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1.
Front Immunol ; 12: 700184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34408749

RESUMO

Coronavirus disease 2019 (COVID-19), which has high incidence rates with rapid rate of transmission, is a pandemic that spread across the world, resulting in more than 3,000,000 deaths globally. Currently, several drugs have been used for the clinical treatment of COVID-19, such as antivirals (radecivir, baritinib), monoclonal antibodies (tocilizumab), and glucocorticoids (dexamethasone). Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are essential regulators of virus infections and antiviral immune responses including biological processes that are involved in the regulation of COVID-19 and subsequent disease states. Upon viral infections, cellular lncRNAs directly regulate viral genes and influence viral replication and pathology through virus-mediated changes in the host transcriptome. Additionally, several host lncRNAs could help the occurrence of viral immune escape by inhibiting type I interferons (IFN-1), while others could up-regulate IFN-1 production to play an antiviral role. Consequently, understanding the expression and function of lncRNAs during severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection will provide insights into the development of lncRNA-based methods. In this review, we summarized the current findings of lncRNAs in the regulation of the strong inflammatory response, immune dysfunction and thrombosis induced by SARS-CoV-2 infection, discussed the underlying mechanisms, and highlighted the therapeutic challenges of COVID-19 treatment and its future research directions.


Assuntos
COVID-19/imunologia , Interações entre Hospedeiro e Microrganismos/genética , Imunidade Inata/genética , RNA Longo não Codificante/metabolismo , Trombose/imunologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Biomarcadores/análise , COVID-19/complicações , COVID-19/tratamento farmacológico , COVID-19/genética , Teste para COVID-19/métodos , Citocinas/genética , Citocinas/metabolismo , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/imunologia , Interações entre Hospedeiro e Microrganismos/efeitos dos fármacos , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Evasão da Resposta Imune/genética , Pandemias/prevenção & controle , RNA Longo não Codificante/análise , RNA Longo não Codificante/antagonistas & inibidores , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Trombose/genética , Trombose/virologia , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/imunologia
2.
Sci Rep ; 11(1): 16145, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373516

RESUMO

The genetic element s2m has been acquired through horizontal transfer by many distantly related viruses, including the SARS-related coronaviruses. Here we show that s2m is evolutionarily conserved in these viruses. Though several lineages of severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) devoid of the element can be found, these variants seem to have been short lived, indicating that they were less evolutionary fit than their s2m-containing counterparts. On a species-level, however, there do not appear to be any losses and this pattern strongly suggests that the s2m element is essential to virus replication in SARS-CoV-2 and related viruses. Further experiments are needed to elucidate the function of s2m.


Assuntos
Coronaviridae/genética , Sequências Repetitivas Dispersas/genética , RNA Viral/genética , SARS-CoV-2/genética , Replicação Viral/genética , Animais , Sequência de Bases , COVID-19/virologia , Coronaviridae/classificação , Evolução Molecular , Transferência Genética Horizontal , Humanos , Filogenia , SARS-CoV-2/fisiologia , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie
3.
Viruses ; 13(6)2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205979

RESUMO

There are over 100 different chemical RNA modifications, collectively known as the epitranscriptome. N6-methyladenosine (m6A) is the most commonly found internal RNA modification in cellular mRNAs where it plays important roles in the regulation of the mRNA structure, stability, translation and nuclear export. This modification is also found in viral RNA genomes and in viral mRNAs derived from both RNA and DNA viruses. A growing body of evidence indicates that m6A modifications play important roles in regulating viral replication by interacting with the cellular m6A machinery. In this review, we will exhaustively detail the current knowledge on m6A modification, with an emphasis on its function in virus biology.


