Publications by authors named "Nicola G A Abrescia"

51 Publications

Bacteriophage PRD1 as a nanoscaffold for drug loading.

Nanoscale 2021 Dec 13;13(47):19875-19883. Epub 2021 Dec 13.

Structure and Cell Biology of Viruses Lab, Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, Derio, Spain.

Viruses are very attractive biomaterials owing to their capability as nanocarriers of genetic material. Efforts have been made to functionalize self-assembling viral protein capsids on their exterior or interior to selectively take up different payloads. PRD1 is a double-stranded DNA bacteriophage comprising an icosahedral protein outer capsid and an inner lipidic vesicle. Here, we report the three-dimensional structure of PRD1 in complex with the antipsychotic drug chlorpromazine (CPZ) by cryo-electron microscopy. We show that the jellyrolls of the viral major capsid protein P3, protruding outwards from the capsid shell, serve as scaffolds for loading heterocyclic CPZ molecules. Additional X-ray studies and molecular dynamics simulations show the binding modes and organization of CPZ molecules when complexed with P3 only and onto the virion surface. Collectively, we provide a proof of concept for the possible use of the lattice-like organisation and the quasi-symmetric morphology of virus capsomers for loading heterocyclic drugs with defined properties.
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http://dx.doi.org/10.1039/d1nr04153cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8667075PMC
December 2021

Mitochondrial bioenergetics boost macrophage activation, promoting liver regeneration in metabolically compromised animals.

Hepatology 2021 Sep 12. Epub 2021 Sep 12.

Transplant Coordination Unit, Marqués de Valdecilla University Hospital-IDIVAL, Cantabria University, Santander, Spain.

Background And Aims: Hepatic ischemia-reperfusion injury (IRI) is the leading cause of early posttransplantation organ failure as mitochondrial respiration and ATP production are affected. A shortage of donors has extended liver donor criteria, including aged or steatotic livers, which are more susceptible to IRI. Given the lack of an effective treatment and the extensive transplantation waitlist, we aimed at characterizing the effects of an accelerated mitochondrial activity by silencing methylation-controlled J protein (MCJ) in three preclinical models of IRI and liver regeneration, focusing on metabolically compromised animal models.

Approach And Results: Wild-type (WT), MCJ knockout (KO), and Mcj silenced WT mice were subjected to 70% partial hepatectomy (Phx), prolonged IRI, and 70% Phx with IRI. Old and young mice with metabolic syndrome were also subjected to these procedures. Expression of MCJ, an endogenous negative regulator of mitochondrial respiration, increases in preclinical models of Phx with or without vascular occlusion and in donor livers. Mice lacking MCJ initiate liver regeneration 12 h faster than WT and show reduced ischemic injury and increased survival. MCJ knockdown enables a mitochondrial adaptation that restores the bioenergetic supply for enhanced regeneration and prevents cell death after IRI. Mechanistically, increased ATP secretion facilitates the early activation of Kupffer cells and production of TNF, IL-6, and heparin-binding EGF, accelerating the priming phase and the progression through G /S transition during liver regeneration. Therapeutic silencing of MCJ in 15-month-old mice and in mice fed a high-fat/high-fructose diet for 12 weeks improves mitochondrial respiration, reduces steatosis, and overcomes regenerative limitations.

Conclusions: Boosting mitochondrial activity by silencing MCJ could pave the way for a protective approach after major liver resection or IRI, especially in metabolically compromised, IRI-susceptible organs.
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http://dx.doi.org/10.1002/hep.32149DOI Listing
September 2021

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers.

Int J Mol Sci 2021 Jun 1;22(11). Epub 2021 Jun 1.

CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, 48162 Derio, Bizkaia, Spain.

The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing -(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.
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http://dx.doi.org/10.3390/ijms22116000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8199549PMC
June 2021

Chemoenzymatic Synthesis of Complex N-Glycans of the Parasite S. mansoni to Examine the Importance of Epitope Presentation on DC-SIGN recognition.

