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1.
Int J Mol Sci ; 22(11)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073578

RESUMO

Ceramide is a lipid messenger at the heart of sphingolipid metabolism. In concert with its metabolizing enzymes, particularly sphingomyelinases, it has key roles in regulating the physical properties of biological membranes, including the formation of membrane microdomains. Thus, ceramide and its related molecules have been attributed significant roles in nearly all steps of the viral life cycle: they may serve directly as receptors or co-receptors for viral entry, form microdomains that cluster entry receptors and/or enable them to adopt the required conformation or regulate their cell surface expression. Sphingolipids can regulate all forms of viral uptake, often through sphingomyelinase activation, and mediate endosomal escape and intracellular trafficking. Ceramide can be key for the formation of viral replication sites. Sphingomyelinases often mediate the release of new virions from infected cells. Moreover, sphingolipids can contribute to viral-induced apoptosis and morbidity in viral diseases, as well as virus immune evasion. Alpha-galactosylceramide, in particular, also plays a significant role in immune modulation in response to viral infections. This review will discuss the roles of ceramide and its related molecules in the different steps of the viral life cycle. We will also discuss how novel strategies could exploit these for therapeutic benefit.


Assuntos
Ceramidas/metabolismo , HIV-1/metabolismo , Vírus da Influenza A/metabolismo , SARS-CoV-2/metabolismo , Viroses/metabolismo , Viroses/virologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Ceramidas/química , Regulação Viral da Expressão Gênica , HIV-1/patogenicidade , Humanos , Imunomodulação , Vírus da Influenza A/patogenicidade , SARS-CoV-2/patogenicidade , Vírion/crescimento & desenvolvimento , Viroses/imunologia , Internalização do Vírus , Replicação Viral/efeitos dos fármacos , Replicação Viral/imunologia
2.
Commun Biol ; 4(1): 715, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112887

RESUMO

While SARS-CoV-2 is causing modern human history's most serious health crisis and upending our way of life, clinical and basic research on the virus is advancing rapidly, leading to fascinating discoveries. Two studies have revealed how the viral virulence factor, nonstructural protein 1 (Nsp1), binds human ribosomes to inhibit host cell translation. Here, we examine the main conclusions on the molecular activity of Nsp1 and its role in suppressing innate immune responses. We discuss different scenarios potentially explaining how the viral RNA can bypass its own translation blockage and speculate on the suitability of Nsp1 as a therapeutic target.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Ribossomos/virologia , SARS-CoV-2/patogenicidade , Proteínas não Estruturais Virais/metabolismo , Regiões 5' não Traduzidas , Regulação Viral da Expressão Gênica , Humanos , Imunidade Inata , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética
3.
Nat Commun ; 12(1): 3287, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078893

RESUMO

The SARS-CoV-2 nsp16/nsp10 enzyme complex modifies the 2'-OH of the first transcribed nucleotide of the viral mRNA by covalently attaching a methyl group to it. The 2'-O methylation of the first nucleotide converts the status of mRNA cap from Cap-0 to Cap-1, and thus, helps the virus evade immune surveillance in host cells. Here, we report two structures of nsp16/nsp10 representing pre- and post-release states of the RNA product (Cap-1). We observe overall widening of the enzyme upon product formation, and an inward twisting motion in the substrate binding region upon product release. These conformational changes reset the enzyme for the next round of catalysis. The structures also identify a unique binding mode and the importance of a divalent metal ion for 2'-O methylation. We also describe underlying structural basis for the perturbed enzymatic activity of a clinical variant of SARS-CoV-2, and a previous SARS-CoV outbreak strain.