Assuntos
Adenosina/análogos & derivados , Adenosina/genética , Epigênese Genética , Regulação Viral da Expressão Gênica , RNA Viral/genética , Adenosina/metabolismo , Animais , Interações Hospedeiro-Patógeno/imunologia , Humanos , Metilação , RNA Viral/metabolismo , Especificidade da Espécie , Transcrição Genética , Replicação Viral/genética
4.
Commun Biol ; 4(1): 858, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244608

RESUMO

The single-stranded, negative-sense, viral genomic RNA (vRNA) of influenza A virus is encapsidated by viral nucleoproteins (NPs) and an RNA polymerase to form a ribonucleoprotein complex (vRNP) with a helical, rod-shaped structure. The vRNP is responsible for transcription and replication of the vRNA. However, the vRNP conformation during RNA synthesis is not well understood. Here, using high-speed atomic force microscopy and cryo-electron microscopy, we investigated the native structure of influenza A vRNPs during RNA synthesis in vitro. Two distinct types of vRNPs were observed in association with newly synthesized RNAs: an intact, helical rod-shaped vRNP connected with a folded RNA and a deformed vRNP associated with a looped RNA. Interestingly, the looped RNA was a double-stranded RNA, which likely comprises a nascent RNA and the template RNA detached from NPs of the vRNP. These results suggest that while some vRNPs keep their helical structures during RNA synthesis, for the repeated cycle of RNA synthesis, others accidentally become structurally deformed, which likely results in failure to commence or continue RNA synthesis. Thus, our findings provide the ultrastructural feature of vRNPs during RNA synthesis.


Assuntos
Vírus da Influenza A/metabolismo , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Microscopia Crioeletrônica/métodos , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Influenza Humana/virologia , Microscopia de Força Atômica/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/ultraestrutura , Proteínas Virais/genética , Proteínas Virais/ultraestrutura , Replicação Viral/genética
5.
PLoS Biol ; 19(7): e3001333, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34252080

RESUMO

SARS-CoV-2 infections are characterized by viral proliferation and clearance phases and can be followed by low-level persistent viral RNA shedding. The dynamics of viral RNA concentration, particularly in the early stages of infection, can inform clinical measures and interventions such as test-based screening. We used prospective longitudinal quantitative reverse transcription PCR testing to measure the viral RNA trajectories for 68 individuals during the resumption of the 2019-2020 National Basketball Association season. For 46 individuals with acute infections, we inferred the peak viral concentration and the duration of the viral proliferation and clearance phases. According to our mathematical model, we found that viral RNA concentrations peaked an average of 3.3 days (95% credible interval [CI] 2.5, 4.2) after first possible detectability at a cycle threshold value of 22.3 (95% CI 20.5, 23.9). The viral clearance phase lasted longer for symptomatic individuals (10.9 days [95% CI 7.9, 14.4]) than for asymptomatic individuals (7.8 days [95% CI 6.1, 9.7]). A second test within 2 days after an initial positive PCR test substantially improves certainty about a patient's infection stage. The effective sensitivity of a test intended to identify infectious individuals declines substantially with test turnaround time. These findings indicate that SARS-CoV-2 viral concentrations peak rapidly regardless of symptoms. Sequential tests can help reveal a patient's progress through infection stages. Frequent, rapid-turnaround testing is needed to effectively screen individuals before they become infectious.


Assuntos
Teste de Ácido Nucleico para COVID-19/estatística & dados numéricos , COVID-19/diagnóstico , RNA Viral/genética , SARS-CoV-2/genética , Replicação Viral/genética , Eliminação de Partículas Virais/genética , Adulto , Atletas , Basquetebol , COVID-19/epidemiologia , COVID-19/patologia , COVID-19/virologia , Convalescença , Humanos , Masculino , Estudos Prospectivos , Saúde Pública/métodos , SARS-CoV-2/crescimento & desenvolvimento , Índice de Gravidade de Doença , Estados Unidos/epidemiologia
6.
Nat Commun ; 12(1): 4598, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34312390

RESUMO

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.