Angew Chem Int Ed Engl 2021 08 16;60(35):19287-19296. Epub 2021 Jul 16.

Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Bijvoet Center for Biomolecular Research, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands.

The importance of multivalency for N-glycan-protein interactions has primarily been studied by attachment of minimal epitopes to artificial multivalent scaffold and not in the context of multi-antennary glycans. N-glycans can be modified by bisecting GlcNAc, core xylosides and fucosides, and extended N-acetyl lactosamine moieties. The impact of such modifications on glycan recognition are also not well understood. We describe here a chemoenzymatic methodology that can provide N-glycans expressed by the parasitic worm S. mansoni having unique epitopes at each antenna and containing core xyloside. NMR, computational and electron microscopy were employed to investigate recognition of the glycans by the human lectin DC-SIGN. It revealed that core xyloside does not influence terminal epitope recognition. The multi-antennary glycans bound with higher affinity to DC-SIGN compared to mono-valent counterparts, which was attributed to proximity-induced effective concentration. The multi-antennary glycans cross-linked DC-SIGN into a dense network, which likely is relevant for antigen uptake and intracellular routing.
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http://dx.doi.org/10.1002/anie.202105647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8456914PMC
August 2021

Minimal epitope for Mannitou IgM on paucimannose-carrying glycoproteins.

Glycobiology 2021 Sep;31(8):1005-1017

Unité de Glycobiologie Structurale et Fonctionnelle, UMR 8576 du CNRS et Université de Lille, 50 Avenue Halley, 59650 Villeneuve d'Ascq, France.

Paucimannosidic glycans are restricted to the core structure [Man1-3GlcNAc2Fuc0-1] of N-glycans and are rarely found in mammalian tissues. Yet, especially [Man2-3GlcNAc2Fuc1] have been found significantly upregulated in tumors, including in colorectal and liver cancer. Mannitou IgM is a murine monoclonal antibody that was previously shown to recognize Man3GlcNAc2 with an almost exclusive selectivity. Here, we have sought the definition of the minimal glycan epitope of Mannitou IgM, initiated by screening on a newly designed paucimannosidic glycan microarray; among the best binders were Man3GlcNAc2 and its α1,6 core-fucosylated variant, Man3GlcNAc2Fuc1. Unexpectedly and in contrast to earlier findings, Man5GlcNAc2-type structures bind equally well and a large tolerance was observed for substitutions on the α1,6 arm. It was confirmed that any substitution on the single α1,3-linked mannose completely abolishes binding. Surface plasmon resonance for kinetic measurements of Mannitou IgM binding, either directly on the glycans or as presented on omega-1 and kappa-5 soluble egg antigens from the helminth parasite Schistosoma mansoni, showed submicromolar affinities. To characterize the epitope in greater and atomic detail, saturation transfer difference nuclear magnetic resonance spectroscopy was performed with the Mannitou antigen-binding fragment. The STD-NMR data demonstrated the strongest interactions with the aliphatic protons H1 and H2 of the α1-3-linked mannose and weaker imprints on its H3, H4 and H5 protons. In conclusion, Mannitou IgM binding requires a nonsubstituted α1,3-linked mannose branch of paucimannose also on proteins, making it a highly specific tool for the distinction of concurrent human tumor-associated carbohydrate antigens.
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http://dx.doi.org/10.1093/glycob/cwab027DOI Listing
September 2021

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals.

Commun Biol 2021 04 20;4(1):486. Epub 2021 Apr 20.

Cancer Immunology and Immunotherapy Lab, CIC bioGUNE, Basque Research and Technology Alliance (BRTA), Bizkaia, Spain.

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.
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http://dx.doi.org/10.1038/s42003-021-02011-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058339PMC
April 2021

A novel Schmallenberg virus subunit vaccine candidate protects IFNAR mice against virulent SBV challenge.

Sci Rep 2020 11 23;10(1):18725. Epub 2020 Nov 23.

Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, 48160, Derio, Spain.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.
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http://dx.doi.org/10.1038/s41598-020-73424-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7684302PMC
November 2020

Superimposition of Viral Protein Structures: A Means to Decipher the Phylogenies of Viruses.

Viruses 2020 10 9;12(10). Epub 2020 Oct 9.

Center for Cooperative Research in Biosciences (CIC bioGUNE), Basque Research and Technology Alliance (BRTA), Bizkaia Technology Park, 48160 Derio, Spain.

Superimposition of protein structures is key in unravelling structural homology across proteins whose sequence similarity is lost. Structural comparison provides insights into protein function and evolution. Here, we review some of the original findings and thoughts that have led to the current established structure-based phylogeny of viruses: starting from the original observation that the major capsid proteins of plant and animal viruses possess similar folds, to the idea that each virus has an innate "self". This latter idea fueled the conceptualization of the PRD1-adenovirus lineage whose members possess a major capsid protein (innate "self") with a double jelly roll fold. Based on this approach, long-range viral evolutionary relationships can be detected allowing the virosphere to be classified in four structure-based lineages. However, this process is not without its challenges or limitations. As an example of these hurdles, we finally touch on the difficulty of establishing structural "self" traits for enveloped viruses showcasing the coronaviruses but also the power of structure-based analysis in the understanding of emerging viruses.
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http://dx.doi.org/10.3390/v12101146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600307PMC
October 2020

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins.

Angew Chem Int Ed Engl 2020 12 22;59(52):23763-23771. Epub 2020 Oct 22.

CIC bioGUNE, Basque Research and Technology Alliance, BRTA, Bizkaia Technology Park, 48162, Derio, Spain.

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.
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http://dx.doi.org/10.1002/anie.202011015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7894318PMC
December 2020

The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes.

J Biol Chem 2020 01 24;295(1):1-12. Epub 2019 Oct 24.

Molecular Recognition and Host-Pathogen Interactions Programme, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain. Electronic address:

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease currently present in many countries of Africa, Europe, and Asia. Despite intense research efforts, relevant gaps in the architecture of the infectious virus particle remain. Here, we used single-particle cryo-EM to analyze the three-dimensional structure of the mature ASFV particle. Our results show that the ASFV virion, with a radial diameter of ∼2,080 Å, encloses a genome-containing nucleoid surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes. The outer capsid forms a hexagonal lattice (triangulation number = 277) composed of 8,280 copies of the double jelly-roll major capsid protein (MCP) p72, arranged in trimers displaying a pseudo-hexameric morphology, and of 60 copies of a penton protein at the vertices. The inner protein layer, organized as a = 19 capsid, confines the core shell, and it is composed of the mature products derived from the ASFV polyproteins pp220 and pp62. Also, an icosahedral membrane lies between the two protein layers, whereas a pleomorphic envelope wraps the outer capsid. This high-level organization confers to ASFV a unique architecture among the NCLDVs that likely reflects the complexity of its infection process and may help explain current challenges in controlling it.
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http://dx.doi.org/10.1074/jbc.AC119.011196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952596PMC
January 2020

Membrane-Containing Icosahedral Bacteriophage PRD1: The Dawn of Viral Lineages.

Adv Exp Med Biol 2019 ;1215:85-109

Molecular Recognition and Host-Pathogen Interactions Programme, CIC bioGUNE, CIBERehd, Derio, Spain.

Membrane-containing enterobacterial phage PRD1 was isolated from sewage more than 40 years ago. At that time none would have expected the impact that unravelling its biology would have on modern virology and on the way we understand virus assembly, evolution and classification today. PRD1 structural analyses have provided a framework for understanding some aspects of virus evolution-introducing the concept of "viral lineages"-where the three-dimensional structures of virus capsids represent the fingerprint for evolutionary relationship which cannot be traced from the sequence data. In this review we summarise those findings that have led to the notion of viral lineages and the multidisciplinary efforts made in elucidating PRD1 life cycle. These studies have rendered PRD1 a model system not only for the family Tectiviridae to which it belongs, but more generally to complex DNA viruses enclosing a membrane vesicle beneath the capsid shell.
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http://dx.doi.org/10.1007/978-3-030-14741-9_5DOI Listing
September 2019

Structural basis for assembly of vertical single β-barrel viruses.

