucleoporins of the nuclear pore complex (NPC) to access the host nucleus. In this review, we discuss the viral factors and the host factors involved in the nuclear import and export of viral components. As nucleocytoplasmic shuttling is vital for the replication of many viruses, we also review several drugs that target the host nuclear transport machinery and discuss their feasibility for use in antiviral treatment.


Assuntos
Núcleo Celular/metabolismo , Núcleo Celular/virologia , SARS-CoV-2/fisiologia , Fenômenos Fisiológicos Virais , Replicação Viral/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , COVID-19/metabolismo , COVID-19/virologia , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Internalização do Vírus , Vírus/patogenicidade
3.
Nat Commun ; 12(1): 4270, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257311

RESUMO

The recent dramatic appearance of variants of concern of SARS-coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape from host immunity and antiviral therapeutics. Here, we employ genome-wide computational prediction and single-nucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus-free models. Further, optimized and multiplexed Cas13b CRISPR RNAs (crRNAs) suppress viral replication in mammalian cells infected with replication-competent SARS-CoV-2, including the recently emerging dominant variant of concern B.1.1.7. The comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches does not impair the capacity of a potent single crRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 strains in infected mammalian cells, including the Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint for antiviral drug development to suppress and prevent a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.


Assuntos
Mutação , SARS-CoV-2/fisiologia , Replicação Viral/fisiologia , Animais , Antivirais/farmacologia , COVID-19/tratamento farmacológico , COVID-19/virologia , Sistemas CRISPR-Cas , Chlorocebus aethiops , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Desenvolvimento de Medicamentos , Genoma Viral , Células HEK293 , Humanos , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Células Vero , Replicação Viral/genética
4.
Front Immunol ; 12: 698578, 2021.
Artigo em Inglês | MEDLINE | ID: covidwho-1278398

RESUMO

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children's health.


Assuntos
COVID-19/imunologia , COVID-19/transmissão , Transmissão Vertical de Doenças Infecciosas , Placenta/virologia , Complicações Infecciosas na Gravidez/imunologia , Adulto , Feminino , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Placenta/imunologia , Gravidez , SARS-CoV-2/imunologia , Replicação Viral/fisiologia
5.
Emerg Microbes Infect ; 10(1): 1156-1168, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: covidwho-1249264

RESUMO

ABSTRACTThe risk of secondary infection with SARS-CoV-2 and influenza A virus is becoming a practical problem that must be addressed as the flu season merges with the COVID-19 pandemic. As SARS-CoV-2 and influenza A virus have been found in patients, understanding the in vivo characteristics of the secondary infection between these two viruses is a high priority. Here, hACE2 transgenic mice were challenged with the H1N1 virus at a nonlethal dose during the convalescent stage on 7 and 14 days post SARS-CoV-2 infection, and importantly, subsequent H1N1 infection showed enhanced viral shedding and virus tissue distribution. Histopathological observation revealed an extensive pathological change in the lungs related to H1N1 infection in mice recovered from SARS-CoV-2 infection, with severe inflammation infiltration and bronchiole disruption. Moreover, upon H1N1 exposure on 7 and 14 dpi of SARS-CoV-2 infection, the lymphocyte population activated at a lower level with T cell suppressed in both PBMC and lung. These findings will be valuable for evaluating antiviral therapeutics and vaccines as well as guiding public health work.


Assuntos
Lesão Pulmonar Aguda/patologia , Enzima de Conversão de Angiotensina 2/genética , COVID-19/patologia , Infecções por Orthomyxoviridae/patologia , Lesão Pulmonar Aguda/virologia , Animais , COVID-19/terapia , Coinfecção/patologia , Coinfecção/virologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Pulmão/patologia , Contagem de Linfócitos , Linfócitos/imunologia , Camundongos , Camundongos Transgênicos , Infecções por Orthomyxoviridae/terapia , SARS-CoV-2/isolamento & purificação , Carga Viral , Replicação Viral/fisiologia , Eliminação de Partículas Virais/fisiologia
6.
Mol Cell ; 81(13): 2851-2867.e7, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34118193

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control its life cycle remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively the cellular and viral RBPs that are involved in SARS-CoV-2 infection. We reveal that SARS-CoV-2 infection profoundly remodels the cellular RNA-bound proteome, which includes wide-ranging effects on RNA metabolic pathways, non-canonical RBPs, and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Among them are several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.


