Cryo-EM structure of enteric adenovirus HAdV-F41 highlights structural variations among human adenoviruses

Enteric adenoviruses, one of the main causes of viral gastroenteritis in the world, must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. We report the 4-Å-resolution structure of a human enteric adenovirus, HAdV-F41, and compare it with that of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms. While the overall structures of hexon, penton base, and internal minor coat proteins IIIa and VIII are conserved, we observe partially ordered elements reinforcing the vertex region, which suggests their role in enhancing the physicochemical capsid stability of HAdV-F41. Unexpectedly, we find an organization of the external minor coat protein IX different from all previously characterized human and nonhuman mastadenoviruses. Knowledge of the structure of enteric adenoviruses provides a starting point for the design of vectors suitable for oral delivery or intestinal targetingThis work was supported by grants PID2019-104098GB-I00/AEI/10.13039/501100011033 and BFU2016-74868-P, cofunded by the Spanish State Research Agency and the European Regional Development Fund; BFU2013-41249-P and BIO2015-68990-REDT (the Spanish Adenovirus Network, AdenoNet) from the Spanish Ministry of Economy, Industry, and Competitiveness; and the Agencia Estatal CSIC (2019AEP045) to C.S.M. The CNB-CSIC is further supported by a Severo Ochoa Excellence grant (SEV 2017-0712). Work in M.B’s. lab was supported by grant 194562-08 from the Natural Sciences and Engineering Research Council of is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. holds a predoctoral contract from La Caixa Foundation (ID 100010434), under agreement LCF/BQ/SO16/52270032. Access to CEITEC was supported by iNEXT, project number 653706, funded by the Horizon 2020 Programme of the European Union. The CEITEC Cryo-electron Microscopy and Tomography core facility is supported by MEYS CR (LM2018127

Acidification induces condensation of the adenovirus core

The adenovirus (AdV) icosahedral capsid encloses a nucleoprotein core formed by the dsDNA genome bound to numerous copies of virus-encoded, positively charged proteins. For an efficient delivery of its genome, AdV must undergo a cascade of dismantling events from the plasma membrane to the nuclear pore. Throughout this uncoating process, the virion moves across potentially disruptive environments whose influence in particle stability is poorly understood. In this work we analyze the effect of acidic conditions on AdV particles by exploring their mechanical properties, genome accessibility and capsid disruption. Our results show that under short term acidification the AdV virion becomes softer and its genome less accessible to an intercalating dye, even in the presence of capsid openings. The AFM tip penetrates deeper in virions at neutral pH, and mechanical properties of genome-less particles are not altered upon acidification. Altogether, these results indicate that the main effect of acidification is the compaction of the nucleoproteic core, revealing a previously unknown role for chemical cues in AdV uncoating. Statement of significance: Studying the behavior of virus particles under changing environmental conditions is key to understand cell entry and propagation. One such change is the acidification undergone in certain cell compartments, which is thought to play a role in the programmed uncoating of virus genomes. Mild acidification in the early endosome has been proposed as a trigger signal for human AdV uncoating. However, the actual effect of low pH in AdV stability and entry is not well defined. Understanding the consequences of acidification in AdV structure and stability is also relevant to define storage conditions for therapeutic vectors, or design AdV variants resistant to intestinal conditions for oral administration of vaccinesWe thank M. G. Mateu (CBMSO-CSIC-UAM) for careful reading and insightful comments on early drafts, M. Castellanos and L. A. Campos (CNB-CSIC) for advice with fluorescence measurements and analyses, and M.I. Laguna (CNB-CSIC) for expert technical help. This work was supported by grants from the Spanish Ministry of Economy, Industry and Competitiveness (FIS2017- 89549- R; “Maria de Maeztu” Program for Units of Excellence in R&D MDM-2014-0377; and FIS2017-90701-REDT) and from the Human Frontiers Science Program (HFSPO RGP0012/2018) to P.J.P.; as well as grants PID2019-104098GB-I00/AEI/10.13039/501100011033 and BFU2016-74868-P, co-funded by the Spanish State Research Agency and the European Regional Development Fund, and 2019AEP045 from the Agencia Estatal CSIC to C.S.M. The CNB-CSIC is further supported by a Severo Ochoa Excellence grant (SEV 2017-0712). MM was funded by grant RTI2018-099985-B-I00, (MICINN/FEDER, UE) and the Ciber of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII). M.H.-P. was a recipient of a Juan de la Cierva Incorporation postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. held a predoctoral contract from La Caixa Foundation (ID 100010434, under agreement LCF/BQ/SO16/52270032). J. G. is a re cipient of a FPI predoctoral contract (BES-2017-079868) funded by the Spanish State Research Agenc

