RESUMO
Positive-sense single-stranded RNA viruses, such as coronaviruses, flaviviruses and alphaviruses, carry out transcription and replication inside virus-induced membranous organelles within host cells1-7. The remodelling of the host-cell membranes for the formation of these organelles is coupled to the membrane association of viral replication complexes and to RNA synthesis. These viral niches allow for the concentration of metabolites and proteins for the synthesis of viral RNA, and prevent the detection of this RNA by the cellular innate immune system8. Here we present the cryo-electron microscopy structure of non-structural protein 1 (nsP1) of the alphavirus chikungunya virus, which is responsible for RNA capping and membrane binding of the viral replication machinery. The structure shows the enzyme in its active form, assembled in a monotopic membrane-associated dodecameric ring. The structure reveals the structural basis of the coupling between membrane binding, oligomerization and allosteric activation of the capping enzyme. The stoichiometry-with 12 active sites in a single complex-redefines viral replication complexes as RNA synthesis reactors. The ring shape of the complex implies it has a role in controlling access to the viral organelle and ensuring the exit of properly capped viral RNA. Our results provide high-resolution information about the membrane association of the replication machinery of positive-sense single-stranded RNA viruses, and open up avenues for the further characterization of viral replication on cell membranes and the generation of antiviral agents.
Assuntos
Membrana Celular/metabolismo , Vírus Chikungunya/crescimento & desenvolvimento , Vírus Chikungunya/ultraestrutura , Microscopia Crioeletrônica , Capuzes de RNA/metabolismo , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Domínio Catalítico , Linhagem Celular , Membrana Celular/química , Vírus Chikungunya/química , Vírus Chikungunya/genética , Modelos Moleculares , Capuzes de RNA/química , RNA Viral/biossíntese , RNA Viral/química , RNA Viral/metabolismo , Proteínas não Estruturais Virais/ultraestruturaRESUMO
Long-terminal repeat (LTR) retrotransposons are genetic elements that, like retroviruses, replicate by reverse transcription of an RNA intermediate into a complementary DNA (cDNA) that is next integrated into the host genome by their own integrase. The Ty1 LTR retrotransposon has proven to be a reliable working model to investigate retroelement integration site preference. However, the low yield of recombinant Ty1 integrase production reported so far has been a major obstacle for structural studies. Here we analyze the biophysical and biochemical properties of a stable and functional recombinant Ty1 integrase highly expressed in E.coli. The recombinant protein is monomeric and has an elongated shape harboring the three-domain structure common to all retroviral integrases at the N-terminal half, an extra folded region, and a large intrinsically disordered region at the C-terminal half. Recombinant Ty1 integrase efficiently catalyzes concerted integration in vitro, and the N-terminal domain displays similar activity. These studies that will facilitate structural analyses may allow elucidating the molecular mechanisms governing Ty1 specific integration into safe places in the genome.
Assuntos
Integrases/química , Proteínas Intrinsicamente Desordenadas/química , Retroelementos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Integrases/genética , Integrases/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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 fármacosRESUMO
Strains of uropathogenic Escherichia coli (UPEC) are the major causative agent of urinary tract infections (UTI), the most common infectious diseases in the world. Their ability to attach and enter into cells in the urinary tract is a limiting step for their pathogenicity. Many studies are thus focussing on these key mechanisms to propose new therapeutic strategies. To facilitate such studies, we developed a fast and high-throughput assay which makes it possible to monitor the interaction of UPEC with cultured human uroepithelial cells. This assay allows measurement of the in vitro association of fluorescently labelled clinical isolates with bladder epithelial cells using flow cytometry in a microplate format. The assay was sensitive enough to detect variations between isolates expressing different adhesins and virulence factors and the inhibitory effect of proanthocyanidins. Thus we have developed a fast and robust assay which allows us to measure variations in the adhesion properties of UPEC to human bladder cells. This novel assay will be valuable for the study of initial steps of pathogenesis in UTI and for the screening or validation of inhibitory molecules.
Assuntos
Aderência Bacteriana/fisiologia , Bexiga Urinária/microbiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/fisiologia , Aderência Bacteriana/efeitos dos fármacos , Técnicas Bacteriológicas/métodos , Células Cultivadas , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Citometria de Fluxo/métodos , Humanos , Fitoterapia , Extratos Vegetais/farmacologia , Bexiga Urinária/citologia , Escherichia coli Uropatogênica/patogenicidade , Vaccinium macrocarpon , VirulênciaRESUMO
Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host.