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1.
Int J Biol Sci ; 16(13): 2464-2476, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760213

RESUMO

In 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused infections worldwide. However, the correlation between the immune infiltration and coronavirus disease 2019 (COVID-19) susceptibility or severity in cancer patients remains to be fully elucidated. ACE2 expressions in normal tissues, cancers and cell lines were comprehensively assessed. Furthermore, we compared ACE2 expression between cancers and matched normal tissues through Gene Expression Profiling Interactive Analysis (GEPIA). In addition, we performed gene set enrichment analysis (GSEA) to investigate the related signaling pathways. Finally, the correlations between ACE2 expression and immune infiltration were investigated via Tumor Immune Estimation Resource (TIMER) and GEPIA. We found that ACE2 was predominantly expressed in both adult and fetal tissues from the digestive, urinary and male reproductive tracts; moreover, ACE2 expressions in corresponding cancers were generally higher than that in matched healthy tissues. GSEA showed that various metabolic and immune-related pathways were significantly associated with ACE2 expression across multiple cancer types. Intriguingly, we found that ACE2 expression correlated significantly with immune cell infiltration in both normal and cancer tissues, especially in the stomach and colon. These findings proposed a possible fecal-oral and maternal-fetal transmission of SARS-CoV-2 and suggested that cancers of the respiratory, digestive or urinary tracts would be more vulnerable to SARS-CoV-2 infection.


Assuntos
Biologia Computacional , Infecções por Coronavirus/imunologia , Neoplasias/imunologia , Pneumonia Viral/imunologia , Adulto , Betacoronavirus , Infecções por Coronavirus/complicações , Enterócitos/metabolismo , Células Epiteliais/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Genótipo , Células Caliciformes/metabolismo , Hepatócitos/metabolismo , Humanos , Sistema Imunitário , Túbulos Renais/embriologia , Masculino , Neoplasias/complicações , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/complicações , Prognóstico , RNA-Seq , Transdução de Sinais
2.
Genes (Basel) ; 11(8)2020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32785186

RESUMO

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.


Assuntos
Galinhas/genética , Galinhas/virologia , Infecções por Coronavirus/veterinária , Interações Hospedeiro-Patógeno/genética , Proteínas de Membrana/genética , Animais , Infecções por Coronavirus/genética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno/fisiologia , Vírus da Bronquite Infecciosa/patogenicidade , Vírus da Bronquite Infecciosa/fisiologia , Técnicas de Cultura de Órgãos , Doenças das Aves Domésticas/etiologia , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , Carga Viral , Tropismo Viral
3.
Proc Natl Acad Sci U S A ; 117(29): 17240-17248, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32632017

RESUMO

Probabilistic bet hedging, a strategy to maximize fitness in unpredictable environments by matching phenotypic variability to environmental variability, is theorized to account for the evolution of various fate-specification decisions, including viral latency. However, the molecular mechanisms underlying bet hedging remain unclear. Here, we report that large variability in protein abundance within individual herpesvirus virion particles enables probabilistic bet hedging between viral replication and latency. Superresolution imaging of individual virions of the human herpesvirus cytomegalovirus (CMV) showed that virion-to-virion levels of pp71 tegument protein-the major viral transactivator protein-exhibit extreme variability. This super-Poissonian tegument variability promoted alternate replicative strategies: high virion pp71 levels enhance viral replicative fitness but, strikingly, impede silencing, whereas low virion pp71 levels reduce fitness but promote silencing. Overall, the results indicate that stochastic tegument packaging provides a mechanism enabling probabilistic bet hedging between viral replication and latency.


