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
Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early and late stages of elongation with the RdRp core accommodating a 10-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.
Assuntos
Phlebovirus , Febre Grave com Síndrome de Trombocitopenia , Humanos , Febre Grave com Síndrome de Trombocitopenia/genética , Microscopia Crioeletrônica , RNA Viral/genética , RNA Polimerase Dependente de RNA/metabolismo , Phlebovirus/genética , Replicação Viral , Genoma ViralRESUMO
Lassa virus is endemic in West Africa and can cause severe hemorrhagic fever. The viral L protein transcribes and replicates the RNA genome via its RNA-dependent RNA polymerase activity. Here, we present nine cryo-EM structures of the L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved 3' and 5' promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and early elongation states, the endonuclease is inhibited by two distinct L protein peptides, whereas in the pre-initiation state it is uninhibited. In the early elongation state, a template-product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide and the full C-terminal region of the L protein, including the putative cap-binding domain, is well-ordered. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated.
Assuntos
Vírus Lassa/genética , RNA Viral/química , RNA Polimerase Dependente de RNA/química , Transcrição Gênica , Proteínas Virais/química , Motivos de Aminoácidos , Domínio Catalítico , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Vírus Lassa/química , Vírus Lassa/enzimologia , Modelos Moleculares , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , RNA Viral/biossíntese , RNA Viral/genética , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Interrupted dimeric coiled coil segments are found in a broad range of proteins and generally confer selective functional properties such as binding to specific ligands. However, there is only one documented case of a basic-helix-loop-helix leucine zipper transcription factor-microphthalmia-associated transcription factor (MITF)-in which an insertion of a three-residue stammer serves as a determinant of conditional partner selectivity. To unravel the molecular principles of this selectivity, we have analyzed the high-resolution structures of stammer-containing MITF and an engineered stammer-less MITF variant, which comprises an uninterrupted symmetric coiled coil. Despite this fundamental difference, both MITF structures reveal identical flanking in-phase coiled coil arrangements, gained by helical over-winding and local asymmetry in wild-type MITF across the stammer region. These conserved structural properties allow the maintenance of a proper functional readout in terms of nuclear localization and binding to specific DNA-response motifs regardless of the presence of the stammer. By contrast, MITF heterodimer formation with other bHLH-Zip transcription factors is only permissive when both factors contain either the same type of inserted stammer or no insert. Our data illustrate a unique principle of conditional partner selectivity within the wide arsenal of transcription factors with specific partner-dependent functional readouts.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/química , Núcleo Celular/química , Fator de Transcrição Associado à Microftalmia/química , Conformação Proteica , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Ligantes , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Ligação Proteica , Domínios Proteicos/genética , Multimerização ProteicaRESUMO
PURPOSE: Tuberculosis is still a major threat to global health. New tools and strategies to produce disease-related proteins are quintessential for the development of novel vaccines and diagnostic markers. EXPERIMENTAL DESIGN: To obtain recombinant proteins from Mycobacterium tuberculosis (Mtb) for use in clinical applications, a standardized procedure was developed that includes subcloning, protein expression in Mycobacterium smegmatis and protein purification using chromatography. The potential for the different protein targets to serve as diagnostic markers for tuberculosis was established using multiplex immunoassays. RESULTS: Twelve soluble proteins from Mtb, including one protein complex, were purified to near-homogeneity following recombinant expression in M. smegmatis. Protein purity was assessed both by size exclusion chromatography and MS. Multiplex serological testing of the final protein preparations showed that all but one protein displayed a clear antibody response in serum samples from 278 tuberculosis patients. CONCLUSION AND CLINICAL RELEVANCE: The established workflow comprises a simple, cost-effective, and scalable pipeline for production of soluble proteins from Mtb and can be used to prioritize immunogenic proteins suitable for use as diagnostic markers.
Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteômica/normas , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Humanos , Espectrometria de Massas , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/fisiologia , Padrões de Referência , Solubilidade , Tuberculose/sangueRESUMO
Microphthalmia-associated transcription factor (MITF) is a master regulator of melanocyte development and an important oncogene in melanoma. MITF heterodimeric assembly with related basic helix-loop-helix leucine zipper transcription factors is highly restricted, and its binding profile to cognate DNA sequences is distinct. Here, we determined the crystal structure of MITF in its apo conformation and in the presence of two related DNA response elements, the E-box and M-box. In addition, we investigated mouse and human Mitf mutations to dissect the functional significance of structural features. Owing to an unusual three-residue shift in the leucine zipper register, the MITF homodimer shows a marked kink in one of the two zipper helices to allow an out-of-register assembly. Removal of this insertion relieves restricted heterodimerization by MITF and permits assembly with the transcription factor MAX. Binding of MITF to the M-box motif is mediated by an unusual nonpolar interaction by Ile212, a residue that is mutated in mice and humans with Waardenburg syndrome. As several related transcription factors have low affinity for the M-box sequence, our analysis unravels how these proteins discriminate between similar target sequences. Our data provide a rational basis for targeting MITF in the treatment of important hereditary diseases and cancer.
Assuntos
Zíper de Leucina , Fator de Transcrição Associado à Microftalmia/química , Fator de Transcrição Associado à Microftalmia/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Animais , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Elementos Facilitadores Genéticos/genética , Humanos , Zíper de Leucina/genética , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Dados de Sequência Molecular , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Síndrome de Waardenburg/genéticaRESUMO
The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C-terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured alpha-helical domain binds PMP-targeting signal (mPTS) sequences with about 10 muM affinity. Removal of a conserved N-terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS-binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N-terminal and C-terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo-targeting signal recognition and additional functions.