Nat Commun 2019 03 12;10(1):1184. Epub 2019 Mar 12.

Molecular Recognition and Host-pathogen Interactions Programme, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160, Derio, Spain.

The vertical double β-barrel major capsid protein (MCP) fold, fingerprint of the PRD1-adeno viral lineage, is widespread in many viruses infecting organisms across the three domains of life. The discovery of PRD1-like viruses with two MCPs challenged the known assembly principles. Here, we present the cryo-electron microscopy (cryo-EM) structures of the archaeal, halophilic, internal membrane-containing Haloarcula californiae icosahedral virus 1 (HCIV-1) and Haloarcula hispanica icosahedral virus 2 (HHIV-2) at 3.7 and 3.8 Å resolution, respectively. Our structures reveal proteins located beneath the morphologically distinct two- and three-tower capsomers and homopentameric membrane proteins at the vertices that orchestrate the positioning of pre-formed vertical single β-barrel MCP heterodimers. The cryo-EM based structures together with the proteomics data provide insights into the assembly mechanism of this type of viruses and into those with membrane-less double β-barrel MCPs.
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http://dx.doi.org/10.1038/s41467-019-08927-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414509PMC
March 2019

Rules of engagement between αvβ6 integrin and foot-and-mouth disease virus.

Nat Commun 2017 05 23;8:15408. Epub 2017 May 23.

Division of Structural Biology, The Nuffield Department of Medicine, University of Oxford, The Henry Wellcome Building for Genomic Medicine, Headington, Oxford OX3 7BN, UK.

Foot-and-mouth disease virus (FMDV) mediates cell entry by attachment to an integrin receptor, generally αvβ6, via a conserved arginine-glycine-aspartic acid (RGD) motif in the exposed, antigenic, GH loop of capsid protein VP1. Infection can also occur in tissue culture adapted virus in the absence of integrin via acquired basic mutations interacting with heparin sulphate (HS); this virus is attenuated in natural infections. HS interaction has been visualized at a conserved site in two serotypes suggesting a propensity for sulfated-sugar binding. Here we determined the interaction between αvβ6 and two tissue culture adapted FMDV strains by cryo-electron microscopy. In the preferred mode of engagement, the fully open form of the integrin, hitherto unseen at high resolution, attaches to an extended GH loop via interactions with the RGD motif plus downstream hydrophobic residues. In addition, an N-linked sugar of the integrin attaches to the previously identified HS binding site, suggesting a functional role.
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http://dx.doi.org/10.1038/ncomms15408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457520PMC
May 2017

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.

Nat Commun 2017 05 2;8:15075. Epub 2017 May 2.

Department of Molecular and Cellular Biochemistry, Indiana University, Simon Hall MSB, 212 S Hawthorne Dr, Bloomington, Indiana 47405, USA.

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
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http://dx.doi.org/10.1038/ncomms15075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418573PMC
May 2017

DNA vaccination regimes against Schmallenberg virus infection in IFNAR mice suggest two targets for immunization.

Antiviral Res 2017 05 21;141:107-115. Epub 2017 Feb 21.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain. Electronic address:

Schmallenberg virus (SBV) is an RNA virus of the Bunyaviridae family, genus Orthobunyavirus that infects wild and livestock species of ruminants. While inactivated and attenuated vaccines have been shown to prevent SBV infection, little is known about their mode of immunity; specifically, which components of the virus are responsible for inducing immunological responses in the host. As previous DNA vaccination experiments on other bunyaviruses have found that glycoproteins, as well as modified (i.e. ubiquitinated) nucleoproteins (N) can confer immunity against virulent viral challenge, constructs encoding for fragments of SBV glycoproteins G and G, as well as ubiquitinated and non-ubiquitinated N were cloned in mammalian expression vectors, and vaccinated intramuscularly in IFNAR mice. Upon viral challenge with virulent SBV, disease progression was monitored. Both the ubiquitinated and non-ubiquitinated nucleoprotein candidates elicited high titers of antibodies against SBV, but only the non-ubiquitinated candidate induced statistically significant protection of the vaccinated mice from viral challenge. Another construct encoding for a putative ectodomain of glycoprotein G (segment aa. 678-947) also reduced the SBV-viremia in mice after SBV challenge. When compared to other experimental groups, both the nucleoprotein and G-ectodomain vaccinated groups displayed significantly reduced viremia, as well as exhibiting no clinical signs of SBV infection. These results show that both the nucleoprotein and the putative G-ectodomain can serve as protective immunological targets against SBV infection, highlighting that viral glycoproteins, as well as nucleoproteins are potent targets in vaccination strategies against bunyaviruses.
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http://dx.doi.org/10.1016/j.antiviral.2017.02.013DOI Listing
May 2017

Neurotropic alphaviruses can propagate without capsid.

Oncotarget 2017 Feb;8(6):8999-9000

Division of Gene Therapy, CIMA, University of Navarra, Pamplona, Spain.

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http://dx.doi.org/10.18632/oncotarget.13993DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354702PMC
February 2017

Membrane-assisted viral DNA ejection.

Biochim Biophys Acta Gen Subj 2017 Mar 16;1861(3):664-672. Epub 2016 Dec 16.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain. Electronic address:

Genome packaging and delivery are fundamental steps in the replication cycle of all viruses. Icosahedral viruses with linear double-stranded DNA (dsDNA) usually package their genome into a preformed, rigid procapsid using the power generated by a virus-encoded packaging ATPase. The pressure and stored energy due to this confinement of DNA at a high density is assumed to drive the initial stages of genome ejection. Membrane-containing icosahedral viruses, such as bacteriophage PRD1, present an additional architectural complexity by enclosing their genome within an internal membrane vesicle. Upon adsorption to a host cell, the PRD1 membrane remodels into a proteo-lipidic tube that provides a conduit for passage of the ejected linear dsDNA through the cell envelope. Based on volume analyses of PRD1 membrane vesicles captured by cryo-electron tomography and modeling of the elastic properties of the vesicle, we propose that the internal membrane makes a crucial and active contribution during infection by maintaining the driving force for DNA ejection and countering the internal turgor pressure of the host. These novel functions extend the role of the PRD1 viral membrane beyond tube formation or the mere physical confinement of the genome. The presence and assistance of an internal membrane might constitute a biological advantage that extends also to other viruses that package their linear dsDNA to high density within an internal vesicle.
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http://dx.doi.org/10.1016/j.bbagen.2016.12.013DOI Listing
March 2017

Mechanism of Structural Tuning of the Hepatitis C Virus Human Cellular Receptor CD81 Large Extracellular Loop.

Structure 2017 01 1;25(1):53-65. Epub 2016 Dec 1.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Derio 48160, Spain; IKERBASQUE, Basque Foundation for Science, Bilbao 48013, Spain. Electronic address:

Hepatitis C virus (HCV) enters into human hepatocytes via tetraspanin hCD81. HCV glycoprotein E2 recognizes the "head" subdomain of the large extracellular loop (LEL) of CD81 (hCD81), but the precise mechanism of virus cell attachment and entry remains elusive. Here, by combining the structural analysis of a conspicuous number of crystallized CD81 molecules with molecular dynamics simulations, we show that the conformational plasticity of the hCD81 head subdomain is a molecular property of the receptor. The observed closed, intermediate, and open conformations of the head subdomain provide distinct binding platforms. Simulations at pH 7.4 and 4.0 indicate that this dynamism is pH modulated. The crystallized double conformation of the disulfide bridge C157-C175 at the base of the head subdomain identifies this bond as the molecular zipper of the plasticity of hCD81. We propose that this conformational dependence of hCD81, which is finely tuned by pH and redox conditions, enables the virus-receptor interactions to diversely re-engage at endosomal conditions.
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http://dx.doi.org/10.1016/j.str.2016.11.003DOI Listing
January 2017

The democratization of cryo-EM.