Assuntos
Anticorpos Neutralizantes/imunologia , Vacinas contra COVID-19/imunologia , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Adulto , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/genética , Feminino , Células HEK293 , Humanos , Soros Imunes/imunologia , Masculino , Pessoa de Meia-Idade , Mutação , Testes de Neutralização , SARS-CoV-2/genética , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Vacinação/métodos , Vírus da Estomatite Vesicular Indiana/genética , Vírus da Estomatite Vesicular Indiana/imunologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética , Replicação Viral/imunologia
7.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199658

RESUMO

Influenza A virus (IAV) causes seasonal epidemics and sporadic pandemics, therefore is an important research subject for scientists around the world. Despite the high variability of its genome, the structure of viral RNA (vRNA) possesses features that remain constant between strains and are biologically important for virus replication. Therefore, conserved structural motifs of vRNA can represent a novel therapeutic target. Here, we focused on the presence of G-rich sequences within the influenza A/California/07/2009(H1N1) genome and their ability to form RNA G-quadruplex structures (G4s). We identified 12 potential quadruplex-forming sequences (PQS) and determined their conservation among the IAV strains using bioinformatics tools. Then we examined the propensity of PQS to fold into G4s by various biophysical methods. Our results revealed that six PQS oligomers could form RNA G-quadruplexes. However, three of them were confirmed to adopt G4 structures by all utilized methods. Moreover, we showed that these PQS motifs are present within segments encoding polymerase complex proteins indicating their possible role in the virus biology.


Assuntos
Quadruplex G , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A/genética , Influenza Humana/genética , Biologia Computacional , Genoma Viral/efeitos dos fármacos , Genoma Viral/genética , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , RNA Viral/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genética
8.
EMBO J ; 40(17): e107776, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34232536

RESUMO

Host-virus protein-protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity-labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Further characterization of these interactions and comparison to other large-scale study of cellular responses to SARS-CoV-2 infection elucidated how distinct SARS-CoV-2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID-19. The interactomes of two key SARS-CoV-2-encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS-CoV-2 provides valuable resources for the understanding and treating of this disease.


Assuntos
COVID-19/genética , Proteínas do Nucleocapsídeo de Coronavírus/genética , SARS-CoV-2/genética , Proteínas não Estruturais Virais/genética , COVID-19/patologia , COVID-19/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Mapas de Interação de Proteínas/genética , SARS-CoV-2/patogenicidade , Replicação Viral/genética
9.
Nat Commun ; 12(1): 4314, 2021 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-34262047

RESUMO

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.


Assuntos
Pneumopatias/genética , SARS-CoV-2/fisiologia , Transcriptoma , Internalização do Vírus , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/genética , COVID-19/patologia , Doença Crônica , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/patologia , Imunidade Inata/genética , Inflamação/genética , Pulmão/metabolismo , Pulmão/patologia , Pneumopatias/patologia , SARS-CoV-2/patogenicidade , Replicação Viral/genética
10.
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
11.
J Biol Chem ; 297(1): 100856, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097873

RESUMO

The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.


Assuntos
Imunidade Inata/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/genética , Viroses/genética , Transporte Ativo do Núcleo Celular/genética , Transporte Ativo do Núcleo Celular/imunologia , Vírus de DNA/genética , Vírus de DNA/patogenicidade , Humanos , Evasão da Resposta Imune/genética , Evasão da Resposta Imune/imunologia , NF-kappa B/genética , Poro Nuclear/imunologia , Complexo de Proteínas Formadoras de Poros Nucleares/imunologia , Vírus de RNA/genética , Vírus de RNA/patogenicidade , Proteínas não Estruturais Virais/genética , Viroses/imunologia , Viroses/virologia , Replicação Viral/genética , Replicação Viral/imunologia
12.
Viruses ; 13(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064911

