RESUMO
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a human pathogen that is now endemic to several East Asian countries. The viral large (L) protein catalyzes viral transcription by stealing host mRNA caps via a process known as cap-snatching. Here, we establish an in vitro cap-snatching assay and present three high-quality electron cryo-microscopy (cryo-EM) structures of the SFTSV L protein in biologically relevant, transcription-specific states. In a priming-state structure, we show capped RNA bound to the L protein cap-binding domain (CBD). The L protein conformation in this priming structure is significantly different from published replication-state structures, in particular the N- and C-terminal domains. The capped-RNA is positioned in a way that it can feed directly into the RNA-dependent RNA polymerase (RdRp) ready for elongation. We also captured the L protein in an early-elongation state following primer-incorporation demonstrating that this priming conformation is retained at least in the very early stages of primer extension. This structural data is complemented by in vitro biochemical and cell-based assays. Together, these insights further our mechanistic understanding of how SFTSV and other bunyaviruses incorporate stolen host mRNA fragments into their viral transcripts thereby allowing the virus to hijack host cell translation machinery.
Assuntos
Interações entre Hospedeiro e Microrganismos , Modelos Moleculares , Phlebovirus , Capuzes de RNA , Transcrição Gênica , Humanos , Microscopia Crioeletrônica , Phlebovirus/química , Phlebovirus/genética , Phlebovirus/ultraestrutura , Conformação Proteica , Capuzes de RNA/química , Capuzes de RNA/metabolismo , Capuzes de RNA/ultraestrutura , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/ultraestrutura , Replicação Viral/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologiaRESUMO
We recently reported the presence of nicotinamide adenine dinucleotide (NAD)-capped RNAs in mammalian cells and a role for DXO and the Nudix hydrolase Nudt12 in decapping NAD-capped RNAs (deNADding) in cells. Analysis of 5'caps has revealed that in addition to NAD, mammalian RNAs also contain other metabolite caps including flavin adenine dinucleotide (FAD) and dephosphoCoA (dpCoA). In the present study we systematically screened all mammalian Nudix proteins for their potential deNADing, FAD cap decapping (deFADding) and dpCoA cap decapping (deCoAping) activity. We demonstrate that Nudt16 is a novel deNADding enzyme in mammalian cells. Additionally, we identified seven Nudix proteins-Nudt2, Nudt7, Nudt8, Nudt12, Nudt15, Nudt16 and Nudt19, to possess deCoAping activity in vitro. Moreover, our screening revealed that both mammalian Nudt2 and Nudt16 hydrolyze FAD-capped RNAs in vitro with Nudt16 regulating levels of FAD-capped RNAs in cells. All decapping activities identified hydrolyze the metabolite cap substrate within the diphosphate linkage. Crystal structure of human Nudt16 in complex with FAD at 2.7 Å resolution provide molecular insights into the binding and metal-coordinated hydrolysis of FAD by Nudt16. In summary, our study identifies novel cellular deNADding and deFADding enzymes and establishes a foundation for the selective functionality of the Nudix decapping enzymes on non-canonical metabolite caps.