Assuntos
Citomegalovirus/genética , Citomegalovirus/fisiologia , Proteínas Virais/metabolismo , Latência Viral/genética , Latência Viral/fisiologia , Evolução Biológica , Infecções por Citomegalovirus , Regulação Viral da Expressão Gênica , Humanos , Monócitos , Vírion/metabolismo , Replicação Viral
4.
Fertil Steril ; 114(1): 33-43, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32622411

RESUMO

OBJECTIVE: To identify cell types in the male and female reproductive systems at risk for SARS-CoV-2 infection because of the expression of host genes and proteins used by the virus for cell entry. DESIGN: Descriptive analysis of transcriptomic and proteomic data. SETTING: Academic research department and clinical diagnostic laboratory. PATIENT(S): Not applicable (focus was on previously generated gene and protein expression data). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Identification of cell types coexpressing the key angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) genes and proteins as well as other candidates potentially involved in SARS-CoV-2 cell entry. RESULT(S): On the basis of single-cell RNA sequencing data, coexpression of ACE2 and TMPRSS2 was not detected in testicular cells, including sperm. A subpopulation of oocytes in nonhuman primate ovarian tissue was found to express ACE2 and TMPRSS2, but coexpression was not observed in ovarian somatic cells. RNA expression of TMPRSS2 in 18 samples of human cumulus cells was shown to be low or absent. There was general agreement between publicly available bulk RNA and protein datasets in terms of ACE2 and TMPRSS2 expression patterns in testis, ovary, endometrial, and placental cells. CONCLUSION(S): These analyses suggest that SARS-CoV-2 infection is unlikely to have long-term effects on male and female reproductive function. Although the results cannot be considered definitive, they imply that procedures in which oocytes are collected and fertilized in vitro are associated with very little risk of viral transmission from gametes to embryos and may indeed have the potential to minimize exposure of susceptible reproductive cell types to infection in comparison with natural conception.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Fertilidade/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Pneumonia Viral/metabolismo , Reprodução/fisiologia , Internalização do Vírus , Adolescente , Adulto , Animais , Betacoronavirus/genética , Linhagem Celular , Infecções por Coronavirus/genética , Feminino , Humanos , Macaca fascicularis , Masculino , Ovário/citologia , Ovário/metabolismo , Ovário/virologia , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Gravidez , Proteômica/métodos , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Testículo/citologia , Testículo/metabolismo , Testículo/virologia , Transcriptoma/fisiologia , Adulto Jovem
5.
Nat Commun ; 11(1): 3345, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620802

RESUMO

Nonsense-mediated mRNA decay (NMD) is an evolutionarily conserved RNA decay mechanism that has emerged as a potent cell-intrinsic restriction mechanism of retroviruses and positive-strand RNA viruses. However, whether NMD is capable of restricting DNA viruses is not known. The DNA virus Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma and primary effusion lymphoma (PEL). Here, we demonstrate that NMD restricts KSHV lytic reactivation. Leveraging high-throughput transcriptomics we identify NMD targets transcriptome-wide in PEL cells and identify host and viral RNAs as substrates. Moreover, we identified an NMD-regulated link between activation of the unfolded protein response and transcriptional activation of the main KSHV transcription factor RTA, itself an NMD target. Collectively, our study describes an intricate relationship between cellular targets of an RNA quality control pathway and KSHV lytic gene expression, and demonstrates that NMD can function as a cell intrinsic restriction mechanism acting upon DNA viruses.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/genética , Degradação do RNAm Mediada por Códon sem Sentido , RNA Viral/metabolismo , Ativação Viral/genética , Linhagem Celular Tumoral , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/virologia , RNA Mensageiro/metabolismo , RNA-Seq , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/virologia , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional , Resposta a Proteínas não Dobradas/genética , Latência Viral/genética
6.
PLoS Pathog ; 16(6): e1008589, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32603362

RESUMO

Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-induced sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is found in all KS-lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of KS-spindle cells that have lost KSHV and proliferate autonomously or via paracrine mechanisms. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although infected cells that lose the KSHV-episome prior to tumor formation lose their tumorigenicity, explanted tumor cells that lost the KSHV-episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV tumorigenesis supporting the possibility of hit and run viral-sarcomagenesis. RNA-sequencing and CpG-methylation analysis were performed on KSHV-positive and KSHV-negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV-positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors occurring en exactly the same position, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV-negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V and other proposed mechanism, could be responsible for driving tumorigenesis in the absence of KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of "hit and run" KSHV-sarcomagenesis and point to the existence of selectable KSHV-induced host mutations that may impact AIDS-KS treatment.


