Computational studies on rep and capsid proteins of CRESS DNA viruses.

The circular rep-encoding single-stranded DNA viruses (CRESS DNA viruses) are among the smallest, with 2-6 kb ssDNA genomes that encode for a coat protein (C) and a replication protein (R). To comprehend the complexity and divergence of the C and R proteins, we have created predictive structural models of representative viruses infecting unique hosts from each family using the neural network-based method AlphaFold2 and carried out molecular dynamic simulations to assess their stability. The structural characteristics indicate that differences in loops and amino-terminus may play a significant role in facilitating adaptations to multiple hosts and vectors. In comparison to the C, the Rs show a high degree of conservation and structural mimicry of the nuclease-helicase domains of plasmids. A phylogenetic analysis based on the structures and sequences of the C and R proteins reveals evolutionary variances. Our study also highlights the conservation of structural components involved in the interaction of R with the conserved intergenic region of the genome. Further, we envisage that the adaptability of R's central linker may be crucial for establishing interactions with multiple protein partners, including C. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-024-00858-x.

Intrinsic disorder in flaviviral capsid proteins and its role in pathogenesis.

A high level of disorder in many viral proteins is a direct consequence of their small genomes, which makes interaction with multiple binding partners a necessity for infection and pathogenicity. A segment of the flaviviral capsid protein (C), also known as the molecular recognition feature (MoRF), undergoes a disorder-toorder transition upon binding to several protein partners. To understand their role in pathogenesis, MoRFs were identified and their occurrence across different flaviviral capsids were studied. Despite lack of sequence similarities, docking studies of Cs with the host proteins indicate conserved interactions involving MoRFs across members of phylogenetic subclades. Additionally, it was observed from the protein-protein networks that some MoRFs preferentially bind proteins that are involved in specialized functions such as ribosome biogenesis. The findings point to the importance of MoRFs in the flaviviral life cycle, with important consequences for disease progression and suppression of the host immune system. Potentially, they might have impacted the way flaviviruses evolved to infect varied hosts using multiple vectors.

Viral nanoparticles: Current advances in design and development.

Viral nanoparticles (VNPs) are self-assembling, adaptable delivery systems for vaccines and other therapeutic agents used in a variety of biomedical applications. The potential of viruses to invade and infect various hosts and cells renders them suitable as potential nanocarriers, possessing distinct functional characteristics, immunogenic properties, and improved biocompatibility and biodegradability. VNPs are frequently produced through precise genetic or chemical engineering, which involves adding diverse sequences or functional payloads to the capsid protein (CP). Several spherical and helical plant viruses, bacteriophages, and animal viruses are currently being used as VNPs, or non-infectious virus-like particles (VLPs). In addition to their broad use in cancer therapy, vaccine technology, diagnostics, and molecular imaging, VNPs have made important strides in the realms of tissue engineering, biosensing, and antimicrobial prophylaxis. They are also being used in energy storage cells due to their binding and piezoelectric properties. The large-scale production of VNPs for research, preclinical testing, and clinical use is fraught with difficulties, such as those relating to cost-effectiveness, scalability, and purity. Consequently, many plants- and microorganism-based platforms are being developed, and newer viruses are being explored. The goal of the current review is to provide an overview of these advances.

Brain Virome in Neurodegenerative Disorders: Insights from Transcriptomic Data Analysis.

Neurodegenerative disorders (NDs) are chronic ailments of the central nervous system that gradually deteriorate the structures and functions of neurons. The etiologies of NDs include genetic factors, aging, infections, starvation, brain trauma, and spinal cord injury, among others. However, it is unclear whether viral infections impact the prognosis of NDs or contribute to their development. Hence, we investigated the prevalence of neurotropic viruses in brain samples by using transcriptomic data. A total of 1635 viral isolates with complete genomic information was used to investigate the incidence of 18 distinct viruses across 129 data sets from healthy and ND subjects. Our findings support the evidence pointing to the existence of a brain virome where certain viruses co-occur. We further hypothesize that distinct virome profiles are linked to different forms of NDs.

SARS-CoV-2: analysis of the effects of mutations in non-structural proteins.

A worldwide pandemic that started in China in late 2019 was caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a single-stranded RNA virus belonging to the family Coronaviridae. Due to its structural variability and mutability, this virus continues to evolve and pose a major health threat around the world. Its characteristics, such as transmissibility, antigenicity, and resistance to drugs and vaccines, are continually altered through mutations. Examining mutational hotspots and their structural repercussions can thus aid in the development of more-effective vaccinations and treatment plans. In this study, we used full genome sequences of SARS-CoV-2 variants to predict structural changes in viral proteins. These sequences were obtained from the Global Initiative on Sharing Avian Influenza Data (GISAID), and a set of significant mutations were identified in each of the non-structural proteins (NSP1-16) and structural proteins, including the envelope, nucleocapsid, membrane, and spike proteins. The mutations were characterized as stabilizing or destabilizing based on their effect on protein dynamics and stability, and their impact on structure and function was evaluated. Among all of the proteins, NSP6 stands out as especially variable. The results of this study augment our understanding of how mutational events influence virus pathogenicity and evolution.