Assuntos
Magnésio/química , Capuzes de RNA/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Viral da Expressão Gênica , Humanos , Magnésio/metabolismo , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Capuzes de RNA/química , Capuzes de RNA/genética , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , SARS-CoV-2/enzimologia , SARS-CoV-2/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética
4.
Comput Biol Chem ; 92: 107486, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33984653

RESUMO

SARS-CoV-2 is a single-stranded RNA (+) virus first identified in China and then became an ongoing global outbreak. In most cases, it is fatal in humans due to respiratory malfunction. Extensive researches are going to find an effective therapeutic technique for the treatment of SARS-CoV-2 infected individuals. In this study, we attempted to design a siRNA molecule to silence the most suitable nucleocapsid(N) gene of SARS-CoV-2, which play a major role during viral pathogenesis, replication, encapsidation and RNA packaging. At first, 270 complete N gene sequences of different strains in Bangladesh of these viruses were retrieved from the NCBI database. Different computational methods were used to design siRNA molecules. A siRNA molecule was built against these strains using the SiDirect 2.0 server. Using Mfold and the OligoCalc server, the siRNA molecule was tested for its secondary structure and GC material. The Clustal Omega tool was employed to evaluate any off-target harmony of the planned siRNA molecule. Herein, we proposed a duplex siRNA molecule that does not fit any off-target sequences for the gene silencing of SARS-CoV-2. To treat SARS-CoV-2 infections, currently, any effective therapy is not available. Our engineered siRNA molecule could give an alternative therapeutic approach against various sequenced SARS-CoV-2 strains in Bangladesh.


Assuntos
COVID-19/virologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacologia , SARS-CoV-2/genética , Bangladesh/epidemiologia , COVID-19/epidemiologia , Simulação por Computador , Proteínas do Nucleocapsídeo de Coronavírus/genética , Regulação Viral da Expressão Gênica , Humanos , Modelos Químicos
5.
Curr Protoc ; 1(5): e146, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34033698

RESUMO

Human papillomaviruses (HPVs), specifically high-risk HPVs, are responsible for up to 3% of all cancers in women and up to 2% of all cancers in men. They have been identified as the etiological agent of cervical cancer and have been increasingly found to be the driver behind head and neck cancers of the oropharynx. A system in which we can simultaneously observe transcriptional changes to both a host's tumor microenvironment and its associated oncogenic driver (e.g., HPV) would be highly valuable for understanding HPV's role in tumorigenesis. This article describes a detailed methodology for utilizing high-throughput RNA analysis to study viral transcription in formalin-fixed, paraffin-embedded clinical tumor samples. Although our lab utilizes these methods for the study of head and neck cancer, the principles contained within are widely applicable to all fields of HPV study. © 2021 Wiley Periodicals LLC. Basic Protocol: HPV16 transcript analysis using NanoString Support Protocol 1: Preparation of RNA from formalin-fixed, paraffin-embedded slides Support Protocol 2: Preparation of RNA from cell lysates Support Protocol 3: Fluorometric RNA concentration and RNA integrity analysis Support Protocol 4: Determination of input RNA based on DV300 calculation.


Assuntos
Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Papillomavirus Humano 16/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Transcrição Genética , Microambiente Tumoral/genética , Fluorometria , Formaldeído , Regulação Viral da Expressão Gênica , Humanos , Inclusão em Parafina , RNA Neoplásico/genética , RNA Neoplásico/isolamento & purificação , Robótica , Fixação de Tecidos
6.
EMBO J ; 40(11): e102277, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33876849

RESUMO

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.


Assuntos
Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Regulação Viral da Expressão Gênica , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Vírus da SARS/fisiologia , SARS-CoV-2/fisiologia , Proteínas não Estruturais Virais/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias , Cromatografia em Gel , Proteases Semelhantes à Papaína de Coronavírus/química , Cristalografia por Raios X , Genes Reporter , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes , Modelos Moleculares , Fatores de Iniciação de Peptídeos/química , Ligação Proteica , Biossíntese de Proteínas , Conformação Proteica , Domínios Proteicos , Mapeamento de Interação de Proteínas , RNA Viral/genética , Proteínas de Ligação a RNA/química , RNA Polimerase Dependente de RNA/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Subunidades Ribossômicas/metabolismo , Vírus da SARS/genética , SARS-CoV-2/genética , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas não Estruturais Virais/química , Difração de Raios X
7.
Int J Mol Sci ; 22(8)2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33919712

