RANBP2 and USP9x regulate nuclear import of adenovirus minor coat protein IIIa.

As intracellular parasites, viruses exploit cellular proteins at every stage of infection. Adenovirus outbreaks are associated with severe acute respiratory illnesses and conjunctivitis, with no specific antiviral therapy available. An adenoviral vaccine based on human adenovirus species D (HAdV-D) is currently in use for COVID-19. Herein, we investigate host interactions of HAdV-D type 37 (HAdV-D37) protein IIIa (pIIIa), identified by affinity purification and mass spectrometry (AP-MS) screens. We demonstrate that viral pIIIa interacts with ubiquitin-specific protease 9x (USP9x) and Ran-binding protein 2 (RANBP2). USP9x binding did not invoke its signature deubiquitination function but rather deregulated pIIIa-RANBP2 interactions. In USP9x-knockout cells, viral genome replication and viral protein expression increased compared to wild type cells, supporting a host-favored mechanism for USP9x. Conversely, RANBP2-knock down reduced pIIIa transport to the nucleus, viral genome replication, and viral protein expression. Also, RANBP2-siRNA pretreated cells appeared to contain fewer mature viral particles. Transmission electron microscopy of USP9x-siRNA pretreated, virus-infected cells revealed larger than typical paracrystalline viral arrays. RANBP2-siRNA pretreatment led to the accumulation of defective assembly products at an early maturation stage. CRM1 nuclear export blockade by leptomycin B led to the retention of pIIIa within cell nuclei and hindered pIIIa-RANBP2 interactions. In-vitro binding analyses indicated that USP9x and RANBP2 bind to C-terminus of pIIIa amino acids 386-563 and 386-510, respectively. Surface plasmon resonance testing showed direct pIIIa interaction with recombinant USP9x and RANBP2 proteins, without competition. Using an alternative and genetically disparate adenovirus type (HAdV-C5), we show that the demonstrated pIIIa interaction is also important for a severe respiratory pathogen. Together, our results suggest that pIIIa hijacks RANBP2 for nuclear import and subsequent virion assembly. USP9x counteracts this interaction and negatively regulates virion synthesis. This analysis extends the scope of known adenovirus-host interactions and has potential implications in designing new antiviral therapeutics.

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.

ICTV Virus Taxonomy Profile: Adenoviridae 2022.

The family Adenoviridae includes non-enveloped viruses with linear dsDNA genomes of 25-48 kb and medium-sized icosahedral capsids. Adenoviruses have been discovered in vertebrates from fish to humans. The family is divided into six genera, each of which is more common in certain animal groups. The outcome of infection may vary from subclinical to lethal disease. This is a summary of the ICTV Report on the family Adenoviridae, which is available at ictv.global/report/adenoviridae.

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.

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.

Regulation of the Ebola Virus VP24 Protein by SUMO.

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center.

Adenovirus (AdV) morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM) to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

Virus Maturation.

A virus particle consists of a genome contained within a protein shell. This shell (the capsid) plays multiple roles throughout the infectious cycle, from genome protection to host recognition to successful genome delivery. When capsids first assemble in the cell, most often an initial product is obtained that has not achieved its fully infectious form. To do so, it must undergo a final process called maturation. Virus maturation entails conformational and stability changes. These changes are often driven by proteolytic cleavages, and their main purpose is to ensure successful delivery of the virus genome to a new host cell. Recent advances in molecular, structural, and physical virology techniques are providing a wealth of detailed information and new points of view to understand the principles of virus maturation. Evidence showing that viral capsids are built with a limited set of structural solutions has prompted a new virus classification in structural lineages deriving from a few initial ancestors. This chapter summarizes the current knowledge on maturation for the main virus structural lineages, as well as for other relevant viruses not assigned to any particular lineage yet.

The so far farthest reaches of the double jelly roll capsid protein fold.

BACKGROUND: During the last two decades, structural biology analyses have shown that viruses infecting hosts far apart in evolution share similar architectural features, prompting a new virus classification based on structural lineages. Until recently, only a few prokaryotic viruses had been described for one of the lineages, whose main characteristic is a capsid protein with a perpendicular double jelly roll. MAIN BODY: Metagenomics analyses are showing that the variety of prokaryotic viruses encoding double jelly roll capsid proteins is much larger than previously thought. The newly discovered viruses have novel genome organisations with interesting implications for virus structure, function and evolution. There are also indications of their having a significant ecological impact. CONCLUSION: Viruses with double jelly roll capsid proteins that infect prokaryotic hosts form a large part of the virosphere that had so far gone unnoticed. Their discovery by metagenomics is only a first step towards many more exciting findings. Work needs to be invested in isolating these viruses and their hosts, characterizing the structure and function of the proteins their genomes encode, and eventually access the wealth of biological information they may hold.

Role of Condensing Particles in Polymer Confinement: A Model for Virus-Packed "Minichromosomes".