Assuntos
Transformação Celular Viral , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8 , Neoplasias Experimentais , Plasmídeos , Sarcoma de Kaposi , Animais , Linhagem Celular , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/virologia , Plasmídeos/genética , Plasmídeos/metabolismo , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/patologia , Sarcoma de Kaposi/virologia
7.
Fertil Steril ; 114(2): 223-232, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641214

RESUMO

OBJECTIVE: To determine the susceptibility of the endometrium to infection by-and thereby potential damage from-SARS-CoV-2. DESIGN: Analysis of SARS-Cov-2 infection-related gene expression from endometrial transcriptomic data sets. SETTING: Infertility research department affiliated with a public hospital. PATIENT(S): Gene expression data from five studies in 112 patients with normal endometrium collected throughout the menstrual cycle. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Gene expression and correlation between viral infectivity genes and age throughout the menstrual cycle. RESULT(S): Gene expression was high for TMPRSS4, CTSL, CTSB, FURIN, MX1, and BSG; medium for TMPRSS2; and low for ACE2. ACE2, TMPRSS4, CTSB, CTSL, and MX1 expression increased toward the window of implantation. TMPRSS4 expression was positively correlated with ACE2, CTSB, CTSL, MX1, and FURIN during several cycle phases; TMPRSS2 was not statistically significantly altered across the cycle. ACE2, TMPRSS4, CTSB, CTSL, BSG, and MX1 expression increased with age, especially in early phases of the cycle. CONCLUSION(S): Endometrial tissue is likely safe from SARS-CoV-2 cell entry based on ACE2 and TMPRSS2 expression, but susceptibility increases with age. Further, TMPRSS4, along with BSG-mediated viral entry into cells, could imply a susceptible environment for SARS-CoV-2 entry via different mechanisms. Additional studies are warranted to determine the true risk of endometrial infection by SARS-CoV-2 and implications for fertility treatments.


Assuntos
Betacoronavirus/metabolismo , Infecções por Coronavirus/metabolismo , Endométrio/metabolismo , Endométrio/virologia , Regulação Viral da Expressão Gênica , Pneumonia Viral/metabolismo , Adulto , Fatores Etários , Betacoronavirus/genética , Infecções por Coronavirus/genética , Feminino , Humanos , Ciclo Menstrual , Pessoa de Meia-Idade , Pandemias , Peptidil Dipeptidase A/biossíntese , Peptidil Dipeptidase A/genética , Pneumonia Viral/genética , Medição de Risco/métodos , Internalização do Vírus , Adulto Jovem
8.
PLoS Pathog ; 16(7): e1008671, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32614923

RESUMO

Viral infection outcomes are governed by the complex and dynamic interplay between the infecting virus population and the host response. It is increasingly clear that both viral and host cell populations are highly heterogeneous, but little is known about how this heterogeneity influences infection dynamics or viral pathogenicity. To dissect the interactions between influenza A virus (IAV) and host cell heterogeneity, we examined the combined host and viral transcriptomes of thousands of individual cells, each infected with a single IAV virion. We observed complex patterns of viral gene expression and the existence of multiple distinct host transcriptional responses to infection at the single cell level. We show that human H1N1 and H3N2 strains differ significantly in patterns of both viral and host anti-viral gene transcriptional heterogeneity at the single cell level. Our analyses also reveal that semi-infectious particles that fail to express the viral NS can play a dominant role in triggering the innate anti-viral response to infection. Altogether, these data reveal how patterns of viral population heterogeneity can serve as a major determinant of antiviral gene activation.


Assuntos
Regulação Viral da Expressão Gênica/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Células A549 , Humanos , Imunidade Inata/imunologia , Proteínas não Estruturais Virais/imunologia
9.
J Biol Chem ; 295(31): 10741-10748, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32571880