RESUMO

The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Proteínas de Choque Térmico/genética , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Regulação para Cima , Sequência de Bases , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , DNA Viral/biossíntese , Estresse do Retículo Endoplasmático , Endorribonucleases/metabolismo , Regulação Viral da Expressão Gênica , Células HEK293 , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Humanos , Modelos Biológicos , Regiões Promotoras Genéticas , Transporte Proteico , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Ativação Transcricional/genética , Resposta a Proteínas não Dobradas , Regulação para Cima/genética , eIF-2 Quinase/metabolismo
8.
Int J Mol Sci ; 22(9)2021 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-33923223

RESUMO

Herpes Simplex Virus Type-1 (HSV-1) forms progeny in the nucleus within distinct membrane-less inclusions, the viral replication compartments (VRCs), where viral gene expression, DNA replication, and packaging occur. The way in which the VRCs maintain spatial integrity remains unresolved. Here, we demonstrate that the essential viral transcription factor ICP4 is an intrinsically disordered protein (IDP) capable of driving protein condensation and liquid-liquid phase separation (LLPS) in transfected cells. Particularly, ICP4 forms nuclear liquid-like condensates in a dose- and time-dependent manner. Fluorescence recovery after photobleaching (FRAP) assays revealed rapid exchange rates of EYFP-ICP4 between phase-separated condensates and the surroundings, akin to other viral IDPs that drive LLPS. Likewise, HSV-1 VRCs revealed by EYFP-tagged ICP4 retained their liquid-like nature, suggesting that they are phase-separated condensates. Individual VRCs homotypically fused when reaching close proximity and grew over the course of infection. Together, the results of this study demonstrate that the HSV-1 transcription factor ICP4 has characteristics of a viral IDP, forms condensates in the cell nucleus by LLPS, and can be used as a proxy for HSV-1 VRCs with characteristics of liquid-liquid phase-separated condensates.


Assuntos
Regulação Viral da Expressão Gênica , Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Compartimentos de Replicação Viral , Animais , Núcleo Celular/metabolismo , Chlorocebus aethiops , Herpes Simples/genética , Herpes Simples/metabolismo , Proteínas Imediatamente Precoces/genética , Proteínas Intrinsicamente Desordenadas/genética , Extração Líquido-Líquido , Transição de Fase , Células Vero
9.
Front Cell Infect Microbiol ; 11: 603309, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33816328

RESUMO

Herpesviruses utilize various host factors to establish latent infection, survival, and spread disease in the host. These factors include host cellular machinery, host proteins, gene expression, multiple transcription factors, cellular signal pathways, immune cell activation, transcription factors, cytokines, angiogenesis, invasion, and factors promoting metastasis. The knowledge and understanding of host genes, protein products, and biochemical pathways lead to discovering safe and effective antivirals to prevent viral reactivation and spread infection. Here, we focus on the contribution of pro-inflammatory, anti-inflammatory, and resolution lipid metabolites of the arachidonic acid (AA) pathway in the lifecycle of herpesvirus infections. We discuss how various herpesviruses utilize these lipid pathways to their advantage and how we target them to combat herpesvirus infection. We also summarize recent development in anti-herpesvirus therapeutics and new strategies proposed or under clinical trials. These anti-herpesvirus therapeutics include inhibitors blocking viral life cycle events, engineered anticancer agents, epigenome influencing factors, immunomodulators, and therapeutic compounds from natural extracts.


Assuntos
Infecções por Herpesviridae , Herpesviridae , Regulação Viral da Expressão Gênica , Humanos , Ativação Viral , Latência Viral , Replicação Viral
10.
Arch Virol ; 166(6): 1795-1799, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33839920

RESUMO

In this study, bacteriophage BSP7, a novel Bacillus subtilis-infecting member of the family Siphoviridae, was isolated from a Korean soybean-based fermented food, Deonjang, using B. subtilis ATCC 21336 as a host. The genome is 55,455 bp long with 39.92% G+C content. A total of 70 ORFs with no tRNA were detected in the genome. A distinct feature of the BSP7 genome among B. subtilis-infecting Siphoviridae family phages is the presence of putative ORFs related to biosynthesis of 7-cyano-7-deazaguanine (PreQ0), a precursor of queuosine and archaeosine biosynthesis. Bioinformatic analysis revealed that the genome of BSP7 does not exhibit any significant similarities to other phages with sequences in the NCBI database. A comparative genomic analysis also confirmed the uniqueness of BSP7 within the family Siphoviridae.