Confined mixtures of a polymer and nonspecifically binding particles (condensers) are studied as models for viruses containing double-stranded DNA (polymer) and condensing proteins (particles). We explore a model in which all interactions between the packed content (polymer and particles) and its confinement are purely repulsive, with only a short-range attraction between the condensers and polymer to simulate binding. In the range of physical parameters applicable to viruses, the model predicts reduction of pressure in the system effected by the condensers, despite the reduction in free volume. Condensers are found to be interspersed throughout the spherical confinement and only partially wrapped in the polymer, which acts as an effective medium for the condenser interactions. Crowding of the viral interior influences the DNA and protein organization, producing a picture inconsistent with a chromatin-like, beads-on-a-string structure. The model predicts an organization of the confined interior compatible with experimental data on unperturbed adenoviruses and polyomaviruses, at the same time providing insight into the role of condensing proteins in the viral infectious cycles of related viral families.

Distribution of DNA-condensing protein complexes in the adenovirus core.

Genome packing in adenovirus has long evaded precise description, since the viral dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the virus protein coating (capsid). We show that useful insights regarding the organization of the core can be inferred from the analysis of spatial distributions of the DNA and condensing protein units (adenosomes). These were obtained from the inspection of cryo-electron tomography reconstructions of individual human adenovirus particles. Our analysis shows that the core lacks symmetry and strict order, yet the adenosome distribution is not entirely random. The features of the distribution can be explained by modeling the condensing proteins and the part of the genome in each adenosome as very soft spheres, interacting repulsively with each other and with the capsid, producing a minimum outward pressure of ∼0.06 atm. Although the condensing proteins are connected by DNA in disrupted virion cores, in our models a backbone of DNA linking the adenosomes is not required to explain the experimental results in the confined state. In conclusion, the interior of an adenovirus infectious particle is a strongly confined and dense phase of soft particles (adenosomes) without a strictly defined DNA backbone.

Structures of Adenovirus Incomplete Particles Clarify Capsid Architecture and Show Maturation Changes of Packaging Protein L1 52/55k.

UNLABELLED: Adenovirus is one of the most complex icosahedral, nonenveloped viruses. Even after its structure was solved at near-atomic resolution by both cryo-electron microscopy and X-ray crystallography, the location of minor coat proteins is still a subject of debate. The elaborated capsid architecture is the product of a correspondingly complex assembly process, about which many aspects remain unknown. Genome encapsidation involves the concerted action of five virus proteins, and proteolytic processing by the virus protease is needed to prime the virion for sequential uncoating. Protein L1 52/55k is required for packaging, and multiple cleavages by the maturation protease facilitate its release from the nascent virion. Light-density particles are routinely produced in adenovirus infections and are thought to represent assembly intermediates. Here, we present the molecular and structural characterization of two different types of human adenovirus light particles produced by a mutant with delayed packaging. We show that these particles lack core polypeptide V but do not lack the density corresponding to this protein in the X-ray structure, thereby adding support to the adenovirus cryo-electron microscopy model. The two types of light particles present different degrees of proteolytic processing. Their structures provide the first glimpse of the organization of L1 52/55k protein inside the capsid shell and of how this organization changes upon partial maturation. Immature, full-length L1 52/55k is poised beneath the vertices to engage the virus genome. Upon proteolytic processing, L1 52/55k disengages from the capsid shell, facilitating genome release during uncoating. IMPORTANCE: Adenoviruses have been extensively characterized as experimental systems in molecular biology, as human pathogens, and as therapeutic vectors. However, a clear picture of many aspects of their basic biology is still lacking. Two of these aspects are the location of minor coat proteins in the capsid and the molecular details of capsid assembly. Here, we provide evidence supporting one of the two current models for capsid architecture. We also show for the first time the location of the packaging protein L1 52/55k in particles lacking the virus genome and how this location changes during maturation. Our results contribute to clarifying standing questions in adenovirus capsid architecture and provide new details on the role of L1 52/55k protein in assembly.

Processing of the l1 52/55k protein by the adenovirus protease: a new substrate and new insights into virion maturation.

Late in adenovirus assembly, the viral protease (AVP) becomes activated and cleaves multiple copies of three capsid and three core proteins. Proteolytic maturation is an absolute requirement to render the viral particle infectious. We show here that the L1 52/55k protein, which is present in empty capsids but not in mature virions and is required for genome packaging, is the seventh substrate for AVP. A new estimate on its copy number indicates that there are about 50 molecules of the L1 52/55k protein in the immature virus particle. Using a quasi-in vivo situation, i.e., the addition of recombinant AVP to mildly disrupted immature virus particles, we show that cleavage of L1 52/55k is DNA dependent, as is the cleavage of the other viral precursor proteins, and occurs at multiple sites, many not conforming to AVP consensus cleavage sites. Proteolytic processing of L1 52/55k disrupts its interactions with other capsid and core proteins, providing a mechanism for its removal during viral maturation. Our results support a model in which the role of L1 52/55k protein during assembly consists in tethering the viral core to the icosahedral shell and in which maturation proceeds simultaneously with packaging, before the viral particle is sealed.