Assuntos
COVID-19/metabolismo , Proteoma/metabolismo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/fisiologia , Proteínas Virais/metabolismo , Replicação Viral/fisiologia , Células A549 , COVID-19/genética , Humanos , Proteoma/genética , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Proteínas Virais/genética
7.
Emerg Microbes Infect ; 10(1): 1156-1168, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34060982

RESUMO

ABSTRACTThe risk of secondary infection with SARS-CoV-2 and influenza A virus is becoming a practical problem that must be addressed as the flu season merges with the COVID-19 pandemic. As SARS-CoV-2 and influenza A virus have been found in patients, understanding the in vivo characteristics of the secondary infection between these two viruses is a high priority. Here, hACE2 transgenic mice were challenged with the H1N1 virus at a nonlethal dose during the convalescent stage on 7 and 14 days post SARS-CoV-2 infection, and importantly, subsequent H1N1 infection showed enhanced viral shedding and virus tissue distribution. Histopathological observation revealed an extensive pathological change in the lungs related to H1N1 infection in mice recovered from SARS-CoV-2 infection, with severe inflammation infiltration and bronchiole disruption. Moreover, upon H1N1 exposure on 7 and 14 dpi of SARS-CoV-2 infection, the lymphocyte population activated at a lower level with T cell suppressed in both PBMC and lung. These findings will be valuable for evaluating antiviral therapeutics and vaccines as well as guiding public health work.


Assuntos
Lesão Pulmonar Aguda/patologia , Enzima de Conversão de Angiotensina 2/genética , COVID-19/patologia , Infecções por Orthomyxoviridae/patologia , Lesão Pulmonar Aguda/virologia , Animais , COVID-19/terapia , Coinfecção/patologia , Coinfecção/virologia , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Humanos , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Pulmão/patologia , Contagem de Linfócitos , Linfócitos/imunologia , Camundongos , Camundongos Transgênicos , Infecções por Orthomyxoviridae/terapia , SARS-CoV-2/isolamento & purificação , Carga Viral , Replicação Viral/fisiologia , Eliminação de Partículas Virais/fisiologia
8.
Proc Natl Acad Sci U S A ; 118(25)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34099577

RESUMO

Coronaviruses are pathogens of pandemic potential. Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. More than 70% of MERS-CoV-infected dromedaries are found in East, North, and West Africa, but zoonotic MERS disease is only reported from the Arabian Peninsula. We compared viral replication competence of clade A and B viruses from the Arabian Peninsula with genetically diverse clade C viruses found in East (Egypt, Kenya, and Ethiopia), North (Morocco), and West (Nigeria and Burkina Faso) Africa. Viruses from Africa had lower replication competence in ex vivo cultures of the human lung and in lungs of experimentally infected human-DPP4 (hDPP4) knockin mice. We used lentivirus pseudotypes expressing MERS-CoV spike from Saudi Arabian clade A prototype strain (EMC) or African clade C1.1 viruses and demonstrated that clade C1.1 spike was associated with reduced virus entry into the respiratory epithelial cell line Calu-3. Isogenic EMC viruses with spike protein from EMC or clade C1.1 generated by reverse genetics showed that the clade C1.1 spike was associated with reduced virus replication competence in Calu-3 cells in vitro, in ex vivo human bronchus, and in lungs of hDPP4 knockin mice in vivo. These findings may explain why zoonotic MERS disease has not been reported from Africa so far, despite exposure to and infection with MERS-CoV.


Assuntos
Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Zoonoses/virologia , África , Animais , Arábia , Linhagem Celular , Dipeptidil Peptidase 4/metabolismo , Técnicas de Introdução de Genes , Humanos , Cinética , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Fenótipo , Filogenia , Glicoproteína da Espícula de Coronavírus/metabolismo , Replicação Viral/fisiologia
9.
Front Immunol ; 12: 698578, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34149740