Assuntos
Bacillus subtilis/virologia , Genoma Viral , Guanina/análogos & derivados , Siphoviridae/genética , Sequência de Bases , DNA Viral/genética , Regulação Viral da Expressão Gênica/fisiologia , Guanina/biossíntese , Siphoviridae/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
Nucleic Acids Res ; 49(8): 4386-4401, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823541

RESUMO

Bacteria persist under constant threat of predation by bacterial viruses (phages). Bacteria-phage conflicts result in evolutionary arms races often driven by mobile genetic elements (MGEs). One such MGE, a phage satellite in Vibrio cholerae called PLE, provides specific and robust defense against a pervasive lytic phage, ICP1. The interplay between PLE and ICP1 has revealed strategies for molecular parasitism allowing PLE to hijack ICP1 processes in order to mobilize. Here, we describe the mechanism of PLE-mediated transcriptional manipulation of ICP1 structural gene transcription. PLE encodes a novel DNA binding protein, CapR, that represses ICP1's capsid morphogenesis operon. Although CapR is sufficient for the degree of capsid repression achieved by PLE, its activity does not hinder the ICP1 lifecycle. We explore the consequences of repression of this operon, demonstrating that more stringent repression achieved through CRISPRi restricts both ICP1 and PLE. We also discover that PLE transduces in modified ICP1-like particles. Examination of CapR homologs led to the identification of a suite of ICP1-encoded homing endonucleases, providing a putative origin for the satellite-encoded repressor. This work unveils a facet of the delicate balance of satellite-mediated inhibition aimed at blocking phage production while successfully mobilizing in a phage-derived particle.


Assuntos
Proteínas de Bactérias/metabolismo , Bacteriófagos/crescimento & desenvolvimento , DNA Satélite/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Regulação Viral da Expressão Gênica , Sequências Repetitivas Dispersas , Vibrio cholerae/virologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bacteriófagos/genética , Sítios de Ligação , Sistemas CRISPR-Cas , Proteínas do Capsídeo/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Óperon/genética , Domínios Proteicos , Transdução Genética , Vibrio cholerae/enzimologia , Vibrio cholerae/genética , Vírion/genética , Vírion/crescimento & desenvolvimento
12.
Mar Drugs ; 19(5)2021 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-33922914

RESUMO

The high risk of morbidity and mortality associated with SARS-CoV-2 has accelerated the development of many potential vaccines. However, these vaccines are designed against SARS-CoV-2 isolated in Wuhan, China, and thereby may not be effective against other SARS-CoV-2 variants such as the United Kingdom variant (VUI-202012/01). The UK SARS-CoV-2 variant possesses D614G mutation in the Spike protein, which impart it a high rate of infection. Therefore, newer strategies are warranted to design novel vaccines and drug candidates specifically designed against the mutated forms of SARS-CoV-2. One such strategy is to target ACE2 (angiotensin-converting enzyme2)-Spike protein RBD (receptor binding domain) interaction. Here, we generated a homology model of Spike protein RBD of SARS-CoV-2 UK strain and screened a marine seaweed database employing different computational approaches. On the basis of high-throughput virtual screening, standard precision, and extra precision molecular docking, we identified BE011 (Dieckol) as the most potent compounds against RBD. However, Dieckol did not display drug-like properties, and thus different derivatives of it were generated in silico and evaluated for binding potential and drug-like properties. One Dieckol derivative (DK07) displayed good binding affinity for RBD along with acceptable physicochemical, pharmacokinetic, drug-likeness, and ADMET properties. Analysis of the RBD-DK07 interaction suggested the formation of hydrogen bonds, electrostatic interactions, and hydrophobic interactions with key residues mediating the ACE2-RBD interaction. Molecular dynamics simulation confirmed the stability of the RBD-DK07 complex. Free energy calculations suggested the primary role of electrostatic and Van der Waals' interaction in stabilizing the RBD-DK07 complex. Thus, DK07 may be developed as a potential inhibitor of the RBD-ACE2 interaction. However, these results warrant further validation by in vitro and in vivo studies.