Altered Lysosomal Function Manipulates Cellular Biosynthetic Capacity By Remodeling Intracellular Cholesterol Distribution.

Lysosomes are known to influence cholesterol trafficking into endoplasmic reticulum (ER) membranes. Though intracellular cholesterol levels are known to influence the lipid biosynthetic responses in ER, the specific effects of lysosomal modulation on these outcomes is not known. To demonstrate this, C2C12 cells were treated with chloroquine, a lysosomotropic agent, and its effects on cellular biosynthetic capacity, structural and functional status of ER was determined. In addition to its known effects on autophagy reduction, chloroquine treatment induced accumulation of total cellular lipid and ER-specific cholesterol content. It was also observed that chloroquine caused an increase in smooth-ER content with defects in overall protein turnover. Further, since ER and mitochondria function in close association through ER membrane contact sites, it is likely that lysosomal modulation also brings about associated changes in mitochondria. In this regard, we found that chloroquine reduces mitochondrial membrane potential and mitochondrial dynamics. Collectively, the differential biosynthetic response of rise in lipid content, but not protein content, cannot be accounted by merely considering that chloroquine induced suppression of autophagy causes defects in organelle function. In this defective autophagy scenario, both biosynthetic responses such as lipid and protein synthesis are expected to be reduced rather than only the latter, as observed with chloroquine. These findings suggest that cholesterol trafficking/distribution within cellular organelles could act as an intracellular mediator of differential biosynthetic remodelling in interconnected organelles.

Revisiting Viral RNA-Dependent RNA Polymerases: Insights from Recent Structural Studies.

RNA-dependent RNA polymerases (RdRPs) represent a distinctive yet versatile class of nucleic acid polymerases encoded by RNA viruses for the replication and transcription of their genome. The structure of the RdRP is comparable to that of a cupped right hand consisting of fingers, palm, and thumb subdomains. Despite the presence of a common structural core, the RdRPs differ significantly in the mechanistic details of RNA binding and polymerization. The present review aims at exploring these incongruities in light of recent structural studies of RdRP complexes with diverse cofactors, RNA moieties, analogs, and inhibitors.

Insights into the capsid structure of banana bunchy top virus.

Banana is the major staple food crop for approximately 400 million people. Bunchy top disease of banana is one of the most devastating diseases caused by banana bunchy top virus (BBTV), which results in stunting of plants, bunchy appearance of leaves and a significant loss of yield. While many isolates of BBTV from various regions of India have been characterized by different groups, no structural study exists for this important virus. To bridge this gap, the pET28a clone of the coat protein (CP) gene from BBTV isolate of Hill banana grown in lower Pulney Hills (Virupakshi) of Tamilnadu was expressed in BL21 (DE3) pLysS. Purification of the CP was achieved by Ni-NTA affinity chromatography. In vitro capsid assembly studied using sucrose density gradient centrifugation suggested that the CP did not assemble as a virus-like particle (VLP), but remained as smaller oligomers. Studies using dynamic light scattering (DLS) indicate that the purified protein is poly-dispersed, represented majorly as pentamers. Homology modeling studies provided useful insights into the probable fold of the CP suggesting that it is a ß-sandwich, similar to that seen in the majority of plant viruses. In silico capsid reconstruction aided the understanding of the quaternary organization of subunits in the capsid and their molecular interactions. The location of the aphid-binding EAG motif was identified on the surface loops close to the pentameric axis indicating its role in vector-mediated transmission. Comparison with the CP and capsid structure of geminiviruses provided useful insights into the mode of nucleic acid binding and the role of genome during capsid assembly. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03204-4.

Analysis of host protein interactions in plant viruses: an in silico study using Sesbania mosaic virus.

