Structural studies on adenovirus: the human adenovirus packaging motor, and characterization of two reptilian adenoviruses

Tesis doctoral inédita, leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 21-03-201

Structure and uncoating of immature adenovirus

Maturation via proteolytical processing is a common trait in the viral world, and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins, but ends with proteolytically processed versions in the mature virion; and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytical processing are not infectious. We present the 3D structure of immature adenovirus particles, as represented by the thermosensitive mutant Ad2 ts1 grown under nonpermissive conditions, and compare it with the mature capsid. Our 3DEM maps at subnanometer resolution indicate that adenovirus maturation does not involve large scale conformational changes in the capsid. Difference maps reveal the location of unprocessed peptides pIIIa and pVI and help to define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (BFU2007-60228 to C.S.M. and BIO2007-67150-C03-03 to R.M.), the Comunidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas (CCG08-CSIC/SAL-3442 to C.S.M.) and the National Institutes of Health (5R01CA111569 to D.T.C., R0141599 to W.F.M. and GM037705 to S.J.F.). R.M.-C. is a recipient of a PFIS fellowship from the Instituto de Salud Carlos III of Spain. A.J.P.-B. holds a CSIC JAE-Doc postdoctoral position, partially funded by the European Social FundPeer reviewe

Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins

International audienceOf five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast-and atadenoviruses, induced by the presence of an extended polypeptide. This polypeptide, and -helical clusters beneath the facet, likely correspond to genus-specific proteins LH2 and p32k. Another genus-specific protein, LH3, with a fold typical of bacteriophage tailspikes, contacts the capsid surface via a triskelion structure identical to that used by mastadenovirus protein IX, revealing a conserved capsid-binding motif and an ancient gene duplication event. Our data also suggest that mastadenovirus E1B-55 K was exapted from the atadenoviruslike LH3 protein. This work provides new information on the evolution of adenoviruses, emphasizing the importance of minor coat proteins for determining specific physicochemical properties of virions and most likely their tropism

Crystal Structure of the Fibre Head Domain of the Atadenovirus Snake Adenovirus 1

<div><p>Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest.</p></div

Structure and uncoating of immature adenovirus

Maturation via proteolytical processing is a common trait in the viral world, and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins, but ends with proteolytically processed versions in the mature virion; and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytical processing are not infectious. We present the 3D structure of immature adenovirus particles, as represented by the thermosensitive mutant Ad2 ts1 grown under nonpermissive conditions, and compare it with the mature capsid. Our 3DEM maps at subnanometer resolution indicate that adenovirus maturation does not involve large scale conformational changes in the capsid. Difference maps reveal the location of unprocessed peptides pIIIa and pVI and help to define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (BFU2007-60228 to C.S.M. and BIO2007-67150-C03-03 to R.M.), the Comunidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas (CCG08-CSIC/SAL-3442 to C.S.M.) and the National Institutes of Health (5R01CA111569 to D.T.C., R0141599 to W.F.M. and GM037705 to S.J.F.). R.M.-C. is a recipient of a PFIS fellowship from the Instituto de Salud Carlos III of Spain. A.J.P.-B. holds a CSIC JAE-Doc postdoctoral position, partially funded by the European Social FundPeer reviewe

Structure of the Snake Atadenovirus 1 fibre head.

<p>A. Monomer structure. The beta-strands are labeled. B. Topology. The ABCJ and GHID beta-sheets are coloured yellow, the alpha-helix in the DG-loop is shown in red. C. Trimer structure, side view, with the three monomers coloured differently. D. Trimer structure, top view.</p

Structural comparison with other viral receptor-binding proteins.

<p>Superposition of the SnAdV-1 fibre head monomer structure (green) onto the bacteriophage TP901-1 receptor-binding protein monomer (A; PDB entry 2F0C; red), onto the avian reovirus fibre head domain monomer (B; PDB entry 2BT7; blue) and the HAdV-37 fibre head monomer (C; PDB entry 2WBW; yellow). The SnAdV-1 fibre head monomer is in the same orientation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114373#pone-0114373-g001" target="_blank">Fig. 1A</a>. The top of the trimer is indicated with * and the side with #.</p

The role of capsid maturation on adenovirus priming for sequential uncoating

14 pags, 6 figs, 5 tabsIncluye material complementario en la web del editorAdenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus.This work was also supportedby Ministry of Science and Innovation of Spain Grants BFU2010-16382/BMC (to C. S. M.), MAT2008-02533, PIB2010US-00233, and FIS2011-29493 (to P. J. P.), FIS2010-10552-E and FIS2011-16090-E (to C. S. M. and P. J. P.), and BFU2009-10052 (to M. M.), and by Local Madrid Government Grant P2009/MAT-1467 (to P. J. P.

The role of capsid maturation on adenovirus priming for sequential uncoating.

Adenovirus assembly concludes with proteolytic processing of several capsid and core proteins. Immature virions containing precursor proteins lack infectivity because they cannot properly uncoat, becoming trapped in early endosomes. Structural studies have shown that precursors increase the network of interactions maintaining virion integrity. Using different biophysical techniques to analyze capsid disruption in vitro, we show that immature virions are more stable than the mature ones under a variety of stress conditions and that maturation primes adenovirus for highly cooperative DNA release. Cryoelectron tomography reveals that under mildly acidic conditions mimicking the early endosome, mature virions release pentons and peripheral core contents. At higher stress levels, both mature and immature capsids crack open. The virus core is completely released from cracked capsids in mature virions, but it remains connected to shell fragments in the immature particle. The extra stability of immature adenovirus does not equate with greater rigidity, because in nanoindentation assays immature virions exhibit greater elasticity than the mature particles. Our results have implications for the role of proteolytic maturation in adenovirus assembly and uncoating. Precursor proteins favor assembly by establishing stable interactions with the appropriate curvature and preventing premature ejection of contents by tightly sealing the capsid vertices. Upon maturation, core organization is looser, particularly at the periphery, and interactions preserving capsid curvature are weakened. The capsid becomes brittle, and pentons are more easily released. Based on these results, we hypothesize that changes in core compaction during maturation may increase capsid internal pressure to trigger proper uncoating of adenovirus