Assuntos
Benzofuranos/química , Benzofuranos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Simulação por Computador , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Estrutura Molecular , Glicoproteína da Espícula de Coronavírus/metabolismo
13.
Int J Biol Sci ; 17(6): 1521-1529, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907515

RESUMO

The COVID-19 pandemic has been raging worldwide for more than a year. Many efforts have been made to create vaccines and develop new antiviral drugs to cope with the disease. Here, we propose the application of short interfering RNAs (siRNAs) to degrade the viral genome, thus reducing viral infection. By introducing the concept of the probability of binding efficiency (PBE) and combining the secondary structures of RNA molecules, we designed 11 siRNAs that target the consensus regions of three key viral genes: the spike (S), nucleocapsid (N) and membrane (M) genes of SARS-CoV-2. The silencing efficiencies of the siRNAs were determined in human lung and endothelial cells overexpressing these viral genes. The results suggested that most of the siRNAs could significantly reduce the expression of the viral genes with inhibition rates above 50% in 24 hours. This work not only provides a strategy for designing potentially effective siRNAs against target genes but also validates several potent siRNAs that can be used in the clinical development of preventative medication for COVID-19 in the future.


Assuntos
COVID-19/virologia , Regulação Viral da Expressão Gênica/fisiologia , Genes Virais , RNA Interferente Pequeno/fisiologia , SARS-CoV-2/genética , Células A549 , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutação , Probabilidade , Glicoproteína da Espícula de Coronavírus/genética
14.
Curr Med Sci ; 41(2): 297-305, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33877545

RESUMO

Since the outbreak of the novel corona virus disease 2019 (COVID-19) at the end of 2019, specific antiviral drugs have been lacking. A Chinese patent medicine Toujiequwen granules has been promoted in the treatment of COVID-19. The present study was designed to reveal the molecular mechanism of Toujiequwen granules against COVID-19. A network pharmacological method was applied to screen the main active ingredients of Toujiequwen granules. Network analysis of 149 active ingredients and 330 drug targets showed the most active ingredient interacting with many drug targets is quercetin. Drug targets most affected by the active ingredients were PTGS2, PTGS1, and DPP4. Drug target disease enrichment analysis showed drug targets were significantly enriched in cardiovascular diseases and digestive tract diseases. An "active ingredient-target-disease" network showed that 57 active ingredients from Toujiequwen granules interacted with 15 key targets of COVID-19. There were 53 ingredients that could act on DPP4, suggesting that DPP4 may become a potential new key target for the treatment of COVID-19. GO analysis results showed that key targets were mainly enriched in the cellular response to lipopolysaccharide, cytokine activity and other functions. KEGG analysis showed they were mainly concentrated in viral protein interaction with cytokine and cytokine receptors and endocrine resistance pathway. The evidence suggests that Toujiequwen granules might play an effective role by improving the symptoms of underlying diseases in patients with COVID-19 and multi-target interventions against multiple signaling pathways related to the pathogenesis of COVID-19.


Assuntos
COVID-19/tratamento farmacológico , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , SARS-CoV-2/genética , Antivirais/química , Antivirais/farmacologia , COVID-19/genética , COVID-19/virologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Dipeptidil Peptidase 4/genética , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/classificação , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Quercetina/genética , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/patogenicidade , Transdução de Sinais/efeitos dos fármacos
15.
Arch Virol ; 166(6): 1633-1642, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33787991

RESUMO

Pestivirus nonstructural protein 3 (NS3) is a multifunctional protein with protease and helicase activities that are essential for virus replication. In this study, we used a combination of biochemical and genetic approaches to investigate the relationship between a positively charged patch on the protease module and NS3 function. The surface patch is composed of four basic residues, R50, K74 and K94 in the NS3 protease domain and H24 in the structurally integrated cofactor NS4APCS. Single-residue or simultaneous four-residue substitutions in the patch to alanine or aspartic acid had little effect on ATPase activity. However, single substitutions of R50, K94 or H24 or a simultaneous four-residue substitution resulted in apparent changes in the helicase activity and RNA-binding ability of NS3. When these mutations were introduced into a classical swine fever virus (CSFV) cDNA clone, a single substitution at K94 or a simultaneous four-residue substitution (Qua_A or Qua_D) impaired the production of infectious virus. Furthermore, the replication efficiency of the CSFV variants was partially correlated with the helicase activity of NS3 in vitro. Our results suggest that the conserved positively charged patch on NS3 plays an important role in modulating the NS3 helicase activity in vitro and CSFV production.