RESUMO

Pregnant women have been carefully observed during the COVID-19 pandemic, as the pregnancy-specific immune adaptation is known to increase the risk for infections. Recent evidence indicates that even though most pregnant have a mild or asymptomatic course, a severe course of COVID-19 and a higher risk of progression to diseases have also been described, along with a heightened risk for pregnancy complications. Yet, vertical transmission of the virus is rare and the possibility of placental SARS-CoV-2 infection as a prerequisite for vertical transmission requires further studies. We here assessed the severity of COVID-19 and onset of neonatal infections in an observational study of women infected with SARS-CoV-2 during pregnancy. Our placental analyses showed a paucity of SARS-CoV-2 viral expression ex vivo in term placentae under acute infection. No viral placental expression was detectable in convalescent pregnant women. Inoculation of placental explants generated from placentas of non-infected women at birth with SARS-CoV-2 in vitro revealed inefficient SARS-CoV-2 replication in different types of placental tissues, which provides a rationale for the low ex vivo viral expression. We further detected specific SARS-CoV-2 T cell responses in pregnant women within a few days upon infection, which was undetectable in cord blood. Our present findings confirm that vertical transmission of SARS-CoV-2 is rare, likely due to the inefficient virus replication in placental tissues. Despite the predominantly benign course of infection in most mothers and negligible risk of vertical transmission, continuous vigilance on the consequences of COVID-19 during pregnancy is required, since the maternal immune activation in response to the SARS-CoV2 infection may have long-term consequences for children's health.


Assuntos
COVID-19/imunologia , COVID-19/transmissão , Transmissão Vertical de Doenças Infecciosas , Placenta/virologia , Complicações Infecciosas na Gravidez/imunologia , Adulto , Feminino , Sangue Fetal/imunologia , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Placenta/imunologia , Gravidez , SARS-CoV-2/imunologia , Replicação Viral/fisiologia
10.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: covidwho-1220249

RESUMO

Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.


Assuntos
COVID-19/genética , COVID-19/virologia , SARS-CoV-2/genética , Integração Viral/genética , Animais , COVID-19/metabolismo , Chlorocebus aethiops , Genoma Viral , Células HEK293 , Humanos , RNA Viral/genética , SARS-CoV-2/metabolismo , Células Vero , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
11.
Naunyn Schmiedebergs Arch Pharmacol ; 394(7): 1383-1402, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1219857

RESUMO

SARS-CoV-2 is an enveloped positive-sense RNA virus, contain crown-like spikes on its surface, exceptional of large RNA genome, and a special replication machinery. Common symptoms of SARS-CoV-2 include cough, common cold, fever, sore throat, and a variety of severe acute respiratory disease (SARD) such as pneumonia. SARS-CoV-2 infects epithelial cells, T-cells, macrophages, and dendritic cells and also influences the production and implantation of pro-inflammatory cytokines and chemokines. Repurposing of various drugs during this emergency condition can reduce the rate of mortality as well as time and cost. Two druggable protein and enzyme targets have been selected in this review article due to their crucial role in the viral life cycle. The eukaryotic translation initiation factor (eIF4A), cyclophilin, nucleocapsid protein, spike protein, Angiotensin-converting enzyme 2 (ACE2), 3-chymotrypsin-like cysteine protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) play significant role in early and late phase of SARS-CoV-2 replication and translation. This review paper is based on the rationale of inhibiting of various SARS-CoV-2 proteins and enzymes as novel therapeutic approaches for the management and treatment of patients with SARS-CoV-2 infection. We also discussed the structural and functional relationship of different proteins and enzymes to develop therapeutic approaches for novel coronavirus SARS-CoV-2.


Assuntos
Antivirais/administração & dosagem , Vacinas contra COVID-19/administração & dosagem , COVID-19/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/metabolismo , COVID-19/metabolismo , Vacinas contra COVID-19/metabolismo , Reposicionamento de Medicamentos , Humanos , SARS-CoV-2/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
12.
J Clin Invest ; 131(13)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34003804

RESUMO

The upper respiratory tract is compromised in the early period of COVID-19, but SARS-CoV-2 tropism at the cellular level is not fully defined. Unlike recent single-cell RNA-Seq analyses indicating uniformly low mRNA expression of SARS-CoV-2 entry-related host molecules in all nasal epithelial cells, we show that the protein levels are relatively high and that their localizations are restricted to the apical side of multiciliated epithelial cells. In addition, we provide evidence in patients with COVID-19 that SARS-CoV-2 is massively detected and replicated within the multiciliated cells. We observed these findings during the early stage of COVID-19, when infected ciliated cells were rapidly replaced by differentiating precursor cells. Moreover, our analyses revealed that SARS-CoV-2 cellular tropism was restricted to the nasal ciliated versus oral squamous epithelium. These results imply that targeting ciliated cells of the nasal epithelium during the early stage of COVID-19 could be an ideal strategy to prevent SARS-CoV-2 propagation.


