Ensamblaje y maduración de la cápsida del virus de la bursitis infecciosa: estructura y función de la proteasa viral VP4

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 08-11-2013Infectious bursal disease virus (IBDV) is a polyploid, bisegmented dsRNA virus with a single, non-enveloped ~70-nm-diameter capsid with a T=13l lattice. The capsid structural units are trimers of a single protein, VP2. The VP2 precursor form is encoded as part of the polyprotein NH2-pVP2-VP4-VP3-COOH; VP4 is the viral protease, able to cleave its own N and C termini, and VP3 is a multifunctional protein with scaffolding and dsRNA-binding activities. Expression of the IBDV viral polyprotein leads to formation of different macromolecular assemblies, depending on the heterologous expression system used. We analyzed IBDV assembly using various baculovirus systems. Whereas expression with the pFastBac vector led to production of rigid pVP2 helical assemblies, expression with the pAcYM1 vector led to assembly of spherical virus-like particles (VLP) and flexible tubes. Western blot and mass spectrometry analyses showed that spherical VLP were formed by the entire polyprotein, the pVP2, and the intermediate precursor VP4-VP3, suggesting that VP4 activity is partially inhibited. Three-dimensional cryo-electron microscopy (3D cryo-EM) of these assemblies showed that, compared to the VLP with a marked polyhedral contour or the virion capsid, the VLP internal surface has extra densities, which would correspond to the protein portions of precursors not removed proteolytically. These results indicate that IBDV capsid assembly proceeds through a procapsid intermediate that requires further proteolytic maturation. Sequence analysis of the polyprotein in the context of the pAcYM1 system detected only the R505H substitution, suggesting that, after excluding any mutations in the VP4 sequence, the context of the processing sites determines VP4 activity. Assembly of IBDV immature capsids from polyprotein precursors in conditions that compromise pVP2/VP4 and VP4/VP3 cleavage resembles the assembly of the Gag precursor in immature HIV virions. Finally, we analyzed VP4 helical structures, which are assembled naturally in IBDV-infected cells, by 3D cryo-EM at subnanometer resolution. VP4 tube is a right-handed three start helix, made of VP4 dimers, with an axial rise of 18.4 Å and an azimutal angle of 40.6º. To better understand the architecture and role of VP4 helical structure, we built a pseudo-atomic structure of VP4 by homology modeling, as the birnavirus VP4 showed a conserved overall structure despite a low level of sequence identity. Analysis of the resulting hybrid structure after flexible docking of the VP4 comparative model showed the intra- and interdimeric interactions that stabilize the VP4 holocomplex. Potential conformational differences provided new information on the mechanism of inactivation, as they showed that the catalytic serine/lysine dyad, located on a surface crevice, is blocked in the assembled VP4. This conformational change is induced by the VP4 C terminus-mediated interdimeric contacts, and suggest that once that has achieved its proteolytic function, VP4 is inactivated by assembly into helical structures to prevent lethal damage to the virus or the host components needed for virus multiplication

An integrative structural biology analysis of von Willebrand factor binding and processing by ADAMTS-13 in solution

Peer reviewe

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.This study was supported in part by grants from Spanish, French, Danish and Catalan public and private bodies (grant/fellowship references PID2019-107725RG-I00, BES-2015-074583, ANR-10-LABX-12-01, 6108-00031B, 8022-00385B, LF18039, NNF18OC0032724, Novo Nordisk Foundation “Bio-MS”, 2017SGR3 and Fundació “La Marató de TV3” 201815). This work was also supported by EPICS-XS, project 823839, funded by the Horizon 2020 programme of the European Union. The CBS is a member of France-BioImaging (FBI) and the French Infrastructure for Integrated Structural Biology (FRISBI), which are national infrastructures supported by the French National Research Agency (grants ANR-10-INBS-04-01 and ANR-10-INBS-05, respectively). Finally, we acknowledge the Structural Mass Spectrometry Unit of CIISB, an Instruct-CZ Centre, which was supported by MEYS CR (LM2018127)

Efecto de la naringenina, hesperetina y sus formas glicosidadas sobre la replicación de la cepa 17D del virus de la fiebre amarilla

Infections produces by flaviviruses are threats to public health worldwide. The absence of effective drugs treatment makesimperative the quest for efficient antivirals. Using a plaque inhibition assay we studied the antiviral action of the non-cytotoxic flavanones naringenin, hesperetin, and their glycoside forms on the replication of the 17D strain of yellow fevervirus (YFV17D). Using plaque assay the glycosylated forms did not show antiviral effect at the highest concentration used,naringenin and hesperetin reduced the plaque size and the infectious titer up to 85.51% and 100% respectively at themaximum concentration employed. The effective dose (ED 50 ) of naringenin (0.0013 M), calculated by plaque assay, wasapproximately tenfold lower than hesperetin (ED 50 0.01 M), and neither showed any virucidal effect. Evaluation of kineticentrance of YFV to cell showed that after 2h of infection 80% of the virus was already into the cell, but in the presence ofnaringenin or hesperetin, this entrance were inhibited to 48.58% and 55.2% respectively (p≤ 0.05). Study of molecularparameters of naringenin and hesperetin demonstrated that naringenin Log P = -1.56 is ten times higher than hesperetin(Log P = -2.56), and therefore less hydrophilic and probably more effective in passing through membranes, which could berelated to its ability to interfere with the viral replication beside the entrance levelLas infecciones producidas por flavivirus representan una amenaza para la salud pública a nivel mundial. La ausencia defármacos eficaces para su tratamiento hace imperativa la búsqueda de antivirales efectivos. Empleando ensayos deinhibición de placa se estudió la acción antiviral de las flavanonas no citotóxicas naringenina, hesperetina, y sus formasglicosidadas sobre la replicación de la cepa 17D del virus de la fiebre amarilla (YFV17D). Las formas glicosidadas nomostraron efecto antiviral con la máxima concentración. La naringenina y hesperetina fueron capaces de reducir el tamañoy número de placas virales en un 80% y 100% respectivamente. La dosis efectiva (ED 50 ) de la naringenina (0.0013 M) fueaproximadamente diez veces menor que la hesperetina (ED 50 0.01 M) y ninguna mostró efecto virucida. Después de 2h deinfección el 80% del virus ha entrado a la célula pero en presencia de naringenina o hesperetina dicho proceso es inhibidoen un 48,58% y 55,2% respectivamente (p≤0,05). El estudio de parámetros moleculares de ambas flavanonas demostró queel Log P=-1.56 de la naringenina es diez veces mayor que el de hesperetina (Log P=-2,56) indicando que es menos hidrofílicoy probablemente más eficaz pasando a través de membranas, lo que podría estar relacionado con su capacidad de interferiren la replicación viral a niveles distintos de la entrada