Assuntos
Pestivirus/fisiologia , RNA Helicases/metabolismo , Proteínas não Estruturais Virais/metabolismo , Adenosina Trifosfatases , Sequência de Aminoácidos , Substituição de Aminoácidos , Escherichia coli , Regulação Viral da Expressão Gênica , Modelos Moleculares , Mutação , Pestivirus/genética , Conformação Proteica , RNA Helicases/genética , Serina Endopeptidases/metabolismo , Replicação Viral
16.
Gene ; 781: 145541, 2021 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-33667607

RESUMO

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.


Assuntos
Baculoviridae/genética , Citomegalovirus/genética , Regulação Viral da Expressão Gênica , Vetores Genéticos , Regiões Promotoras Genéticas/genética , Animais , Sítios de Ligação , Simulação por Computador , DNA Recombinante/genética , DNA Recombinante/metabolismo , Células HEK293 , Células HeLa , Humanos , Células Sf9 , Fatores de Transcrição/metabolismo
17.
PLoS Biol ; 19(3): e3001158, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33780434

RESUMO

Since its emergence in December 2019, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spread globally and become a major public health burden. Despite its close phylogenetic relationship to SARS-CoV, SARS-CoV-2 exhibits increased human-to-human transmission dynamics, likely due to efficient early replication in the upper respiratory epithelium of infected individuals. Since different temperatures encountered in the human upper and lower respiratory tract (33°C and 37°C, respectively) have been shown to affect the replication kinetics of several respiratory viruses, as well as host innate immune response dynamics, we investigated the impact of temperature on SARS-CoV-2 and SARS-CoV infection using the primary human airway epithelial cell culture model. SARS-CoV-2, in contrast to SARS-CoV, replicated to higher titers when infections were performed at 33°C rather than 37°C. Although both viruses were highly sensitive to type I and type III interferon pretreatment, a detailed time-resolved transcriptome analysis revealed temperature-dependent interferon and pro-inflammatory responses induced by SARS-CoV-2 that were inversely proportional to its replication efficiency at 33°C or 37°C. These data provide crucial insight on pivotal virus-host interaction dynamics and are in line with characteristic clinical features of SARS-CoV-2 and SARS-CoV, as well as their respective transmission efficiencies.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação Viral da Expressão Gênica/genética , Vírus da SARS/genética , SARS-CoV-2/genética , Animais , Antivirais/farmacologia , Células Cultivadas , Chlorocebus aethiops , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Humanos , Interferons/farmacologia , Vírus da SARS/efeitos dos fármacos , Vírus da SARS/fisiologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Especificidade da Espécie , Temperatura , Células Vero , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
18.
Infect Dis Poverty ; 10(1): 28, 2021 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-33726861

RESUMO

BACKGROUND: Coronaviruses (CoVs) are distributed worldwide and have various susceptible hosts; CoVs infecting humans are called human coronaviruses (HCoVs). Although HCoV-specific drugs are still lacking, many potent targets for drug discovery are being explored, and many vigorously designed clinical trials are being carried out in an orderly manner. The aim of this review was to gain a comprehensive understanding of the current status of drug development against HCoVs, particularly severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MAIN TEXT: A scoping review was conducted by electronically searching research studies, reviews, and clinical trials in PubMed and the CNKI. Studies on HCoVs and therapeutic drug discovery published between January 2000 and October 2020 and in English or Chinese were included, and the information was summarized. Of the 3248 studies identified, 159 publication were finally included. Advances in drug development against HCoV, especially SARS-CoV-2, are summarized under three categories: antiviral drugs aimed at inhibiting the HCoV proliferation process, drugs acting on the host's immune system, and drugs derived from plants with potent activity. Furthermore, clinical trials of drugs targeting SARS-CoV-2 are summarized. CONCLUSIONS: During the spread of COVID-19 outbreak, great efforts have been made in therapeutic drug discovery against the virus, although the pharmacological effects and adverse reactions of some drugs under study are still unclear. However, well-designed high-quality studies are needed to further study the effectiveness and safety of these potential drugs so as to provide valid recommendations for better control of the COVID-19 pandemic.