Assuntos
COVID-19/virologia , Interações entre Hospedeiro e Microrganismos , Mucosa Nasal/virologia , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , COVID-19/patologia , COVID-19/fisiopatologia , Diferenciação Celular , Cílios/patologia , Cílios/fisiologia , Cílios/virologia , Furina/genética , Furina/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Macaca , Modelos Biológicos , Mucosa Nasal/patologia , Mucosa Nasal/fisiopatologia , Pandemias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA-Seq , SARS-CoV-2/genética , SARS-CoV-2/patogenicidade , SARS-CoV-2/fisiologia , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Células-Tronco/patologia , Células-Tronco/virologia , Internalização do Vírus , Replicação Viral/genética , Replicação Viral/fisiologia
13.
Proc Natl Acad Sci U S A ; 118(21)2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33958444

RESUMO

Prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and recurrence of PCR-positive tests have been widely reported in patients after recovery from COVID-19, but some of these patients do not appear to shed infectious virus. We investigated the possibility that SARS-CoV-2 RNAs can be reverse-transcribed and integrated into the DNA of human cells in culture and that transcription of the integrated sequences might account for some of the positive PCR tests seen in patients. In support of this hypothesis, we found that DNA copies of SARS-CoV-2 sequences can be integrated into the genome of infected human cells. We found target site duplications flanking the viral sequences and consensus LINE1 endonuclease recognition sequences at the integration sites, consistent with a LINE1 retrotransposon-mediated, target-primed reverse transcription and retroposition mechanism. We also found, in some patient-derived tissues, evidence suggesting that a large fraction of the viral sequences is transcribed from integrated DNA copies of viral sequences, generating viral-host chimeric transcripts. The integration and transcription of viral sequences may thus contribute to the detection of viral RNA by PCR in patients after infection and clinical recovery. Because we have detected only subgenomic sequences derived mainly from the 3' end of the viral genome integrated into the DNA of the host cell, infectious virus cannot be produced from the integrated subgenomic SARS-CoV-2 sequences.


Assuntos
COVID-19/genética , COVID-19/virologia , SARS-CoV-2/genética , Integração Viral/genética , Animais , COVID-19/metabolismo , Chlorocebus aethiops , Genoma Viral , Células HEK293 , Humanos , RNA Viral/genética , SARS-CoV-2/metabolismo , Células Vero , Integração Viral/fisiologia , Replicação Viral/genética , Replicação Viral/fisiologia
14.
Naunyn Schmiedebergs Arch Pharmacol ; 394(7): 1383-1402, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33961065

RESUMO

SARS-CoV-2 is an enveloped positive-sense RNA virus, contain crown-like spikes on its surface, exceptional of large RNA genome, and a special replication machinery. Common symptoms of SARS-CoV-2 include cough, common cold, fever, sore throat, and a variety of severe acute respiratory disease (SARD) such as pneumonia. SARS-CoV-2 infects epithelial cells, T-cells, macrophages, and dendritic cells and also influences the production and implantation of pro-inflammatory cytokines and chemokines. Repurposing of various drugs during this emergency condition can reduce the rate of mortality as well as time and cost. Two druggable protein and enzyme targets have been selected in this review article due to their crucial role in the viral life cycle. The eukaryotic translation initiation factor (eIF4A), cyclophilin, nucleocapsid protein, spike protein, Angiotensin-converting enzyme 2 (ACE2), 3-chymotrypsin-like cysteine protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) play significant role in early and late phase of SARS-CoV-2 replication and translation. This review paper is based on the rationale of inhibiting of various SARS-CoV-2 proteins and enzymes as novel therapeutic approaches for the management and treatment of patients with SARS-CoV-2 infection. We also discussed the structural and functional relationship of different proteins and enzymes to develop therapeutic approaches for novel coronavirus SARS-CoV-2.


