The effect of pH on the structure of Bluetongue virus VP5.

The unenveloped Bluetongue virus capsid comprises several structural layers, the inner two comprising a core, which assembles before addition of the outer proteins, VP2 and VP5. Two symmetric trimers of VP5 fit like pegs into two distinct pits on the core and undergo pH conformational changes in the context of the virus, associated with cell entry. Here we show that in isolation VP5 alone undergoes essentially the same changes with pH and confirm a helical transition, indicating that VP5 is a motor during cell entry. In the absence of VP5 the two pits on the core differ from each other, presumably due to the asymmetric underlying structure of VP3, the innermost capsid protein. On insertion of VP5 these pits become closely similar and remain similar at low pH whilst VP5 is present. This natural asymmetry presumably destabilises the attachment of VP5, facilitating ejection upon low pH, membrane penetration and cell entry.

On the geometry of regular icosahedral capsids containing disymmetrons.

Large icosahedral virus capsids are composed of symmetrons, organized arrangements of capsomers. There are three types of symmetrons: disymmetrons, trisymmetrons, and pentasymmetrons, which have different shapes and are centered on the icosahedral 2-fold, 3-fold and 5-fold axes of symmetry, respectively. Sinkovits and Baker (2010) gave a classification of all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons, which requires the triangulation number T to be odd. In the present paper we incorporate disymmetrons to obtain a geometric classification of large icosahedral viruses formed by regular penta-,tri-, and disymmetrons, giving all mathematically consistent and theoretically possible solutions. For every class of solutions, we further provide formulas for symmetron sizes and parity restrictions on h, k, and T numbers. We also present several methods in which invariants may be used to classify a given configuration.

A comparative analysis of the foamy and ortho virus capsid structures reveals an ancient domain duplication.

BACKGROUND: The Spumaretrovirinae (foamy viruses) and the Orthoretrovirinae (e.g. HIV) share many similarities both in genome structure and the sequences of the core viral encoded proteins, such as the aspartyl protease and reverse transcriptase. Similarity in the gag region of the genome is less obvious at the sequence level but has been illuminated by the recent solution of the foamy virus capsid (CA) structure. This revealed a clear structural similarity to the orthoretrovirus capsids but with marked differences that left uncertainty in the relationship between the two domains that comprise the structure. METHODS: We have applied protein structure comparison methods in order to try and resolve this ambiguous relationship. These included both the DALI method and the SAP method, with rigorous statistical tests applied to the results of both methods. For this, we employed collections of artificial fold 'decoys' (generated from the pair of native structures being compared) to provide a customised background distribution for each comparison, thus allowing significance levels to be estimated. RESULTS: We have shown that the relationship of the two domains conforms to a simple linear correspondence rather than a domain transposition. These similarities suggest that the origin of both viral capsids was a common ancestor with a double domain structure. In addition, we show that there is also a significant structural similarity between the amino and carboxy domains in both the foamy and ortho viruses. CONCLUSIONS: These results indicate that, as well as the duplication of the double domain capsid, there may have been an even more ancient gene-duplication that preceded the double domain structure. In addition, our structure comparison methodology demonstrates a general approach to problems where the components have a high intrinsic level of similarity.

The structure of AAVrh32.33, a novel gene delivery vector.

The Adeno-associated viruses (AAVs) are being developed as gene delivery vectors for therapeutic clinical applications. However, the host antibody immune response directed against their capsid, prevalent in ∼40-70% of the general population, depending on serotype, negatively impacts efficacy. AAVrh32.33, a novel vector developed from rhesus macaques isolates, has significantly lower seroprevalence in human populations compared to AAV2 and AAV8, which are both in clinical use. To better understand the capsid determinants of this differential immune response to AAVrh32.33, its structure was determined by X-ray crystallography to 3.5 Å resolution. The capsid viral protein (VP) structure conserves the eight-stranded ß-barrel core and αA helix reported for other parvoviruses and the distinct capsid surface topology of the AAVs: a depression at the icosahedral twofold axis, three protrusions surrounding the threefold axis, and a depression surround a cylindrical channel at the fivefold axis. A comparison to AAV2, AAV4, and AAV8, to which AAVrh32.33 shares ∼61%, ∼81%, and ∼63% identity, respectively, identified differences in previously defined AAV VP structurally variable regions (VR-1 to VR-IX) which function as receptor attachment, transduction efficiency, and/or antigenic determinants. This structure thus provides a 3D platform for capsid engineering in ongoing efforts to develop AAVrh32.33, as well as other AAV serotypes, for tissue targeted gene-therapy applications with vectors that can evade pre-existing antibody responses against the capsid. These features are required for full clinical realization of the promising AAV gene delivery system.