Assuntos
Antivirais/farmacologia , Infecções por Coronavirus/virologia , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Descoberta de Drogas , Antivirais/uso terapêutico , Biomarcadores , COVID-19/tratamento farmacológico , COVID-19/metabolismo , COVID-19/virologia , Coronavirus/classificação , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/metabolismo , Desenvolvimento de Medicamentos , Descoberta de Drogas/métodos , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Medicina Tradicional , Terapia de Alvo Molecular , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
19.
Arch Virol ; 166(6): 1783-1787, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33779811

RESUMO

A novel mycovirus with the proposed name "Magnaporthe oryzae botourmiavirus 9" (MoBV9) was found in the rice blast fungus Magnaporthe oryzae isolate SH05. The virus has a positive single-stranded RNA genome of 2,812 nucleotides and contains a single open reading frame predicted to encode an RNA-dependent RNA polymerase that is closely related to those of some unclassified viruses of the family Botourmiaviridae, including Plasmopara viticola lesion associated ourmia-like virus 44, Plasmopara viticola lesion associated ourmia-like virus 47, and Cladosporium uredinicola ourmiavirus 1. Genome sequence comparisons and phylogenetic analysis supported the notion that MoBV9 is a new member of the family Botourmiaviridae.


Assuntos
Ascomicetos/virologia , Micovírus/genética , Genoma Viral , Vírus de RNA/genética , RNA Viral/genética , Sequência de Aminoácidos , Micovírus/isolamento & purificação , Regulação Viral da Expressão Gênica , Filogenia , RNA Viral/isolamento & purificação , Proteínas Virais/genética , Proteínas Virais/metabolismo
20.
Infect Genet Evol ; 91: 104796, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33667722

RESUMO

SARS-CoV-2 is a betacoronavirus responsible for the COVID-19 pandemic that has affected millions of people worldwide. Pharmaceutical research against COVID-19 and the most frequently used tests for SARS-CoV-2 both depend on the genomic and peptide sequences of the virus for their robustness. Therefore, understanding the mutation rates and content of the virus is critical. Two key proteins for SARS-CoV-2 infection and replication are the S protein, responsible for viral entry into the cells, and RdRp, the RNA polymerase responsible for replicating the viral genome. Due to their roles in the viral cycle, these proteins are crucial for the fitness and infectiousness of the virus. Our previous findings had shown that the two most frequently observed mutations in the SARS-CoV-2 genome, 14408C>T in the RdRp coding region, and 23403A>G in the S gene, are correlated with higher mutation density over time. In this study, we further detail the selection dynamics and the mutation rates of SARS-CoV-2 genes, comparing them between isolates carrying both mutations, and isolates carrying neither. We find that the S gene and the RdRp coding region show the highest variance between the genotypes, and their selection dynamics contrast each other over time. The S gene displays higher tolerance for positive selection in mutant isolates early during the appearance of the double mutant genotype, and undergoes increasing negative selection over time, whereas the RdRp region in the mutant isolates shows strong negative selection throughout the pandemic.


Assuntos
COVID-19/epidemiologia , Genoma Viral , Mutação Puntual , RNA Polimerase Dependente de RNA/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética , COVID-19/transmissão , COVID-19/virologia , Evolução Molecular , Regulação Viral da Expressão Gênica , Genótipo , Humanos , Taxa de Mutação , Fases de Leitura Aberta , SARS-CoV-2/classificação , Seleção Genética , Reino Unido/epidemiologia , Estados Unidos/epidemiologia
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