Nat Methods 2016 07;13(8):607-8

Structural Biology Unit, CICbioGUNE, CIBERehd, Derio, Spain.

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http://dx.doi.org/10.1038/nmeth.3946DOI Listing
July 2016

Capsid-deficient alphaviruses generate propagative infectious microvesicles at the plasma membrane.

Cell Mol Life Sci 2016 10 27;73(20):3897-916. Epub 2016 Apr 27.

Division of Gene Therapy, CIMA, University of Navarra, Pamplona, Spain.

Alphavirus budding is driven by interactions between nucleocapsids assembled in the cytoplasm and envelope proteins present at the plasma membrane. So far, the expression of capsid and envelope proteins in infected cells has been considered an absolute requirement for alphavirus budding and propagation. In the present study, we show that Semliki Forest virus and Sindbis virus lacking the capsid gene can propagate in mammalian and insect cells. This propagation is mediated by the release of infectious microvesicles (iMVs), which are pleomorphic and have a larger size and density than wild-type virus. iMVs, which contain viral RNA inside and viral envelope proteins on their surface, are released at the plasma membrane and infect cells using the endocytic pathway in a similar way to wild-type virus. iMVs are not pathogenic in immunocompetent mice when injected intravenously, but can infect different organs like lungs and heart. Finally, we also show that alphavirus genomes without capsid can mediate the propagation of heterologous genes, making these vectors potentially interesting for gene therapy or vaccination studies. The minimalist infectious system described in this study shows that a self-replicating RNA able to express membrane proteins with binding and fusion properties is able to propagate, providing some insights into virus evolution.
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http://dx.doi.org/10.1007/s00018-016-2230-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079800PMC
October 2016

Insight into the Assembly of Viruses with Vertical Single β-barrel Major Capsid Proteins.

Structure 2015 Oct 27;23(10):1866-1877. Epub 2015 Aug 27.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Bizkaia Technology Park, 48160 Derio, Spain; IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain. Electronic address:

Archaeal viruses constitute the least explored niche within the virosphere. Structure-based approaches have revealed close relationships between viruses infecting organisms from different domains of life. Here, using biochemical and cryo-electron microscopy techniques, we solved the structure of euryarchaeal, halophilic, internal membrane-containing Haloarcula hispanica icosahedral virus 2 (HHIV-2). We show that the density of the two major capsid proteins (MCPs) recapitulates vertical single β-barrel proteins and that disulfide bridges stabilize the capsid. Below, ordered density is visible close to the membrane and at the five-fold vertices underneath the host-interacting vertex complex underpinning membrane-protein interactions. The HHIV-2 structure exemplifies the division of conserved architectural elements of a virion, such as the capsid, from those that evolve rapidly due to selective environmental pressure such as host-recognizing structures. We propose that in viruses with two vertical single β-barrel MCPs the vesicle is indispensable, and membrane-protein interactions serve as protein-railings for guiding the assembly.
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http://dx.doi.org/10.1016/j.str.2015.07.015DOI Listing
October 2015

Lysine methylation mapping of crenarchaeal DNA-directed RNA polymerases by collision-induced and electron-transfer dissociation mass spectrometry.

J Proteome Res 2014 May 27;13(5):2637-48. Epub 2014 Mar 27.

Proteomics Platform, CIC bioGUNE, ProteoRed-ISCIII, CIBERehd, 48160 Derio, Spain.

