RESUMO
SARS-CoV-2 interferes with antigen presentation by downregulating major histocompatibility complex (MHC) II on antigen-presenting cells, but the mechanism mediating this process is unelucidated. Herein, analysis of protein and gene expression in human antigen-presenting cells reveals that MHC II is downregulated by the SARS-CoV-2 main protease, NSP5. This suppression of MHC II expression occurs via decreased expression of the MHC II regulatory protein CIITA. CIITA downregulation is independent of the proteolytic activity of NSP5, and rather, NSP5 delivers HDAC2 to the transcription factor IRF3 at an IRF-binding site within the CIITA promoter. Here, HDAC2 deacetylates and inactivates the CIITA promoter. This loss of CIITA expression prevents further expression of MHC II, with this suppression alleviated by ectopic expression of CIITA or knockdown of HDAC2. These results identify a mechanism by which SARS-CoV-2 limits MHC II expression, thereby delaying or weakening the subsequent adaptive immune response.
Assuntos
Antígenos de Histocompatibilidade Classe II , Histona Desacetilase 2 , Proteínas Nucleares , Regiões Promotoras Genéticas , SARS-CoV-2 , Transativadores , Humanos , Apresentação de Antígeno/genética , Células Apresentadoras de Antígenos/metabolismo , Células Apresentadoras de Antígenos/imunologia , COVID-19/virologia , COVID-19/imunologia , COVID-19/genética , COVID-19/metabolismo , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/genética , Regulação para Baixo/genética , Células HEK293 , Antígenos de Histocompatibilidade Classe II/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Histona Desacetilase 2/metabolismo , Histona Desacetilase 2/genética , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , SARS-CoV-2/imunologia , Transativadores/metabolismo , Transativadores/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/genéticaRESUMO
Live cell microscopy has become a common technique for exploring dynamic biological processes. When combined with fluorescent markers of cellular structures of interest, or fluorescent reporters of a biological activity of interest, live cell microscopy enables precise temporally and spatially resolved quantitation of the biological processes under investigation. However, because living cells are not normally exposed to light, live cell fluorescence imaging is significantly hindered by the effects of photodamage, which encompasses photobleaching of fluorophores and phototoxicity of the cells under observation. In this chapter, we outline several methods for optimizing and maintaining long-term imaging of live cells while simultaneously minimizing photodamage. This protocol demonstrates the intracellular trafficking of early and late endosomes following phagocytosis using both two and three dimensional imaging, but this protocol can easily be modified to image any biological process of interest in nearly any cell type.