RESUMO

Approximately 17 years after the severe acute respiratory syndrome coronavirus (SARS-CoV) epidemic, the world is currently facing the COVID-19 pandemic caused by SARS corona virus 2 (SARS-CoV-2). According to the most optimistic projections, it will take more than a year to develop a vaccine, so the best short-term strategy may lie in identifying virus-specific targets for small molecule-based interventions. All coronaviruses utilize a molecular mechanism called programmed -1 ribosomal frameshift (-1 PRF) to control the relative expression of their proteins. Previous analyses of SARS-CoV have revealed that it employs a structurally unique three-stemmed mRNA pseudoknot that stimulates high -1 PRF rates and that it also harbors a -1 PRF attenuation element. Altering -1 PRF activity impairs virus replication, suggesting that this activity may be therapeutically targeted. Here, we comparatively analyzed the SARS-CoV and SARS-CoV-2 frameshift signals. Structural and functional analyses revealed that both elements promote similar -1 PRF rates and that silent coding mutations in the slippery sites and in all three stems of the pseudoknot strongly ablate -1 PRF activity. We noted that the upstream attenuator hairpin activity is also functionally retained in both viruses, despite differences in the primary sequence in this region. Small-angle X-ray scattering analyses indicated that the pseudoknots in SARS-CoV and SARS-CoV-2 have the same conformation. Finally, a small molecule previously shown to bind the SARS-CoV pseudoknot and inhibit -1 PRF was similarly effective against -1 PRF in SARS-CoV-2, suggesting that such frameshift inhibitors may be promising lead compounds to combat the current COVID-19 pandemic.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/tratamento farmacológico , Desenho de Fármacos , Mudança da Fase de Leitura do Gene Ribossômico/efeitos dos fármacos , Pneumonia Viral/tratamento farmacológico , RNA Viral/genética , Betacoronavirus/química , Regulação Viral da Expressão Gênica , Humanos , Pandemias , RNA Viral/química , Replicação Viral/efeitos dos fármacos
10.
Antiviral Res ; 180: 104860, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32565134

RESUMO

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory in human with high mortality and it has been a challenge to determine optimum treatment for MERS-CoV-induced respiratory infection. Here, we observed the distribution of MERS-CoV receptors using human respiratory mucosa and also evaluated the contribution of interferon-lambdas (IFN-λs) in response to MERS-CoV infection using in vitro normal human nasal epithelial (NHNE) and bronchial epithelial (NHBE) cells. We found that the gene and protein expression of DPPIV, MERS-CoV receptor, were more dominantly located in nasal and bronchial epithelium although human nasal mucosa exhibited relatively lower DPPIV expression than lung parenchymal tissues. The quantitative mRNA level of the MERS-CoV envelope (upE) gene was significantly induced in MERS-CoV-infected cultured NHNE and NHBE cells until 3 days after infection. The induction of IFNs was identified in NHNE and NHBE cells after MERS-CoV infection and IFN-λs were predominantly increased in MERS-CoV-infected respiratory epithelial cells. Inoculation of IFN-λs to NHNE and NHBE cells suppressed MERS-CoV replication and in particular, IFN-λ4 showed a strong therapeutic effect in reducing MERS-CoV infection with higher induction of IFN-stimulated genes. Thus, IFN-λ has a decisive function in the respiratory epithelium that greatly limits MERS-CoV replication, and may be a key cytokine for better therapeutic outcomes against MERS-CoV infection in respiratory tract.


Assuntos
Antivirais/uso terapêutico , Interferons/uso terapêutico , Interleucinas/uso terapêutico , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Mucosa Respiratória/virologia , Replicação Viral/efeitos dos fármacos , Brônquios/virologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Células Epiteliais/virologia , Regulação Viral da Expressão Gênica , Humanos , Imunidade Inata/imunologia , Interferons/biossíntese , Interleucinas/biossíntese , Mucosa Laríngea/virologia , Pulmão/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Reação em Cadeia da Polimerase , Mucosa Respiratória/efeitos dos fármacos
11.
Am J Physiol Cell Physiol ; 319(2): C258-C267, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32510973