Assuntos
Antivirais/administração & dosagem , Vacinas contra COVID-19/administração & dosagem , COVID-19/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , SARS-CoV-2/efeitos dos fármacos , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Antivirais/metabolismo , COVID-19/metabolismo , Vacinas contra COVID-19/metabolismo , Reposicionamento de Medicamentos , Humanos , SARS-CoV-2/metabolismo , Replicação Viral/efeitos dos fármacos , Replicação Viral/fisiologia
15.
Nat Commun ; 12(1): 2530, 2021 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-33953197

RESUMO

Flaviviruses use a ~70 nucleotide stem-loop structure called stem-loop A (SLA) at the 5' end of the RNA genome as a promoter for RNA synthesis. Flaviviral polymerase NS5 specifically recognizes SLA to initiate RNA synthesis and methylate the 5' guanosine cap. We report the crystal structures of dengue (DENV) and Zika virus (ZIKV) SLAs. DENV and ZIKV SLAs differ in the relative orientations of their top stem-loop helices to bottom stems, but both form an intermolecular three-way junction with a neighboring SLA molecule. To understand how NS5 engages SLA, we determined the SLA-binding site on NS5 and modeled the NS5-SLA complex of DENV and ZIKV. Our results show that the gross conformational differences seen in DENV and ZIKV SLAs can be compensated by the differences in the domain arrangements in DENV and ZIKV NS5s. We describe two binding modes of SLA and NS5 and propose an SLA-mediated RNA synthesis mechanism.


Assuntos
Flavivirus/genética , Regiões Promotoras Genéticas , RNA Viral/química , RNA Viral/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Sítios de Ligação , Cristalografia por Raios X , Vírus da Dengue/genética , Ligação Proteica , RNA Viral/metabolismo , Replicação Viral/fisiologia , Zika virus/genética
16.
Nat Commun ; 12(1): 2766, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986255

RESUMO

The escalating global prevalence of arboviral diseases emphasizes the need to improve our understanding of their biology. Research in this area has been hindered by the lack of molecular tools for studying virus-mosquito interactions. Here, we develop an Aedes aegypti cell line which stably expresses Zika virus (ZIKV) capsid proteins in order to study virus-vector protein-protein interactions through quantitative label-free proteomics. We identify 157 interactors and show that eight have potentially pro-viral activity during ZIKV infection in mosquito cells. Notably, silencing of transitional endoplasmic reticulum protein TER94 prevents ZIKV capsid degradation and significantly reduces viral replication. Similar results are observed if the TER94 ortholog (VCP) functioning is blocked with inhibitors in human cells. In addition, we show that an E3 ubiquitin-protein ligase, UBR5, mediates the interaction between TER94 and ZIKV capsid. Our study demonstrates a pro-viral function for TER94/VCP during ZIKV infection that is conserved between human and mosquito cells.


Assuntos
Proteínas do Capsídeo/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/metabolismo , Zika virus/metabolismo , Células A549 , Aedes/virologia , Animais , Capsídeo/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Mapas de Interação de Proteínas , Interferência de RNA , RNA Interferente Pequeno/genética , Proteína com Valosina/genética , Replicação Viral/fisiologia , Zika virus/genética , Infecção por Zika virus/patologia
17.
Stem Cell Reports ; 16(4): 940-953, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: covidwho-1180038

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) usually results in respiratory disease, but extrapulmonary manifestations are of major clinical interest. Intestinal symptoms of COVID-19 are present in a significant number of patients, and include nausea, diarrhea, and viral RNA shedding in feces. Human induced pluripotent stem cell-derived intestinal organoids (HIOs) represent an inexhaustible cellular resource that could serve as a valuable tool to study SARS-CoV-2 as well as other enteric viruses that infect the intestinal epithelium. Here, we report that SARS-CoV-2 productively infects both proximally and distally patterned HIOs, leading to the release of infectious viral particles while stimulating a robust transcriptomic response, including a significant upregulation of interferon-related genes that appeared to be conserved across multiple epithelial cell types. These findings illuminate a potential inflammatory epithelial-specific signature that may contribute to both the multisystemic nature of COVID-19 as well as its highly variable clinical presentation.