Adenovirus major core protein condenses DNA in clusters and bundles, modulating genome release and capsid internal pressure

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore

Estructura de dos cápsidas estables y complejas: adenovirus entéricos y aviares

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 04-11-2021Esta tesis tiene embargado el acceso al texto completo hasta el 04-05-2023Adenoviruses have a non-enveloped icosahedral capsid with a pseudo T=25 geometry. They are relatively large— with a diameter of 900 Å, enclosing a dsDNA genome in the range of 25-49 kbp (depending on the adenovirus genus) accompanied by virus encoded “histone-like” proteins. Adenoviruses infect a broad range of vertebrate hosts and are traditionally studied due to their pathogenicity in immunocompromised human individuals and in certain animal species. In particular human enteric adenoviruses are of interest because they constitute one of the main causes of viral gastroenteritis in the world and must withstand the harsh conditions found in the gut. This requirement suggests that capsid stability must be different from that of other adenoviruses. On the other hand, the use of non-human adenoviruses has been proposed as alternative vectors in terms of evading the pre-existing immune response against human adenovirus-based vectors. In this thesis we have assessed the thermal stability of an enteric human adenovirus (HAdV-F41) and a fowl adenovirus (FAdV-C4). HAdV-F41 and notably FAdV-C4, are more thermostable than the prototype respiratory adenovirus HAdV-C5. We have determined the structure of HAdV-F41 at 4 Å resolution and of FAdV-C4 at 3.3 Å by single particle averaging cryo-electron microscopy. We have compared the structures with those of other adenoviruses with respiratory (HAdV-C5) and ocular (HAdV-D26) tropisms, as well as adenoviruses infecting lizards (LAdV-2) and cows (BAdV-3). Overall, major coat proteins reveal conservation among adenoviruses, although the organization of surface-exposed loop regions is substantially divergent in HAdV-F41 and FAdV-C4 from the prototype HAdV-C5. Unexpectedly, the organization of the external cementing protein (IX) in HAdV-F41 is unique compared to all previously characterized adenoviruses. In FAdV-C4, internal minor capsid proteins (VIII and IIIa) show important rearranged domains, as compared to other adenovirus structures. Finally, we provide preliminary insights on the different organization of the nucleoproteic core of FAdVC4. Variance in the core organization among adenoviruses might play a role in determining the stability features. All this knowledge could inform on modifications to use HAdV-F41-based oral vaccines with a longer “shelf-life” than drugs relying on HAdV-C5. Design of highly stable vectors, with reduced immunogenic response, based on FAdVs would be also of interestThis study has principally received funding from a predoctoral contract from “La Caixa” Foundation (ID 100010434) under the agreement LCF/BQ/SO16/52270032. This work was further supported by the following grants from: Agencia Estatal de Investigación/FEDER (PID2019-104098GB-I00 and BFU2016-74868-P), MINECO (BIO2015-68990-REDT and BFU2013-41249-P), Diamond Light Source (EM15997), iNEXT (project 653706) and EMBO (Applicant 8182

Adenovirus Structure: What Is New?

Adenoviruses are large (~950 Å) and complex non-enveloped, dsDNA icosahedral viruses. They have a pseudo-T = 25 triangulation number with at least 12 different proteins composing the virion. These include the major and minor capsid proteins, core proteins, maturation protease, terminal protein, and packaging machinery. Although adenoviruses have been studied for more than 60 years, deciphering their architecture has presented a challenge for structural biology techniques. An outstanding event was the first near-atomic resolution structure of human adenovirus type 5 (HAdV-C5), solved by cryo-electron microscopy (cryo-EM) in 2010. Discovery of new adenovirus types, together with methodological advances in structural biology techniques, in particular cryo-EM, has lately produced a considerable amount of new, high-resolution data on the organization of adenoviruses belonging to different species. In spite of these advances, the organization of the non-icosahedral core is still a great unknown. Nevertheless, alternative techniques such as atomic force microscopy (AFM) are providing interesting glimpses on the role of the core proteins in genome condensation and virion stability. Here we summarize the current knowledge on adenovirus structure, with an emphasis on high-resolution structures obtained since 2010