RESUMO

Because of the ongoing pandemic around the world, the mechanisms underlying the SARS-CoV-2-induced COVID-19 are subject to intense investigation. Based on available data for the SARS-CoV-1 virus, we suggest how CoV-2 localization of RNA transcripts in mitochondria hijacks the host cell's mitochondrial function to viral advantage. Besides viral RNA transcripts, RNA also localizes to mitochondria. SARS-CoV-2 may manipulate mitochondrial function indirectly, first by ACE2 regulation of mitochondrial function, and once it enters the host cell, open-reading frames (ORFs) such as ORF-9b can directly manipulate mitochondrial function to evade host cell immunity and facilitate virus replication and COVID-19 disease. Manipulations of host mitochondria by viral ORFs can release mitochondrial DNA (mtDNA) in the cytoplasm and activate mtDNA-induced inflammasome and suppress innate and adaptive immunity. We argue that a decline in ACE2 function in aged individuals, coupled with the age-associated decline in mitochondrial functions resulting in chronic metabolic disorders like diabetes or cancer, may make the host more vulnerable to infection and health complications to mortality. These observations suggest that distinct localization of viral RNA and proteins in mitochondria must play essential roles in SARS-CoV-2 pathogenesis. Understanding the mechanisms underlying virus communication with host mitochondria may provide critical insights into COVID-19 pathologies. An investigation into the SARS-CoV-2 hijacking of mitochondria should lead to novel approaches to prevent and treat COVID-19.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , DNA Mitocondrial/genética , Mitocôndrias/genética , Pneumonia Viral/virologia , RNA Viral/genética , Imunidade Adaptativa , Animais , Betacoronavirus/crescimento & desenvolvimento , Betacoronavirus/imunologia , Betacoronavirus/metabolismo , Infecções por Coronavirus/genética , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , DNA Mitocondrial/metabolismo , Regulação Viral da Expressão Gênica , Interações entre Hospedeiro e Microrganismos , Humanos , Imunidade Inata , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Mitocôndrias/virologia , Pandemias , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/genética , Pneumonia Viral/imunologia , Pneumonia Viral/metabolismo , Replicação Viral
12.
PLoS Pathog ; 16(6): e1008308, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32574204

RESUMO

One of the determinants for tissue tropism of hepatitis C virus (HCV) is miR-122, a liver-specific microRNA. Recently, it has been reported that interaction of miR-122 to HCV RNA induces a conformational change of the 5'UTR internal ribosome entry site (IRES) structure to form stem-loop II structure (SLII) and hijack of translating 80S ribosome through the binding of SLIII to 40S subunit, which leads to efficient translation. On the other hand, low levels of HCV-RNA replication have also been detected in some non-hepatic cells; however, the details of extrahepatic replication remain unknown. These observations suggest the possibility that miRNAs other than miR-122 can support efficient replication of HCV-RNA in non-hepatic cells. Here, we identified a number of such miRNAs and show that they could be divided into two groups: those that bind HCV-RNA at two locations (miR-122 binding sites I and II), in a manner similar to miR-122 (miR-122-like), and those that target a single site that bridges sites I and II and masking both G28 and C29 in the 5'UTR (non-miR-122-like). Although the enhancing activity of these non-hepatic miRNAs were lower than those of miR-122, substantial expression was detected in various normal tissues. Furthermore, structural modeling indicated that both miR-122-like and non-miR-122-like miRNAs not only can facilitate the formation of an HCV IRES SLII but also can stabilize IRES 3D structure in order to facilitate binding of SLIII to the ribosome. Together, these results suggest that HCV facilitates miR-122-independent replication in non-hepatic cells through recruitment of miRNAs other than miR-122. And our findings can provide a more detailed mechanism of miR-122-dependent enhancement of HCV-RNA translation by focusing on IRES tertiary structure.


Assuntos
Regulação Viral da Expressão Gênica , Hepacivirus/fisiologia , MicroRNAs/metabolismo , Biossíntese de Proteínas , RNA Viral , Proteínas Virais/biossíntese , Replicação Viral/fisiologia , Regiões 5' não Traduzidas , Linhagem Celular Tumoral , Humanos , MicroRNAs/genética , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Virais/genética
13.
Nat Commun ; 11(1): 2981, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532979

RESUMO

The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.