RESUMO

If the goal of eliminating dog-mediated human rabies by 2030 is to be achieved, effective mass dog vaccination needs to be complemented by effective prophylaxis for individuals exposed to rabies. Aptamers and short-interfering RNAs (siRNAs) have been successful in therapeutics, but few studies have investigated their potential as rabies therapeutics. In this study, siRNAs and aptamers-using a novel selection method-were developed and tested against rabies virus (RABV) in a post-infection (p.i.) scenario. Multiple means of delivery were tested for siRNAs, including the use of Lipofectamine and conjugation with the developed aptamers. One siRNA (N53) resulted in an 80.13% reduction in viral RNA, while aptamer UPRET 2.03 demonstrated a 61.3% reduction when used alone at 2 h p.i. At 24 h p.i., chimera UPRET 2.03-N8 (aptamer-siRNA) resulted in a 36.5% inhibition of viral replication. To our knowledge, this is the first study using siRNAs or aptamers that (1) demonstrated significant inhibition of RABV using an aptamer, (2) tested Lipofectamine RNAi-Max as a means for delivery, and (3) produced significant RABV inhibition at 24 h p.i. This study serves as a proof-of-concept to potentially use aptamers and siRNAs as rabies immunoglobulin (RIG) replacements or therapeutic options for RABV and provides strong evidence towards their further investigation.


Assuntos
Vírus da Raiva , Raiva/prevenção & controle , Raiva/terapia , Animais , Aptâmeros de Nucleotídeos , Células Cultivadas , Ensaios Clínicos como Assunto , Gerenciamento Clínico , Modelos Animais de Doenças , Terapia Genética/métodos , Humanos , Pré-Medicação , Interferência de RNA , RNA Interferente Pequeno/genética , Raiva/virologia , Vírus da Raiva/genética , Técnica de Seleção de Aptâmeros , Replicação Viral/genética
13.
Infect Genet Evol ; 93: 104973, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34147651

RESUMO

SARS-CoV-2 is currently causing major havoc worldwide with its efficient transmission and propagation. To track the emergence as well as the persistence of mutations during the early stage of the pandemic, a comparative analysis of SARS-CoV-2 whole proteome sequences has been performed by considering manually curated 31,389 whole genome sequences from 84 countries. Among the 7 highly recurring (percentage frequency≥10%) mutations (Nsp2:T85I, Nsp6:L37F, Nsp12:P323L, Spike:D614G, ORF3a:Q57H, N protein:R203K and N protein:G204R), N protein:R203K and N protein: G204R are co-occurring (dependent) mutations. Nsp12:P323L and Spike:D614G often appear simultaneously. The highly recurring Spike:D614G, Nsp12:P323L and Nsp6:L37F as well as moderately recurring (percentage frequency between ≥1 and <10%) ORF3a:G251V and ORF8:L84S mutations have led to4 major clades in addition to a clade that lacks high recurring mutations. Further, the occurrence of ORF3a:Q57H&Nsp2:T85I, ORF3a:Q57H and N protein:R203K&G204R along with Nsp12:P323L&Spike:D614G has led to 3 additional sub-clades. Similarly, occurrence of Nsp6:L37F and ORF3a:G251V together has led to the emergence of a sub-clade. Nonetheless, ORF8:L84S does not occur along with ORF3a:G251V or Nsp6:L37F. Intriguingly, ORF3a:G251V and ORF8:L84S are found to occur independent of Nsp12:P323L and Spike:D614G mutations. These clades have evolved during the early stage of the pandemic and have disseminated across several countries. Further, Nsp10 is found to be highly resistant to mutations, thus, it can be exploited for drug/vaccine development and the corresponding gene sequence can be used for the diagnosis. Concisely, the study reports the SARS-CoV-2 antigens diversity across the globe during the early stage of the pandemic and facilitates the understanding of viral evolution.