Molecular characterization of a lizard adenovirus reveals the first atadenovirus with two fiber genes and the first adenovirus with either one short or three long fibers per penton.

UNLABELLED: Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE: Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins-one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.

Transmission electron microscopy and the molecular structure of icosahedral viruses.

The field of structural virology developed in parallel with methodological advances in X-ray crystallography and cryo-electron microscopy. At the end of the 1970s, crystallography yielded the first high resolution structure of an icosahedral virus, the T=3 tomato bushy stunt virus at 2.9Å. It took longer to reach near-atomic resolution in three-dimensional virus maps derived from electron microscopy data, but this was finally achieved, with the solution of complex icosahedral capsids such as the T=25 human adenovirus at ∼3.5Å. Both techniques now work hand-in-hand to determine those aspects of virus assembly and biology that remain unclear. This review examines the trajectory followed by EM imaging techniques in showing the molecular structure of icosahedral viruses, from the first two-dimensional negative staining images of capsids to the latest sophisticated techniques that provide high resolution three-dimensional data, or snapshots of the conformational changes necessary to complete the infectious cycle.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: IV. viral proteinase slides along DNA to locate and process its substrates.

Precursor proteins used in the assembly of adenovirus virions must be processed by the virally encoded adenovirus proteinase (AVP) before the virus particle becomes infectious. An activated adenovirus proteinase, the AVP-pVIc complex, was shown to slide along viral DNA with an extremely fast one-dimensional diffusion constant, 21.0 ± 1.9 × 10(6) bp(2)/s. In principle, one-dimensional diffusion can provide a means for DNA-bound proteinases to locate and process DNA-bound substrates. Here, we show that this is correct. In vitro, AVP-pVIc complexes processed a purified virion precursor protein in a DNA-dependent reaction; in a quasi in vivo environment, heat-disrupted ts-1 virions, AVP-pVIc complexes processed five different precursor proteins in DNA-dependent reactions. Sliding of AVP-pVIc complexes along DNA illustrates a new biochemical mechanism by which a proteinase can locate its substrates, represents a new paradigm for virion maturation, and reveals a new way of exploiting the surface of DNA.

Regulation of a viral proteinase by a peptide and DNA in one-dimensional space: II. adenovirus proteinase is activated in an unusual one-dimensional biochemical reaction.

Late in an adenovirus infection, the viral proteinase (AVP) becomes activated to process virion precursor proteins used in virus assembly. AVP is activated by two cofactors, the viral DNA and pVIc, an 11-amino acid peptide originating from the C terminus of the precursor protein pVI. There is a conundrum in the activation of AVP in that AVP and pVI are sequence-independent DNA-binding proteins with nm equilibrium dissociation constants such that in the virus particle, they are predicted to be essentially irreversibly bound to the viral DNA. Here, we resolve that conundrum by showing that activation of AVP takes place on the one-dimensional contour of DNA. In vitro, pVI, a substrate, slides on DNA via one-dimensional diffusion, D(1) = 1.45 × 10(6) bp(2)/s, until it binds to AVP also on the same DNA molecule. AVP, partially activated by being bound to DNA, excises pVIc, which binds to the AVP molecule that cut it out. pVIc then forms a disulfide bond with AVP forming the fully active AVP-pVIc complex bound to DNA. In vivo, in heat-disrupted immature virus, AVP was also activated by pVI in DNA-dependent reactions. This activation mechanism illustrates a new paradigm for virion maturation and a new way, by sliding on DNA, for bimolecular complexes to form among proteins not involved in DNA metabolism.

Structure and assembly of complex viruses.

Viral particles consist essentially of a proteinaceous capsid protecting a genome and involved also in many functions during the virus life cycle. In simple viruses, the capsid consists of a number of copies of the same, or a few different proteins organized into a symmetric oligomer. Structurally complex viruses present a larger variety of components in their capsids than simple viruses. They may contain accessory proteins with specific architectural or functional roles; or incorporate non-proteic elements such as lipids. They present a range of geometrical variability, from slight deviations from the icosahedral symmetry to complete asymmetry or even pleomorphism. Putting together the many different elements in the virion requires an extra effort to achieve correct assembly, and thus complex viruses require sophisticated mechanisms to regulate morphogenesis. This chapter provides a general view of the structure and assembly of complex viruses.

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.

Crystallization of the C-terminal domain of the fibre protein from snake adenovirus 1, an atadenovirus.

Adenovirus fibre proteins play an important role in determining viral tropism. The C-terminal domain of the fibre protein from snake adenovirus type 1, a member of the Atadenovirus genus, has been expressed, purified and crystallized. Crystals were obtained belonging to space groups P2(1)2(1)2(1) (two different forms), I2(1)3 and F23. The best of these diffracted synchrotron radiation to a resolution of 1.4 Å. As the protein lacks methionines or cysteines, site-directed mutagenesis was performed to change two leucine residues to methionines. Crystals of selenomethionine-derivatized crystals of the I2(1)3 form were also obtained and a multi-wavelength anomalous dispersion data set was collected.