Assuntos
Bacteriófago T7/genética , DNA/genética , Regulação Viral da Expressão Gênica , Código Genético , Regiões Promotoras Genéticas/genética , Algoritmos , DNA de Cadeia Simples/genética , Modelos Genéticos , Transcrição Genética
14.
Infect Genet Evol ; 83: 104353, 2020 09.
Artigo em Inglês | MEDLINE | ID: covidwho-175958

RESUMO

In December 2019, a novel human-infecting coronavirus (SARS-CoV-2) was recognized in China. In a few months, SARS-CoV-2 has caused thousands of disease cases and deaths in several countries. Phylogenetic analyses indicated that SARS-CoV-2 clusters with SARS-CoV in the Sarbecovirus subgenus and viruses related to SARS-CoV-2 were identified from bats and pangolins. Coronaviruses have long and complex genomes with high plasticity in terms of gene content. To date, the coding potential of SARS-CoV-2 remains partially unknown. We thus used available sequences of bat and pangolin viruses to determine the selective events that shaped the genome structure of SARS-CoV-2 and to assess its coding potential. By searching for signals of significantly reduced variability at synonymous sites (dS), we identified six genomic regions, one of these corresponding to the programmed -1 ribosomal frameshift. The most prominent signal of dS reduction was observed within the E gene. A genome-wide analysis of conserved RNA structures indicated that this region harbors a putative functional RNA element that is shared with the SARS-CoV lineage. Additional signals of reduced dS indicated the presence of internal ORFs. Whereas the presence ORF9a (internal to N) was previously proposed by homology with a well characterized protein of SARS-CoV, ORF3h (for hypothetical, within ORF3a) was not previously described. The predicted product of ORF3h has 90% identity with the corresponding predicted product of SARS-CoV and displays features suggestive of a viroporin. Finally, analysis of the putative ORF10 revealed high dN/dS (3.82) in SARS-CoV-2 and related coronaviruses. In the SARS-CoV lineage, the ORF is predicted to encode a truncated protein and is neutrally evolving. These data suggest that ORF10 encodes a functional protein in SARS-CoV-2 and that positive selection is driving its evolution. Experimental analyses will be necessary to validate and characterize the coding and non-coding functional elements we identified.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Animais , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Pandemias , Filogenia , Recombinação Genética , Alinhamento de Sequência
15.
Infect Genet Evol ; 83: 104353, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32387562

RESUMO

In December 2019, a novel human-infecting coronavirus (SARS-CoV-2) was recognized in China. In a few months, SARS-CoV-2 has caused thousands of disease cases and deaths in several countries. Phylogenetic analyses indicated that SARS-CoV-2 clusters with SARS-CoV in the Sarbecovirus subgenus and viruses related to SARS-CoV-2 were identified from bats and pangolins. Coronaviruses have long and complex genomes with high plasticity in terms of gene content. To date, the coding potential of SARS-CoV-2 remains partially unknown. We thus used available sequences of bat and pangolin viruses to determine the selective events that shaped the genome structure of SARS-CoV-2 and to assess its coding potential. By searching for signals of significantly reduced variability at synonymous sites (dS), we identified six genomic regions, one of these corresponding to the programmed -1 ribosomal frameshift. The most prominent signal of dS reduction was observed within the E gene. A genome-wide analysis of conserved RNA structures indicated that this region harbors a putative functional RNA element that is shared with the SARS-CoV lineage. Additional signals of reduced dS indicated the presence of internal ORFs. Whereas the presence ORF9a (internal to N) was previously proposed by homology with a well characterized protein of SARS-CoV, ORF3h (for hypothetical, within ORF3a) was not previously described. The predicted product of ORF3h has 90% identity with the corresponding predicted product of SARS-CoV and displays features suggestive of a viroporin. Finally, analysis of the putative ORF10 revealed high dN/dS (3.82) in SARS-CoV-2 and related coronaviruses. In the SARS-CoV lineage, the ORF is predicted to encode a truncated protein and is neutrally evolving. These data suggest that ORF10 encodes a functional protein in SARS-CoV-2 and that positive selection is driving its evolution. Experimental analyses will be necessary to validate and characterize the coding and non-coding functional elements we identified.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , RNA Viral/genética , Animais , Regulação Viral da Expressão Gênica , Genoma Viral , Humanos , Pandemias , Filogenia , Recombinação Genética , Alinhamento de Sequência
16.
Proc Natl Acad Sci U S A ; 117(23): 13044-13055, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32434920