Assuntos
COVID-19/virologia , Mutação , SARS-CoV-2/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Evolução Biológica , COVID-19/epidemiologia , Hospitalização , Humanos , Proteoma/genética , Proteoma/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/genética , Replicação Viral/genética , Sequenciamento Completo do Genoma
14.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
15.
Cell ; 184(13): 3474-3485.e11, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34143953

RESUMO

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.


Assuntos
RNA-Polimerase RNA-Dependente de Coronavírus/genética , Exorribonucleases/genética , Metiltransferases/genética , SARS-CoV-2/genética , Sequência de Aminoácidos , COVID-19/virologia , Humanos , RNA Mensageiro/genética , RNA Viral/genética , Alinhamento de Sequência , Transcrição Genética/genética , Replicação Viral/genética
16.
Cells ; 10(6)2021 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-34070971

RESUMO

The recent SARS-CoV-2 pandemic has refocused attention to the betacoronaviruses, only eight years after the emergence of another zoonotic betacoronavirus, the Middle East respiratory syndrome coronavirus (MERS-CoV). While the wild source of SARS-CoV-2 may be disputed, for MERS-CoV, dromedaries are considered as source of zoonotic human infections. Testing 100 immune-response genes in 121 dromedaries from United Arab Emirates (UAE) for potential association with present MERS-CoV infection, we identified candidate genes with important functions in the adaptive, MHC-class I (HLA-A-24-like) and II (HLA-DPB1-like), and innate immune response (PTPN4, MAGOHB), and in cilia coating the respiratory tract (DNAH7). Some of these genes previously have been associated with viral replication in SARS-CoV-1/-2 in humans, others have an important role in the movement of bronchial cilia. These results suggest similar host genetic pathways associated with these betacoronaviruses, although further work is required to better understand the MERS-CoV disease dynamics in both dromedaries and humans.


Assuntos
Imunidade Adaptativa/genética , Camelus/virologia , Doenças Transmissíveis Emergentes/imunologia , Infecções por Coronavirus/imunologia , Imunidade Inata/genética , Zoonoses/imunologia , Animais , Anticorpos Antivirais , Brônquios/citologia , Brônquios/fisiologia , COVID-19/genética , COVID-19/imunologia , COVID-19/virologia , Camelus/genética , Camelus/imunologia , Cílios/fisiologia , Doenças Transmissíveis Emergentes/genética , Doenças Transmissíveis Emergentes/transmissão , Doenças Transmissíveis Emergentes/virologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Reservatórios de Doenças/virologia , Feminino , Predisposição Genética para Doença , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Masculino , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Mucosa Respiratória/citologia , Mucosa Respiratória/fisiologia , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Emirados Árabes Unidos , Replicação Viral/genética , Replicação Viral/imunologia , Zoonoses/genética , Zoonoses/transmissão , Zoonoses/virologia
17.
J Biol Chem ; 297(1): 100845, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34052228

RESUMO

Alzheimer's disease (AD) is a devastating fatal neurodegenerative disease. An alternative to the amyloid cascade hypothesis is that a viral infection is key to the etiology of late-onset AD, with ß-amyloid (Aß) peptides playing a protective role. In the current study, young 5XFAD mice that overexpress mutant human amyloid precursor protein with the Swedish, Florida, and London familial AD mutations were infected with one of two strains of herpes simplex virus 1 (HSV-1), 17syn+ and McKrae, at three different doses. Contrary to previous work, 5XFAD genotype failed to protect mice against HSV-1 infection. The region- and cell-specific tropisms of HSV-1 were not affected by the 5XFAD genotype, indicating that host-pathogen interactions were not altered. Seven- to ten-month-old 5XFAD animals in which extracellular Aß aggregates were abundant showed slightly better survival rate relative to their wild-type (WT) littermates, although the difference was not statistically significant. In these 5XFAD mice, HSV-1 replication centers were partially excluded from the brain areas with high densities of Aß aggregates. Aß aggregates were free of HSV-1 viral particles, and the limited viral invasion to areas with a high density of Aß aggregates was attributed to phagocytic activity of reactive microglia. In the oldest mice (12-15 months old), the survival rate did not differ between 5XFAD and WT littermates. While the current study questions the antiviral role of Aß, it neither supports nor refutes the viral etiology hypothesis of late-onset AD.