The dynamics of interactions of viral proteins with their host are pivotal in establishing a successful infection and ensuring systemic spread. To uncover these, an in silico analysis of the interactions between the coat protein (CP) of Sesbania mosaic virus (SeMV), a group IV virus with single-stranded positive-sense RNA genome was carried out with the known crystal structures of proteins belonging to the Fabaceae family, which is its natural host. SeMV is an isometric plant virus which infects Sesbania grandiflora, a member of Fabaceae, and causes mosaic symptoms. Earlier results have indicated that the assembly and disassembly events of SeMV favor the formation of CP dimers. Hence, the ability and strength of interactions of CP dimer with the host proteins were assessed using in silico protein-protein docking approaches. A set of 61 unique crystal structures of native proteins belonging to Fabaceae were downloaded from the Protein Data Bank (PDB) and docked with the CP dimer of SeMV. From the docking scores and interaction analysis, the host proteins were ranked according to their strength and significance of interactions with the CP dimers. The leads that were identified present themselves as strong candidates for developing antivirals against not only SeMV but also other related viruses that infect Fabaceae. The study is a prototype to understand host protein interactions in viruses and hosts.

Recent advances in understanding the replication initiator protein of the ssDNA plant viruses of the family Nanoviridae.

The families of viruses possessing single-stranded (ss) circular genome employ a dedicated replication initiator protein (Rep) for making copies of their genome through the process of rolling circle replication. The replication begins at conserved nonanucleotide sequence at the intergenic region. The Rep protein seems to be the most conserved amongst the available proteins of the nanovirids and comprises of the N-terminal endonuclease domain and the C-terminal helicase domain. The structural studies of Faba bean necrotic yellows virus endonuclease domain suggests a α + ß fold comprising of central ß sheet built from five antiparallel ß strands surrounded by outer short α helices. The catalysis is mediated by a conserved Tyr residue and employs divalent metal ions (Mn2+). On one hand, the Reps associate with each other and oligomerize and on the other hand interact with varied host and vector associated proteins for successful infection. The sequence analysis of Reps from previously known nanovirids and the newly found ones from metagenomics data shed light on the evolutionary pattern of nanovirids in comparison to other plant infecting ssDNA viruses.

Revised Crystal Structure of Human Adenovirus Reveals the Limits on Protein IX Quasi-Equivalence and on Analyzing Large Macromolecular Complexes.

We report the revised crystal structure of a pseudo-typed human adenovirus at 3.8-Å resolution that is consistent with the atomic models of minor proteins determined by cryo-electron microscopy. The diffraction data from multiple crystals were rescaled and merged to increase the data completeness. The densities for the minor proteins were initially identified in the phase-refined omit maps that were further improved by the phases from docked poly-alanine models to build atomic structures. While the trimeric fiber molecules are disordered due to flexibility and imposition of 5-fold symmetry, the remaining major capsid proteins hexon and penton base are clearly ordered, with the exception of hypervariable region 1 of hexons, the RGD containing loop, and the N-termini of the penton base. The exterior minor protein IX together with the interior minor proteins IIIa and VIII stabilizes the adenovirus virion. A segment of N-terminal pro-peptide of VI is found in the interior cavities of peripentonal hexons, and the rest of VI is disordered. While the triskelion substructures formed by the N-termini of IX conform to excellent quasi 3-fold symmetry, the tetrameric coiled-coils formed by the C-termini and organized in parallel and anti-parallel arrangement do not exhibit any quasi-symmetry. This observation also conveys the pitfalls of using the quasi-equivalence as validation criteria for the structural analysis of extended (non-modular) capsid proteins such as IX. Together, these results remedy certain discrepancies in the previous X-ray model in agreement with the cryo-electron microscopy models.

RNA Dependent RNA Polymerases: Insights from Structure, Function and Evolution.

RNA dependent RNA polymerase (RdRp) is one of the most versatile enzymes of RNA viruses that is indispensable for replicating the genome as well as for carrying out transcription. The core structural features of RdRps are conserved, despite the divergence in their sequences. The structure of RdRp resembles that of a cupped right hand and consists of fingers, palm and thumb subdomains. The catalysis involves the participation of conserved aspartates and divalent metal ions. Complexes of RdRps with substrates, inhibitors and metal ions provide a comprehensive view of their functional mechanism and offer valuable insights regarding the development of antivirals. In this article, we provide an overview of the structural aspects of RdRps and their complexes from the Group III, IV and V viruses and their structure-based phylogeny.

VIPERdb2: an enhanced and web API enabled relational database for structural virology.

VIPERdb (http://viperdb.scripps.edu) is a relational database and a web portal for icosahedral virus capsid structures. Our aim is to provide a comprehensive resource specific to the needs of the virology community, with an emphasis on the description and comparison of derived data from structural and computational analyses of the virus capsids. In the current release, VIPERdb(2), we implemented a useful and novel method to represent capsid protein residues in the icosahedral asymmetric unit (IAU) using azimuthal polar orthographic projections, otherwise known as Phi-Psi (Phi-Psi) diagrams. In conjunction with a new Application Programming Interface (API), these diagrams can be used as a dynamic interface to the database to map residues (categorized as surface, interface and core residues) and identify family wide conserved residues including hotspots at the interfaces. Additionally, we enhanced the interactivity with the database by interfacing with web-based tools. In particular, the applications Jmol and STRAP were implemented to visualize and interact with the virus molecular structures and provide sequence-structure alignment capabilities. Together with extended curation practices that maintain data uniformity, a relational database implementation based on a schema for macromolecular structures and the APIs provided will greatly enhance the ability to do structural bioinformatics analysis of virus capsids.