RESUMO

Epstein-Barr virus (EBV) is associated with epithelial and lymphoid malignancies, establishes latent infection in memory B cells, and intermittently produces infectious virions through lytic replication. Released virions play a key role in latent reservoir maintenance and transmission. Lytic EBV transcription differs from cellular transcription in requiring a virus-encoded preinitiation complex that binds to TATT motifs unique to EBV late lytic promoters. Expression of 15 late lytic genes that are important for virion production and infectivity is particularly dependent on the EBV SM protein, a nuclear protein expressed early during lytic reactivation that binds to viral RNAs and enhances RNA stability. We recently discovered that spironolactone blocks EBV virion production by inhibiting EBV SM function. Since spironolactone causes degradation of xeroderma pigmentosum group B-complementing protein (XPB), a component of human transcription factor TFIIH, in both B lymphocytes and epithelial cells, we hypothesized that SM utilizes XPB to specifically activate transcription of SM target promoters. While EBV SM has been thought to act posttranscriptionally, we provide evidence that SM also facilitates EBV gene transcription. We demonstrate that SM binds and recruits XPB to EBV promoters during lytic replication. Depletion of XPB protein, by spironolactone treatment or by siRNA transfection, inhibits SM-dependent late lytic gene transcription but not transcription of other EBV genes or cellular genes. These data indicate that SM acts as a transcriptional activator that has co-opted XPB to specifically target 15 EBV promoters that have uniquely evolved to require XPB for activity, providing an additional mechanism to differentially regulate EBV gene expression.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno/genética , Fosfoproteínas/metabolismo , Transativadores/metabolismo , Linhagem Celular Tumoral , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Infecções por Vírus Epstein-Barr/tratamento farmacológico , Infecções por Vírus Epstein-Barr/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas/genética , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Espironolactona/farmacologia , Espironolactona/uso terapêutico , Ativação Transcricional/efeitos dos fármacos , Vírion/efeitos dos fármacos , Vírion/metabolismo
17.
Microbes Infect ; 22(4-5): 182-187, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32446902

RESUMO

Envelope protein of coronaviruses is a structural protein existing in both monomeric and homo-pentameric form. It has been related to a multitude of roles including virus infection, replication, dissemination and immune response stimulation. In the present study, we employed an immunoinformatic approach to investigate the major immunogenic domains of the SARS-CoV-2 envelope protein and map them among the homologue proteins of coronaviruses with tropism for animal species that are closely inter-related with the human beings population all over the world. Also, when not available, we predicted the envelope protein structural folding and mapped SARS-CoV-2 epitopes. Envelope sequences alignment provides evidence of high sequence homology for some of the investigated virus specimens; while the structural mapping of epitopes resulted in the interesting maintenance of the structural folding and epitope sequence localization also in the envelope proteins scoring a lower alignment score. In line with the One-Health approach, our evidences provide a molecular structural rationale for a potential role of taxonomically related coronaviruses in conferring protection from SARS-CoV-2 infection and identifying potential candidates for the development of diagnostic tools and prophylactic-oriented strategies.


Assuntos
Betacoronavirus/metabolismo , Biologia Computacional/métodos , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , Proteínas do Envelope Viral/imunologia , Animais , Betacoronavirus/classificação , Betacoronavirus/genética , Betacoronavirus/imunologia , Mapeamento de Epitopos , Regulação Viral da Expressão Gênica , Humanos , Modelos Moleculares , Saúde Única , Pandemias , Filogenia , Conformação Proteica , Alinhamento de Sequência , Análise de Sequência de Proteína
18.
PLoS Pathog ; 16(5): e1008537, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32365141

RESUMO

Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.