Exploring Canine Picornavirus Diversity in the USA Using Wastewater Surveillance: From High-Throughput Genomic Sequencing to Immuno-Informatics and Capsid Structure Modeling.

The SARS-CoV-2 pandemic resulted in a scale-up of viral genomic surveillance globally. However, the wet lab constraints (economic, infrastructural, and personnel) of translating novel virus variant sequence information to meaningful immunological and structural insights that are valuable for the development of broadly acting countermeasures (especially for emerging and re-emerging viruses) remain a challenge in many resource-limited settings. Here, we describe a workflow that couples wastewater surveillance, high-throughput sequencing, phylogenetics, immuno-informatics, and virus capsid structure modeling for the genotype-to-serotype characterization of uncultivated picornavirus sequences identified in wastewater. Specifically, we analyzed canine picornaviruses (CanPVs), which are uncultivated and yet-to-be-assigned members of the family Picornaviridae that cause systemic infections in canines. We analyzed 118 archived (stored at -20 °C) wastewater (WW) samples representing a population of ~700,000 persons in southwest USA between October 2019 to March 2020 and October 2020 to March 2021. Samples were pooled into 12 two-liter volumes by month, partitioned (into filter-trapped solids [FTSs] and filtrates) using 450 nm membrane filters, and subsequently concentrated to 2 mL (1000×) using 10,000 Da MW cutoff centrifugal filters. The 24 concentrates were subjected to RNA extraction, CanPV complete capsid single-contig RT-PCR, Illumina sequencing, phylogenetics, immuno-informatics, and structure prediction. We detected CanPVs in 58.3% (14/24) of the samples generated 13,824,046 trimmed Illumina reads and 27 CanPV contigs. Phylogenetic and pairwise identity analyses showed eight CanPV genotypes (intragenotype divergence <14%) belonging to four clusters, with intracluster divergence of <20%. Similarity analysis, immuno-informatics, and virus protomer and capsid structure prediction suggested that the four clusters were likely distinct serological types, with predicted cluster-distinguishing B-cell epitopes clustered in the northern and southern rims of the canyon surrounding the 5-fold axis of symmetry. Our approach allows forgenotype-to-serotype characterization of uncultivated picornavirus sequences by coupling phylogenetics, immuno-informatics, and virus capsid structure prediction. This consequently bypasses a major wet lab-associated bottleneck, thereby allowing resource-limited settings to leapfrog from wastewater-sourced genomic data to valuable immunological insights necessary for the development of prophylaxis and other mitigation measures.

Approximation of virus structure by icosahedral tilings.

Viruses are remarkable examples of order at the nanoscale, exhibiting protein containers that in the vast majority of cases are organized with icosahedral symmetry. Janner used lattice theory to provide blueprints for the organization of material in viruses. An alternative approach is provided here in terms of icosahedral tilings, motivated by the fact that icosahedral symmetry is non-crystallographic in three dimensions. In particular, a numerical procedure is developed to approximate the capsid of icosahedral viruses by icosahedral tiles via projection of high-dimensional tiles based on the cut-and-project scheme for the construction of three-dimensional quasicrystals. The goodness of fit of our approximation is assessed using techniques related to the theory of polygonal approximation of curves. The approach is applied to a number of viral capsids and it is shown that detailed features of the capsid surface can indeed be satisfactorily described by icosahedral tilings. This work complements previous studies in which the geometry of the capsid is described by point sets generated as orbits of extensions of the icosahedral group, as such point sets are by construction related to the vertex sets of icosahedral tilings. The approximations of virus geometry derived here can serve as coarse-grained models of viral capsids as a basis for the study of virus assembly and structural transitions of viral capsids, and also provide a new perspective on the design of protein containers for nanotechnology applications.