Assuntos
Peptídeos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Interações Hospedeiro-Patógeno/genética , Viroses/genética , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Doença de Alzheimer/virologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Herpes Simples/genética , Herpes Simples/patologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidade , Humanos , Camundongos , Camundongos Transgênicos , Microglia/patologia , Microglia/virologia , Presenilina-1/genética , Viroses/complicações , Viroses/patologia , Viroses/virologia , Replicação Viral/genética
18.
J Virol ; 95(15): e0097120, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011540

RESUMO

HIV-1 encodes several accessory proteins-Nef, Vif, Vpr, and Vpu-whose functions are to modulate the cellular environment to favor immune evasion and viral replication. While Vpr was shown to mediate a G2/M cell cycle arrest and provide a replicative advantage during infection of myeloid cells, the mechanisms underlying these functions remain unclear. In this study, we defined HIV-1 Vpr proximity interaction network using the BioID proximity labeling approach and identified 352 potential Vpr partners/targets, including several complexes, such as the cell cycle-regulatory anaphase-promoting complex/cyclosome (APC/C). Herein, we demonstrate that both the wild type and cell cycle-defective mutants of Vpr induce the degradation of APC1, an essential APC/C scaffolding protein, and show that this activity relies on the recruitment of DCAF1 by Vpr and the presence of a functional proteasome. Vpr forms a complex with APC1, and the APC/C coactivators Cdh1 and Cdc20 are associated with these complexes. Interestingly, we found that Vpr encoded by the prototypic HIV-1 NL4.3 does not interact efficiently with APC1 and is unable to mediate its degradation as a result of a N28S-G41N amino acid substitution. In contrast, we show that APC1 degradation is a conserved feature of several primary Vpr variants from transmitted/founder virus. Functionally, Vpr-mediated APC1 degradation did not impact the ability of the protein to induce a G2 cell cycle arrest during infection of CD4+ T cells or enhance HIV-1 replication in macrophages, suggesting that this conserved activity may be important for other aspects of HIV-1 pathogenesis. IMPORTANCE The function of the Vpr accessory protein during HIV-1 infection remains poorly defined. Several cellular targets of Vpr were previously identified, but their individual degradation does not fully explain the ability of Vpr to impair the cell cycle or promote HIV-1 replication in macrophages. Here, we used the unbiased proximity labeling approach, called BioID, to further define the Vpr proximity interaction network and identified several potentially new Vpr partners/targets. We validated our approach by focusing on a cell cycle master regulator, the APC/C complex, and demonstrated that Vpr mediated the degradation of a critical scaffolding component of APC/C called APC1. Furthermore, we showed that targeting of APC/C by Vpr did not impact the known activity of Vpr. Since degradation of APC1 is a conserved feature of several primary variants of Vpr, it is likely that the interplay between Vpr and APC/C governs other aspects of HIV-1 pathogenesis.


Assuntos
Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Infecções por HIV/patologia , HIV-1/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Replicação Viral/genética , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/metabolismo , Linfócitos T CD4-Positivos/virologia , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Células HEK293 , HIV-1/metabolismo , Células HeLa , Humanos , Macrófagos/virologia , Interferência de RNA , RNA Interferente Pequeno/genética , Espectrometria de Massas em Tandem , Produtos do Gene vpr do Vírus da Imunodeficiência Humana/genética
19.
J Virol ; 95(15): e0013121, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011541