Assuntos
COVID-19/patologia , Colo/patologia , Mucosa Intestinal/patologia , Organoides/patologia , Linhagem Celular , Colo/virologia , Células Epiteliais/virologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Inflamação/virologia , Mucosa Intestinal/virologia , Modelos Biológicos , Organoides/citologia , Organoides/virologia , SARS-CoV-2 , Replicação Viral/fisiologia
18.
J Med Virol ; 93(7): 4258-4264, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: covidwho-1173823

RESUMO

The recent coronavirus disease 2019 (COVID-19), causing a global pandemic with devastating effects on healthcare and social-economic systems, has no special antiviral therapies available for human coronaviruses (CoVs). The severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) possesses a nonstructural protein (nsp14), with amino-terminal domain coding for proofreading exoribonuclease (ExoN) that is required for high-fidelity replication. The ability of CoVs during genome replication and transcription to proofread and exclude mismatched nucleotides has long hindered the development of anti-CoV drugs. The resistance of SARS-CoV-2 to antivirals, especially nucleoside analogs (NAs), shows the need to identify new CoV inhibition targets. Therefore, this review highlights the importance of nsp14-ExoN as a target for inhibition. Also, nucleoside analogs could be used in combination with existing anti-CoV therapeutics to target the proofreading mechanism.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Exorribonucleases/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , Replicação Viral/efeitos dos fármacos , Exorribonucleases/efeitos dos fármacos , Exorribonucleases/metabolismo , Genoma Viral/genética , Humanos , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/fisiologia , RNA Viral/genética , SARS-CoV-2/efeitos dos fármacos , Proteínas não Estruturais Virais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/fisiologia
19.
Int J Mol Sci ; 22(5)2021 Mar 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1129734

RESUMO

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (-gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the -gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.


Assuntos
Quadruplex G , Proteínas de Ligação a RNA/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Dicroísmo Circular , Biologia Computacional/métodos , Bases de Dados Genéticas , Ensaio de Desvio de Mobilidade Eletroforética , Genoma Viral/fisiologia , Humanos , Espectroscopia de Prótons por Ressonância Magnética , Replicação Viral/fisiologia
20.
PLoS Pathog ; 17(4): e1009500, 2021 04.
Artigo em Inglês | MEDLINE | ID: covidwho-1197396

RESUMO

The high transmissibility of SARS-CoV-2 is related to abundant replication in the upper airways, which is not observed for the other highly pathogenic coronaviruses SARS-CoV and MERS-CoV. We here reveal features of the coronavirus spike (S) protein, which optimize the virus towards the human respiratory tract. First, the S proteins exhibit an intrinsic temperature preference, corresponding with the temperature of the upper or lower airways. Pseudoviruses bearing the SARS-CoV-2 spike (SARS-2-S) were more infectious when produced at 33°C instead of 37°C, a property shared with the S protein of HCoV-229E, a common cold coronavirus. In contrast, the S proteins of SARS-CoV and MERS-CoV favored 37°C, in accordance with virus preference for the lower airways. Next, SARS-2-S-driven entry was efficiently activated by not only TMPRSS2, but also the TMPRSS13 protease, thus broadening the cell tropism of SARS-CoV-2. Both proteases proved relevant in the context of authentic virus replication. TMPRSS13 appeared an effective spike activator for the virulent coronaviruses but not the low pathogenic HCoV-229E virus. Activation of SARS-2-S by these surface proteases requires processing of the S1/S2 cleavage loop, in which both the furin recognition motif and extended loop length proved critical. Conversely, entry of loop deletion mutants is significantly increased in cathepsin-rich cells. Finally, we demonstrate that the D614G mutation increases SARS-CoV-2 stability, particularly at 37°C, and, enhances its use of the cathepsin L pathway. This indicates a link between S protein stability and usage of this alternative route for virus entry. Since these spike properties may promote virus spread, they potentially explain why the spike-G614 variant has replaced the early D614 variant to become globally predominant. Collectively, our findings reveal adaptive mechanisms whereby the coronavirus spike protein is adjusted to match the temperature and protease conditions of the airways, to enhance virus transmission and pathology.


Assuntos
COVID-19/metabolismo , Sistema Respiratório/metabolismo , Sistema Respiratório/virologia , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , COVID-19/transmissão , Coronavirus Humano 229E/metabolismo , Furina/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeo Hidrolases/metabolismo , Serina Endopeptidases/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Temperatura , Internalização do Vírus , Replicação Viral/fisiologia
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