Enzymatic machineries fundamental for information processing (e.g., transcription, replication, translation) in Archaea are simplified versions of their eukaryotic counterparts. This is clearly noticeable in the conservation of sequence and structure of corresponding enzymes (see for example the archaeal DNA-directed RNA polymerase (RNAP)). In Eukarya, post-translational modifications (PTMs) often serve as functional regulatory factors for various enzymes and complexes. Among the various PTMs, methylation and acetylation have been recently attracting most attention. Nevertheless, little is known about such PTMs in Archaea, and cross-methodological studies are scarce. We examined methylation and N-terminal acetylation of endogenously purified crenarchaeal RNA polymerase from Sulfolobus shibatae (Ssh) and Sulfolobus acidocaldarius (Sac). In-gel and in-solution protein digestion methods were combined with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) mass spectrometry analysis. Overall, 20 and 26 methyl-lysines for S. shibatae and S. acidocaldarius were identified, respectively. Furthermore, two N-terminal acetylation sites for each of these organisms were assessed. As a result, we generated a high-confidence data set for the mapping of methylation and acetylation sites in both Sulfolobus species, allowing comparisons with the data previously obtained for RNAP from Sulfolobus solfataricus (Sso). We confirmed that all observed methyl-lysines are on the surface of the RNAP.
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http://dx.doi.org/10.1021/pr500084pDOI Listing
May 2014

From lows to highs: using low-resolution models to phase X-ray data.

Acta Crystallogr D Biol Crystallogr 2013 Nov 18;69(Pt 11):2257-65. Epub 2013 Oct 18.

Division of Structural Biology, The Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington, Oxford OX3 7BN, England.

The study of virus structures has contributed to methodological advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ~13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.
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http://dx.doi.org/10.1107/S0907444913022336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817700PMC
November 2013

Mechanism of membranous tunnelling nanotube formation in viral genome delivery.

PLoS Biol 2013 Sep 24;11(9):e1001667. Epub 2013 Sep 24.

Structural Biology Unit, CIC bioGUNE, CIBERehd, Derio, Spain.

In internal membrane-containing viruses, a lipid vesicle enclosed by the icosahedral capsid protects the genome. It has been postulated that this internal membrane is the genome delivery device of the virus. Viruses built with this architectural principle infect hosts in all three domains of cellular life. Here, using a combination of electron microscopy techniques, we investigate bacteriophage PRD1, the best understood model for such viruses, to unveil the mechanism behind the genome translocation across the cell envelope. To deliver its double-stranded DNA, the icosahedral protein-rich virus membrane transforms into a tubular structure protruding from one of the 12 vertices of the capsid. We suggest that this viral nanotube exits from the same vertex used for DNA packaging, which is biochemically distinct from the other 11. The tube crosses the capsid through an aperture corresponding to the loss of the peripentonal P3 major capsid protein trimers, penton protein P31 and membrane protein P16. The remodeling of the internal viral membrane is nucleated by changes in osmolarity and loss of capsid-membrane interactions as consequence of the de-capping of the vertices. This engages the polymerization of the tail tube, which is structured by membrane-associated proteins. We have observed that the proteo-lipidic tube in vivo can pierce the gram-negative bacterial cell envelope allowing the viral genome to be shuttled to the host cell. The internal diameter of the tube allows one double-stranded DNA chain to be translocated. We conclude that the assembly principles of the viral tunneling nanotube take advantage of proteo-lipid interactions that confer to the tail tube elastic, mechanical and functional properties employed also in other protein-membrane systems.
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http://dx.doi.org/10.1371/journal.pbio.1001667DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782422PMC
September 2013

Combined approaches to study virus structures.

Subcell Biochem 2013 ;68:203-46

Structural Biology Unit, CICbioGUNE, CIBERehd, Bizkaia Technology Park, 48160, Derio, Spain.

A virus particle must work as a safe box for protecting its genome, but at the same time it has to undergo dramatic conformational changes in order to preserve itself by propagating in a cell infection. Thus, viruses are miniaturized wonders whose structural complexity requires them to be investigated by a combination of different techniques that can tackle both static and dynamic processes. In this chapter we will illustrate how major structural techniques such as X-ray crystallography and electron microscopy have been and can be combined with other techniques to determine the structure of complex viruses. The power of these hybrid method approaches are revealed through the various examples provided.
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http://dx.doi.org/10.1007/978-94-007-6552-8_7DOI Listing
July 2014

Archaeal transcription: making up for lost time.