RESUMO

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus of chickens that causes lymphomas in various organs. Most MDV genes are conserved among herpesviruses, while others are unique to MDV and may contribute to pathogenesis and/or tumor formation. High transcript levels of the MDV-specific genes MDV082, RLORF11, and SORF6 were recently detected in lytically infected cells; however, it remained elusive if the respective proteins are expressed and if they play a role in MDV pathogenesis. In this study, we first addressed if these proteins are expressed by inserting FLAG tags at their N or C termini. We could demonstrate that among the three genes tested, MDV082 is the only gene that encodes a protein and is expressed very late in MDV plaques in vitro. To investigate the role of this novel MDV082 protein in MDV pathogenesis, we generated a recombinant virus that lacks expression of the MDV082 protein. Our data revealed that the MDV082 protein contributes to the rapid onset of Marek's disease but is not essential for virus replication, spread, and tumor formation. Taken together, this study sheds light on the expression of MDV-specific genes and unravels the role of the late protein MDV082 in MDV pathogenesis. IMPORTANCE MDV is a highly oncogenic alphaherpesvirus that causes Marek's disease in chickens. The virus causes immense economic losses in the poultry industry due to the high morbidity and mortality, but also the cost of the vaccination. MDV encodes over 100 genes that are involved in various processes of the viral life cycle. Functional characterization of MDV genes is an essential step toward understanding the complex virus life cycle and MDV pathogenesis. Here, we have identified a novel protein encoded by MDV082 and two potential noncoding RNAs (RLORF11 and SORF6). The novel MDV082 protein is not needed for efficient MDV replication and tumor formation. However, our data demonstrate that the MDV082 protein is involved in the rapid onset of Marek's disease.


Assuntos
Transformação Celular Viral/genética , Herpesvirus Galináceo 2/genética , Doença de Marek/virologia , Proteínas Virais/genética , Animais , Linhagem Celular , Galinhas/virologia , Aves Domésticas/virologia , Replicação Viral/genética
20.
J Virol ; 95(15): e0077721, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34011549

RESUMO

Venezuelan equine encephalitis virus (VEEV) is a reemerging arthropod-borne virus causing encephalitis in humans and domesticated animals. VEEV possesses a positive single-stranded RNA genome capped at its 5' end. The capping process is performed by the nonstructural protein nsP1, which bears methyl and guanylyltransferase activities. The capping reaction starts with the methylation of GTP. The generated m7GTP is complexed to the enzyme to form an m7GMP-nsP1 covalent intermediate. The m7GMP is then transferred onto the 5'-diphosphate end of the viral RNA. Here, we explore the specificities of the acceptor substrate in terms of length, RNA secondary structure, and/or sequence. Any diphosphate nucleosides but GDP can serve as acceptors of the m7GMP to yield m7GpppA, m7GpppC, or m7GpppU. We show that capping is more efficient on small RNA molecules, whereas RNAs longer than 130 nucleotides are barely capped by the enzyme. The structure and sequence of the short, conserved stem-loop, downstream to the cap, is an essential regulatory element for the capping process. IMPORTANCE The emergence, reemergence, and expansion of alphaviruses (genus of the family Togaviridae) are a serious public health and epizootic threat. Venezuelan equine encephalitis virus (VEEV) causes encephalitis in human and domesticated animals, with a mortality rate reaching 80% in horses. To date, no efficient vaccine or safe antivirals are available for human use. VEEV nonstructural protein 1 (nsP1) is the viral capping enzyme characteristic of the Alphavirus genus. nsP1 catalyzes methyltransferase and guanylyltransferase reactions, representing a good therapeutic target. In the present report, we provide insights into the molecular features and specificities of the cap acceptor substrate for the guanylylation reaction.


Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Capuzes de RNA/genética , RNA Viral/genética , Proteínas não Estruturais Virais/metabolismo , Replicação Viral/genética , Animais , Encefalomielite Equina Venezuelana/patologia , Encefalomielite Equina Venezuelana/virologia , Cavalos , Humanos , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , Nucleotidiltransferases/metabolismo , Proteínas não Estruturais Virais/genética
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