Structure of Seneca Valley Virus-001: an oncolytic picornavirus representing a new genus.

The crystal structure of Seneca Valley Virus-001 (SVV-001), the representative member of a new genus, Senecavirus, is reported at 2.3A resolution. SVV-001 is the first naturally occurring nonpathogenic picornavirus shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+) ssRNA genome of SVV-001 shares closest sequence similarity with the genomes of the members of Cardiovirus. The overall tertiary structure of VP1-VP4 subunits is conserved with the exception of loops, especially those of VP1 that show large deviations relative to the members of the cardioviruses. The surface loops of VP1 and VP2 are predicted to mediate cell tropism of SVV-001. In addition, the organization of the packaged nucleic acid density indicates that certain regions of VP2 and VP4 interact closely with the packaged nucleic acid.

Structural variations in species B adenovirus fibers impact CD46 association.

A majority of species B adenoviruses (Ads) use CD46 as their primary receptor; however, the precise mechanisms involved in the binding of different Ad types to CD46 have not been resolved. Although previous studies indicate close similarities between two members of species B2 Ads in their usage of CD46, our current investigations revealed a surprisingly low CD46 binding affinity of the species B1 Ad16 fiber knob (equilibrium dissociation constant of 437 nM). We determined the crystal structure of the Ad16 fiber knob and constructed a model of this protein in complex with CD46. A comparison of this model to that of the CD46-Ad11 complex revealed structural differences in the FG and IJ loops that are part of the CD46 binding site. An analysis of a panel of recombinant fiber knobs with mutations targeting these regions in Ad16 and Ad11 uncovered a major contribution of the FG loop on CD46 binding. Two extra residues in the FG loop of the Ad16 fiber significantly reduce receptor interaction. Although avidity effects permit the use of CD46 on host cells by Ad16, virus binding occurs with lower efficiency than with B2 Ad types. The longer FG loop of the Ad16 fiber knob also is shared by other species B1 Ad fibers and, thus, may contribute to the low CD46 binding efficiencies observed for these Ad types. Our findings provide a better understanding of how different Ad types associate with CD46 and could aid in the selection of specific Ad fibers for more efficient Ad gene delivery vectors.

Crystallization and preliminary X-ray diffraction studies of Seneca Valley virus-001, a new member of the Picornaviridae family.

Seneca Valley Virus-001 (SVV-001) is a newly found species in the Picornaviridae family. SVV-001 is the first naturally occurring nonpathogenic picornavirus observed to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+)ssRNA genome of SVV-001 shares closest sequence similarity to the genomes of the members of the Cardiovirus genus. However, based on the distinct characteristics of the genome organization and other biochemical properties, it has been suggested that SVV-001 represents a new genus, namely 'Senecavirus', in the Picornaviridae family. In order to understand the oncolytic properties of SVV-001, the native virus was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group R3, with unit-cell parameters (in the hexagonal setting) a = b = 311.5, c = 1526.4 A. Although the SVV crystals diffracted to better than 2.3 A resolution, the data quality is acceptable [I/sigma(I) > 2.0] to 2.6 A resolution. The unit-cell volume and the locked rotation-function analysis suggest that six particles could be accommodated in the unit cell, with two distinct sets of one third of a particle, each containing 20 protomers, occupying the crystallographic asymmetric unit. (ClinicalTrials.gov identifier NCT00314925)

Conservation of fiber structure and CD46 usage by subgroup B2 adenoviruses.

Most subgroup B2 adenoviruses use CD46 as their primary receptor. Recent structural and mutagenesis studies suggested that Ad11 and Ad35 likely engage this receptor in a very similar fashion. However, no comparative studies assessing the cell-associated CD46 binding efficiencies of different Ad fibers have been performed. We solved the crystal structure of Ad35 fiber knob and constructed a model of the fiber knob complexed with CD46. Comparison of our model with that of Ad11-CD46 showed that despite a larger CD46-interacting region in the IJ loop of Ad11, the buried surface area was very similar, suggesting that both fiber knobs might exhibit similar binding. In support of this, cell based competition studies demonstrated almost identical binding efficiencies of Ad11 and Ad35 fibers to cell surface CD46. These findings shed further light on CD46 association by Ad and could impact the selection of novel Ad types for gene transfer.