Análisis multidisciplinar de las relaciones estructura-función de los componentes del virus de la bursitis infecciosa

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 17-12-200

Identification and Characterization of Epithelial Cell-Derived Dense Bodies Produced upon Cytomegalovirus Infection

Dense bodies (DB) are complex, noninfectious particles produced during CMVinfection containing envelope and tegument proteins that may be ideal candidates as vaccines. Although DB were previously described in fibroblasts, no evidence of DB formation has been shown after propagating CMV in epithelial cells. In the present study, both fibroblast MRC-5 and epithelial ARPE-19 cells were used to study DB production during CMV infection. We demonstrate the formation of epithelial cell-derived DB, mostly located as cytoplasmic inclusions in the perinuclear area of the infected cell. DB were gradient-purified, and the nature of the viral particles was confirmed using CMV-specific immunelabeling. Epithelial cell-derived DB had higher density and more homogeneous size (200-300 nm) compared to fibroblast-derived DB (100-600 nm).In agreement with previous results characterizing DB from CMV-infected fibroblasts, the pp65 tegument protein was predominant in the epithelial cell-derived DB. Our results also suggest that epithelial cells had more CMV capsids in the cytoplasm and had spherical bodies compatible with nucleus condensation (pyknosis) in cells undergoing apoptosis that were not detected in MRC-5 infected cells at the tested time post-infection. Our results demonstrate the formation of DB in CMV-infected ARPE-19 epithelial cells that may be suitable candidate to develop a multiprotein vaccine with antigenic properties similar to that of the virions while not including the viral genome.This study was supported by the Spanish Ministry of Science, Innovation and University, Instituto de Salud Carlos III Grant/Award Numbers: PI17CIII-00014 (MPY110/18); PI20CIII-00009 (MPY303/20); DTS18CIII/00006 (MPY127/19). E.G-R is supported by the Sara Borrell Program (CD18CIII/00007), Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades. MJR is supported by the PTA Program (PTA2017-14233-I), Ministerio de Ciencia, Innovación y Universidades.S

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α<inf>2</inf>-macroglobulin

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo-electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus. RnQV1, the type species of the family Quadriviridae, has a multipartite genome consisting of four monocistronic segments. Whereas most dsRNA virus capsids are based on dimers of a single protein, the ~450-Å-diameter, T = 1 RnQV1 capsid is built of P2 and P4 protein heterodimers, each with more than 1000 residues. Despite a lack of sequence similarity between the two proteins, they have a similar α-helical domain, the structural signature shared with the lineage of the dsRNA bluetongue virus-like viruses. Domain insertions in P2 and P4 preferential sites provide additional functions at the capsid outer surface, probably related to enzyme activity. The P2 insertion has a fold similar to that of gelsolin and profilin, two actin-binding proteins with a function in cytoskeleton metabolism, whereas the P4 insertion suggests protease activity involved in cleavage of the P2 383-residue C-terminal region, absent in the mature viral particle. Our results indicate that the intimate virus-fungus partnership has altered the capsid genome-protective and/or receptor-binding functions. Fungal virus evolution has tended to allocate enzyme activities to the virus capsid outer surface

Procedimiento para la producción en levaduras de cápsidas virales vacías compuestas por proteínas derivadas de pVP2 del virus causante de la enfermedad de la bursitis infecciosa (IBDV)

Referencia OEPM: P200401044.-- Fecha de solicitud: 30/04/2004.-- Titulares: Consejo Superior de Investigaciones Científicas (CSIC), Bionostra, S.L.Procedimiento para la producción en levaduras de cápsidas virales vacías compuestas por proteínas derivadas de pVP2 del virus causante de la enfermedad de la bursitis infecciosa (IBDV). Las cápsidas vacías del virus causante de la enfermedad de la bursitis infecciosa (IBDV) están constituidas por ensamblaje de proteínas derivadas de la proteína pVP2 de IBDV, de distinto tamaño y tienen aplicación en la producción de vacunas y en la elaboración de vectores para terapia génica.Peer reviewe

MicroRNA signature from extracellular vesicles of HCV/HIV co-infected individuals differs from HCV mono-infected

