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1.
Nucleic Acids Res ; 47(20): 10914-10930, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31584100

RESUMO

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.


Assuntos
Endonucleases/química , Endonucleases/metabolismo , Capuzes de RNA/metabolismo , Vírus da Febre do Flebótomo Napolitano/enzimologia , Proteínas Virais/química , Proteínas Virais/metabolismo , Biocatálise , Domínio Catalítico , Cátions Bivalentes/farmacologia , Sequência Conservada , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Domínios Proteicos , Eletricidade Estática , Sulfatos/metabolismo , Transcrição Gênica/efeitos dos fármacos
2.
Nat Commun ; 14(1): 2954, 2023 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-37221161

RESUMO

Hantaviruses are causing life-threatening zoonotic infections in humans. Their tripartite negative-stranded RNA genome is replicated by the multi-functional viral RNA-dependent RNA-polymerase. Here we describe the structure of the Hantaan virus polymerase core and establish conditions for in vitro replication activity. The apo structure adopts an inactive conformation that involves substantial folding rearrangement of polymerase motifs. Binding of the 5' viral RNA promoter triggers Hantaan virus polymerase reorganization and activation. It induces the recruitment of the 3' viral RNA towards the polymerase active site for prime-and-realign initiation. The elongation structure reveals the formation of a template/product duplex in the active site cavity concomitant with polymerase core widening and the opening of a 3' viral RNA secondary binding site. Altogether, these elements reveal the molecular specificities of Hantaviridae polymerase structure and uncover the mechanisms underlying replication. They provide a solid framework for future development of antivirals against this group of emerging pathogens.


Assuntos
Vírus Hantaan , Vírus de RNA , Humanos , Nucleotidiltransferases , RNA Viral , Replicação Viral
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