RESUMO
RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.
Assuntos
Grânulos de Ribonucleoproteínas Citoplasmáticas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Rotavirus/genética , Proteínas não Estruturais Virais/metabolismo , Animais , Bovinos , Linhagem Celular , Grânulos de Ribonucleoproteínas Citoplasmáticas/efeitos dos fármacos , Grânulos de Ribonucleoproteínas Citoplasmáticas/ultraestrutura , Grânulos de Ribonucleoproteínas Citoplasmáticas/virologia , Regulação Viral da Expressão Gênica , Genes Reporter , Glicóis/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Haplorrinos , Interações Hospedeiro-Patógeno/genética , Humanos , Concentração Osmolar , Fosforilação , Propilenoglicol/farmacologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Rotavirus/efeitos dos fármacos , Rotavirus/crescimento & desenvolvimento , Rotavirus/ultraestrutura , Transdução de Sinais , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Montagem de Vírus/efeitos dos fármacos , Montagem de Vírus/genética , Replicação Viral/efeitos dos fármacos , Replicação Viral/genéticaRESUMO
Clustered regularly interspaced short palindromic repeats - CRISPR-associated protein (CRISPR-Cas) systems are a critical component of the bacterial adaptive immune response. Since the discovery that they can be reengineered as programmable RNA-guided nucleases, there has been significant interest in using these systems to perform diverse and precise genetic manipulations. Here, we outline recent advances in the mechanistic understanding of CRISPR-Cas9, how these findings have been leveraged in the rational redesign of Cas9 variants with altered activities, and how these novel tools can be exploited for biotechnology and therapeutics. We also discuss the potential of the ubiquitous, yet often-overlooked, multisubunit CRISPR effector complexes for large-scale genomic deletions. Furthermore, we highlight how future structural studies will bolster these technologies.