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus-host interaction. KEY MESSAGES: HCV and HCV/HIV displayed similar plasma-EV size and concentration. EVs- derived miRNA profile was characterized by NGS. 37 SDE miRNAs between HCV and HCV/HIV were observed. SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work has been supported by grants from (1) Institute of Health Carlos III, Spain [PI18CIII/00020/ to AFR], (2) PID2021–126781OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe”, (3) The SPANISH AIDS Research Network RD16CIII/0002/0002 - ISCIII – FEDER, (4) Centro de Investigación en Red en Enfermedades Infecciosas (CIBERINFEC) CB21/13/00044, (5) the National Agency for Scientific and Technology Promotion (ANPCyT) (PICT 2017 Nº713), and (6) the National Research Council (CONICET, PIP 2021-2023). V.C. received funding form the Asociación Universitaria Iberoamericana de Postgrado (AUIP) for the Academic Mobility Scholarship Program. P.V., E.D.M., and M.V.P. are members of the CONICET-Research Career Program. V.C. is a fellow from ANPCyT. The funder’s had no role in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript.S

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media. Our experiments, in combination with electrostatic analyses, reveal a strong interaction between the two outer layers and how it is suppressed by the removal of calcium ions, a key step for transcription initiation. The intermediate layer presents weak hydrophobic interactions with the inner layer that allow the assembly and favor the conformational dynamics needed for transcription. Our work shows how the biophysical properties of the three shells are finely tuned to produce an infective RV virion.This work was supported by grants from the Spanish Ministry of Economy and Competitivity FIS2014-59562-R, FIS2017-89549-R and ‘María de Maeztu’ Program for Units of Excellence in R and D (MDM-2014–0377) to PJP, BFU2013-43149-R to DL and JMR, FIS2015- 67837 P to DR, FIS2015-71108-REDT to PJP, DL, DR, and JMR, and BFU2014-55475-R (Spanish Ministry of Economy and Competitivity) and S2013/MIT-2807 (Comunidad Autónoma de Madrid) to JRC.S

Chimeric Pneumoviridae fusion proteins as immunogens to induce cross‐neutralizing antibody responses

Abstract Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV), two members of the Pneumoviridae family, account for the majority of severe lower respiratory tract infections worldwide in very young children. They are also a frequent cause of morbidity and mortality in the elderly and immunocompromised adults. High levels of neutralizing antibodies, mostly directed against the viral fusion (F) glycoprotein, correlate with protection against either hRSV or hMPV. However, no cross‐neutralization is observed in polyclonal antibody responses raised after virus infection or immunization with purified F proteins. Based on crystal structures of hRSV F and hMPV F, we designed chimeric F proteins in which certain residues of well‐characterized antigenic sites were swapped between the two antigens. The antigenic changes were monitored by ELISA with virus‐specific monoclonal antibodies. Inoculation of mice with these chimeras induced polyclonal cross‐neutralizing antibody responses, and mice were protected against challenge with the virus used for grafting of the heterologous antigenic site. These results provide a proof of principle for chimeric fusion proteins as single immunogens that can induce cross‐neutralizing antibody and protective responses against more than one human pneumovirus

Equilibrium dynamics of a biomolecular complex analyzed at single-amino acid resolution by cryo-electron microscopy

The biological function of macromolecular complexes depends not only on large-scale transitions between conformations, but also on small-scale conformational fluctuations at equilibrium. Information on the equilibrium dynamics of biomolecular complexes could, in principle, be obtained from local resolution (LR) data in cryo-electron microscopy (cryo-EM) maps. However, this possibility had not been validated by comparing, for a same biomolecular complex, LR data with quantitative information on equilibrium dynamics obtained by an established solution technique. In this study we determined the cryo-EM structure of the minute virus of mice (MVM) capsid as a model biomolecular complex. The LR values obtained correlated with crystallographic B factors and with hydrogen/deuterium exchange (HDX) rates obtained by mass spectrometry (HDX-MS), a gold standard for determining equilibrium dynamics in solution. This result validated a LR-based cryo-EM approach to investigate, with high spatial resolution, the equilibrium dynamics of biomolecular complexes. As an application of this approach, we determined the cryo-EM structure of two mutant MVM capsids and compared their equilibrium dynamics with that of the wildtype MVM capsid. The results supported a previously suggested linkage between mechanical stiffening and impaired equilibrium dynamics of a virus particle. Cryo-EM is emerging as a powerful approach for simultaneously acquiring information on the atomic structure and local equilibrium dynamics of biomolecular complexe