RESUMO
Reverse genetics has been an indispensable tool to gain insights into viral pathogenesis and vaccine development. The genomes of large RNA viruses, such as those from coronaviruses, are cumbersome to clone and manipulate in Escherichia coli owing to the size and occasional instability of the genome1-3. Therefore, an alternative rapid and robust reverse-genetics platform for RNA viruses would benefit the research community. Here we show the full functionality of a yeast-based synthetic genomics platform to genetically reconstruct diverse RNA viruses, including members of the Coronaviridae, Flaviviridae and Pneumoviridae families. Viral subgenomic fragments were generated using viral isolates, cloned viral DNA, clinical samples or synthetic DNA, and these fragments were then reassembled in one step in Saccharomyces cerevisiae using transformation-associated recombination cloning to maintain the genome as a yeast artificial chromosome. T7 RNA polymerase was then used to generate infectious RNA to rescue viable virus. Using this platform, we were able to engineer and generate chemically synthesized clones of the virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)4, which has caused the recent pandemic of coronavirus disease (COVID-19), in only a week after receipt of the synthetic DNA fragments. The technical advance that we describe here facilitates rapid responses to emerging viruses as it enables the real-time generation and functional characterization of evolving RNA virus variants during an outbreak.
Assuntos
Betacoronavirus/genética , Clonagem Molecular/métodos , Infecções por Coronavirus/virologia , Genoma Viral/genética , Genômica/métodos , Pneumonia Viral/virologia , Genética Reversa/métodos , Biologia Sintética/métodos , Animais , COVID-19 , China/epidemiologia , Chlorocebus aethiops , Cromossomos Artificiais de Levedura/metabolismo , Infecções por Coronavirus/epidemiologia , RNA Polimerases Dirigidas por DNA/metabolismo , Evolução Molecular , Humanos , Mutação , Pandemias/estatística & dados numéricos , Pneumonia Viral/epidemiologia , Vírus Sinciciais Respiratórios/genética , SARS-CoV-2 , Saccharomyces cerevisiae/genética , Células Vero , Proteínas Virais/metabolismo , Zika virus/genéticaRESUMO
A hallmark of acute classical swine fever is the high interferon (IFN)-α levels found in the serum early after infection, followed by an inflammatory cytokine storm. Plasmacytoid dendritic cells (pDCs) represent the only known cell type that produces IFN-α upon classical swine fever virus (CSFV) infection in vitro. In primary target cells of the virus the viral protein N(pro) inhibits the induction of type I IFN via the degradation of IRF3. We hypothesized that the early systemic pDC-derived IFN-α response sensitizes immune cells for enhanced responsiveness and augment cytokine responses after CSFV infection through the upregulation of IRF7. Therefore, bone marrow-derived granulocyte macrophage-colony stimulating factor (GM-CSF)-induced DCs, were pretreated with IFN-ß or conditioned medium from CSFV-activated enriched pDC, and expression of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, and IFN-α was assessed after infection with wild-type CSFV and with an N(pro) mutant [N(pro)(D(136)N)] unable to interact with IRF3 and IRF7. While type I IFN treatment sensitized the DCs for enhanced IFN and cytokine responses after stimulation with influenza virus, lipopolysaccharide or poly(I):poly(C), this was not observed for CSFV. In contrast, the N(pro)(D(136)N) mutant CSFV induced elevated IFN-α responses in type I IFN-pretreated GM-CSF DCs. These results indicate that CSFV has evolved to prevent type I IFN sensitization in infected cells through the action of the N(pro).
Assuntos
Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/imunologia , Células Dendríticas/imunologia , Endopeptidases/metabolismo , Interferon-alfa/biossíntese , Suínos/imunologia , Proteínas Virais/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Peste Suína Clássica/virologia , Vírus da Febre Suína Clássica/genética , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/virologia , Endopeptidases/genética , Endopeptidases/imunologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interferon Tipo I/imunologia , Interferon-alfa/sangue , Interferon-alfa/genética , Mutação/genética , Ligação Proteica/genética , Ligação Proteica/imunologia , Suínos/virologia , Regulação para Cima/genética , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Pathogen recognition receptors are essential for antiviral host immune responses. These specialized receptors detect conserved viral compounds and induce type I interferons (IFN) and pro-inflammatory cytokines. Here we evaluated the contribution of RIG-I, MDA-5 and TLR3 to the recognition of classical swine fever (CSFV), foot-and-mouth disease virus (FMDV), vesicular stomatitis virus (VSV) and influenza A virus (IAV) to IFN-ß responses in the porcine epithelial cell line PK-15. To this end, we identified porcine gene specific small interfering RNA sequences and employed a lentivirus (LV)-based system to deliver the corresponding short hairpin RNA. With this, gene knockdown cell lines were created and tested with regard to the knockdown levels over time and following IFN-ß stimulation. During several passages of the transduced cells, the expression of both the reporter gene eGFP and the reduced RNA levels of the targeted gene were stable, although the latter was relatively variable. IFN-ß induced IFN-responsive genes such as RIG-I, but the levels of the silenced cell line remained reduced compared to the control cells. Based on virus-induced IFN-ß mRNA responses, our results indicate that in PK-15 cells FMDV-detection is solely mediated by MDA-5, whereas VSV and IAV are mainly detected by RIG-I with a minor contribution of MDA-5, and CSFV is sensed by MDA-5, RIG-I and TLR3.