Biochem Soc Trans 2013 Feb;41(1):356-61

Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain.

In recent years, emerging structural information on the aRNAP (archaeal RNA polymerase) apparatus has shown its strong evolutionary relationship with the eukaryotic counterpart, RNA Pol (polymerase) II. A novel atomic model of SshRNAP (Sulfolobus shibatae RNAP) in complex with dsDNA (double-stranded DNA) constitutes a new piece of information helping the understanding of the mechanisms for DNA stabilization at the position downstream of the catalytic site during transcription. In Archaea, in contrast with Eukarya, downstream DNA stabilization is universally mediated by the jaw domain and, in some species, by the additional presence of the Rpo13 subunit. Biochemical and biophysical data, combined with X-ray structures of apo- and DNA-bound aRNAP, have demonstrated the capability of the Rpo13 C-terminus to bind in a sequence-independent manner to downstream DNA. In the present review, we discuss the recent findings on the aRNAP and focus on the mechanisms by which the RNAP stabilizes the bound DNA during transcription.
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http://dx.doi.org/10.1042/BST20120305DOI Listing
February 2013

Soaking of DNA into crystals of archaeal RNA polymerase achieved by desalting in droplets.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2012 Sep 31;68(Pt 9):1134-8. Epub 2012 Aug 31.

Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain.

Transcription is a fundamental process across the three domains of life and is carried out by multi-subunit enzymatic DNA-directed RNA polymerases (RNAPs). The interaction of RNAP with nucleic acids is tightly controlled for precise and processive RNA synthesis. Whilst a wealth of structural information has been gathered on the eukaryotic Pol II in complex with DNA/RNA, no information exists on its ancestral counterpart archaeal RNAP. Thus, in order to extend knowledge of the archaeal transcriptional apparatus, crystallization of Sulfolobus shibatae RNAP (molecular mass of ~400 kDa) with DNA fragments was pursued. To achieve this goal, crystal growth was first optimized using a nanoseeding technique. An ad hoc soaking protocol was then put into place, which consisted of gently exchanging the high-salt buffer used for apo-RNAP crystal growth into a low-salt buffer necessary for DNA binding to RNAP. Of the various crystals screened, one diffracted to 4.3 Å resolution and structural analysis showed the presence of bound DNA [Wojtas et al. (2012). Nucleic Acids Res. 40, doi:10.1093/nar/gks692].
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http://dx.doi.org/10.1107/S1744309112033507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3433216PMC
September 2012

Structural and functional analyses of the interaction of archaeal RNA polymerase with DNA.

Nucleic Acids Res 2012 Oct 30;40(19):9941-52. Epub 2012 Jul 30.

Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain.

Multi-subunit RNA polymerases (RNAPs) in all three domains of life share a common ancestry. The composition of the archaeal RNAP (aRNAP) is not identical between phyla and species, with subunits Rpo8 and Rpo13 found in restricted subsets of archaea. While Rpo8 has an ortholog, Rpb8, in the nuclear eukaryal RNAPs, Rpo13 lacks clear eukaryal orthologs. Here, we report crystal structures of the DNA-bound and free form of the aRNAP from Sulfolobus shibatae. Together with biochemical and biophysical analyses, these data show that Rpo13 C-terminus binds non-specifically to double-stranded DNA. These interactions map on our RNAP-DNA binary complex on the downstream DNA at the far end of the DNA entry channel. Our findings thus support Rpo13 as a RNAP-DNA stabilization factor, a role reminiscent of eukaryotic general transcriptional factors. The data further yield insight into the mechanisms and evolution of RNAP-DNA interaction.
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http://dx.doi.org/10.1093/nar/gks692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479171PMC
October 2012

Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography.

Virology 2012 Sep 31;430(2):120-6. Epub 2012 May 31.

Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio, Spain.

Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.
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http://dx.doi.org/10.1016/j.virol.2012.05.001DOI Listing
September 2012
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