Assuntos
Infecções por Citomegalovirus , Citomegalovirus/fisiologia , Proteínas Imediatamente Precoces , Imunidade Inata , Proteína da Leucemia Promielocítica , Replicação Viral , Linhagem Celular , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Regulação Viral da Expressão Gênica/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/imunologia , Mutação , Proteína da Leucemia Promielocítica/genética , Proteína da Leucemia Promielocítica/imunologia , Sumoilação/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
19.
J Virol ; 94(14)2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32376622

RESUMO

Porcine deltacoronavirus (PDCoV) is an economically important enteropathogen of swine with worldwide distribution. PDCoV primarily infects the small intestine instead of the large intestine in vivo However, the underlying mechanism of PDCoV tropism to different intestinal segments remains poorly understood as a result of the lack of a suitable in vitro intestinal model that recapitulates the cellular diversity and complex functions of the gastrointestinal tract. Here, we established the PDCoV infection model of crypt-derived enteroids from different intestinal segments. Enteroids were susceptible to PDCoV, and multiple types of different functional intestinal epithelia were infected by PDCoV in vitro and in vivo We further found that PDCoV favorably infected the jejunum and ileum and restrictedly replicated in the duodenum and colon. Mechanistically, enteroids from different intestinal regions displayed a distinct gene expression profile, and the differential expression of primary viral receptor host aminopeptidase N (APN) instead of the interferon (IFN) responses determined the susceptibility of different intestinal segments to PDCoV, although PDCoV substantially elicited antiviral genes production in enteroids after infection. Additional studies showed that PDCoV infection significantly induced the expression of type I and III IFNs at the late stage of infection, and exogenous IFN inhibited PDCoV replication in enteroids. Hence, our results provide critical inputs to further dissect the molecular mechanisms of PDCoV-host interactions and pathogenesis.IMPORTANCE The zoonotic potential of the PDCoV, a coronavirus efficiently infecting cells from a broad range species, including porcine, chicken, and human, emphasizes the urgent need to further study the cell and tissue tropism of PDCoV in its natural host. Herein, we generated crypt stem cell-derived enteroids from porcine different intestinal regions, which well recapitulated the events in vivo of PDCoV infection that PDCoV targeted multiple types of intestinal epithelia and preferably infected the jejunum and ileum over the duodenum and colon. Mechanistically, we demonstrated that the expression of APN receptor rather than the IFN responses determined the susceptibility of different regions of the intestines to PDCoV infection, though PDCoV infection markedly elicited the IFN responses. Our findings provide important insights into how the distinct gene expression profiles of the intestinal segments determine the cell and tissue tropism of PDCoV.


Assuntos
Antígenos CD13/genética , Infecções por Coronavirus/veterinária , Coronavirus/fisiologia , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Tropismo Viral , Animais , Enterocolite/metabolismo , Enterocolite/patologia , Enterocolite/virologia , Interferons/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Suínos , Doenças dos Suínos/patologia , Replicação Viral
20.
J Med Virol ; 92(7): 740-746, 2020 07.
Artigo em Inglês | MEDLINE | ID: covidwho-87806

RESUMO

Confronting the challenge of the outbreak of COVID-19 should sharpen our focus on global drug access as a key issue in antiviral therapy testing. The testing and adoption of effective therapies for novel coronaviruses are hampered by the challenge of conducting controlled studies during a state of emergency. The access to direct antiviral drugs, such as ribavirin, that have an existing inventory and reliable supply chain may be a priority consideration for therapies developed for the 2019-nCoV infection outbreaks and any strain variants that may emerge. On the basis of the direct antiviral activity of ribavirin against 2019-nCoV in vitro and evidence for potency enhancement strategies developed during the prior SARS and MERS outbreaks, ribavirin may significantly impact our ability to end the lingering outbreaks in China and slow outbreaks in other countries. The apparent COVID-19 pandemic provides an opportunity to follow dosage guidelines for treatment with ribavirin, test new therapeutic concepts, and conduct controlled testing to apply the scientific rigor required to address the controversy around this mainstay of antiviral therapy.


Assuntos
Antivirais/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/epidemiologia , Pandemias , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/epidemiologia , RNA Viral/antagonistas & inibidores , Ribavirina/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Ensaios Clínicos como Assunto , Infecções por Coronavirus/virologia , Progressão da Doença , Esquema de Medicação , Regulação Viral da Expressão Gênica , Humanos , Pneumonia Viral/virologia , RNA Viral/biossíntese , RNA Viral/genética , Transdução de Sinais
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