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

9 pags., 7 figs., 1 tab.Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating. We thank the Centro Nacional de Biotecnología and the Centro de Investigaciones Biológicas cryoelectron microscopy facility for data acquisition.This work was supported by Grant BFU2016-74868-P, cofunded by the Spanish State Research Agency and the European Regional Development Fund (to C.S.M.); as well as by grants from the Spanish Ministry of Economy, Industry and Competitiveness BIO2015-68990-REDT (the Spanish Adenovirus Network, AdenoNet) (to C.S.M.), FIS2017-89549-R, “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377), and FIS2017-90701-REDT (to P.J.d.P.). P.H. was funded by National Institutes of Health Grants CA122677 and AI102577. U.F.G. and M.S. were funded by the Swiss National Science Foundation (SNSF 31003A_179256/1) and the Swiss National Science Foundation program Sinergia (CRSII5_170929/1). M.M. was funded by Grant BFU2015-70052R (Spanish Ministry of Economy, Industry and Competitiveness and European Regional Development Fund) and the Centro de Investigación Biomédica en Red de Enfermedades Respiratorias, an initiative from the Spanish Institute of Health Carlos III. M.H.-P. is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish State Research Agency. M.P.-I. holds a predoctoral contract from La Caixa Foundation

Dynamic competition for hexon binding between core protein VII and lytic protein VI promotes adenovirus maturation and entry

Adenovirus minor coat protein VI contains a membrane-disrupting peptide that is inactive when VI is bound to hexon trimers. Protein VI must be released during entry to ensure endosome escape. Hexon:VI stoichiometry has been uncertain, and only fragments of VI have been identified in the virion structure. Recent findings suggest an unexpected relationship between VI and the major core protein, VII. According to the high-resolution structure of the mature virion, VI and VII may compete for the same binding site in hexon; and noninfectious human adenovirus type 5 particles assembled in the absence of VII (Ad5-VII-) are deficient in proteolytic maturation of protein VI and endosome escape. Here we show that Ad5-VII- particles are trapped in the endosome because they fail to increase VI exposure during entry. This failure was not due to increased particle stability, because capsid disruption happened at lower thermal or mechanical stress in Ad5-VII- compared to wild-type (Ad5-wt) particles. Cryoelectron microscopy difference maps indicated that VII can occupy the same binding pocket as VI in all hexon monomers, strongly arguing for binding competition. In the Ad5-VII- map, density corresponding to the immature amino-terminal region of VI indicates that in the absence of VII the lytic peptide is trapped inside the hexon cavity, and clarifies the hexon:VI stoichiometry conundrum. We propose a model where dynamic competition between proteins VI and VII for hexon binding facilitates the complete maturation of VI, and is responsible for releasing the lytic protein from the hexon cavity during entry and stepwise uncoating

Multifunctional carbon nanotubes covalently coated with imine-based covalent organic frameworks: Exploring structure-property relationships through nanomechanics

The assembly of 3-dimensional covalent organic frameworks on the surface of carbon nanotubes is designed and successfully developed for the first time via the hybridization of imine-based covalent organic frameworks (COF-300) and oxidized MWCNTs by one-pot chemical synthesis. The resulting hybrid material ox-MWCNTs@COF exhibits a conformal structure that consists of a uniform amorphous COF layer covering the ox-MWCNT surface The measurements of individual hybrid nanotube mechanical strength performed with atomic force microscopy provide insights into their stability and resistance. The results evidence a very robust hybrid tubular nanostructure that preserves the benefits obtained from COF, such as CO2 adsorption. Further digestion of the organic structure with aniline enables the study of the interplay between the hybrid interface and its nanomechanics. This new hybrid nanomaterial presents exceptional mechanical and electrical properties, merging the properties of the CNT template and COF-300.We thank the Spanish Government (RTI2018-095038-B-I00), “Comunidad de Madrid” and European Structural Funds (S2018/NMT-4367). P. J. P. thanks to “María de Maeztu” Program for Units of Excellence in R&D (MDM-2014-0377), FIS2017-89549-R and Human Frontiers Science Program (HFSPO RGP0012/2018). C. S. M. acknowledges support from the Spanish State Research Agency and European Regional Development Fund [BFU2016-74868-P]. M. H.-P. is a recipient of a Juan de la Cierva postdoctoral contract funded by the Spanish Ministry of Economy and Competitivity. M. P.-I. holds a predoctoral fellowship funded by La Caixa Foundatio