Search Results (193)

Nucleocapsids protect viral genomes and play fundamental roles in viral assembly and infection. While many viruses adopt icosahedral or helical symmetries, negative-strand RNA viruses (NSVs) assemble their nucleocapsids with a distinct translation-based symmetry that is often considered helical because of their curvature. Our study analyzes the structural basis, assembly principles, and functional implications of the linear nucleocapsids. Structural coordinates of viruses were obtained from the Protein Data Bank (PDB) and examined using PyMOL version 1.3 to compare protein folds, RNA–protein interactions, inter-subunit contacts, and curvature properties across multiple nucleocapsids. We found that linear nucleocapsids share a similar 5H + 3H fold in their capsid proteins and encapsidate a fixed number of nucleotides per subunit, though the degree of nucleotide sequestration varies. Their architecture differs in inter-subunit interactions, determining whether empty capsids can assemble and influencing RNase sensitivity. Although these nucleocapsids may appear helical, they lack strict helical symmetry and instead display variable curvature that is modulated by environmental conditions. Relaxation of this curvature is likely required for viral RNA-dependent RNA polymerase to access the sequestered RNA genome during transcription/replication. In conclusion, linear nucleocapsids constitute a class of RNA–protein assemblies with variable curvature. The topologically conserved fold of the capsid protein enables genome protection while regulating exposure of RNA during viral RNA synthesis. Full article

Protein-based nanocarriers have gained considerable attention for targeted cancer theranostic applications due to their inherent biocompatibility, biodegradability, and facile functionalisation. In addition, some of their properties, such as self-assembling nature, low immunogenicity (if species matched), molecular recognition ability, and lack of persistence due to degradation into proteinogenic amino acids, make them highly suitable for oncology-related applications. Each protein-based nanocarrier exhibits unique physicochemical and biological properties. In this review, we summarise recent advances in targeted protein-based nanocarriers, including albumin, lipoproteins, ferritin, viral protein capsids, fibrin type proteins and silk proteins, emphasising receptor-specific targeting mechanisms, the integration of various imaging modalities along with their advantages and limitations, and the importance of employing advanced preclinical models for translational theranostic applications. This review also discusses the most recent and significant studies in the field, providing useful insights into future directions of protein-based nanocarriers for cancer theranostics. Full article

The clinical success of chimeric antigen receptor (CAR) T-cell therapies has revolutionized oncology, yet the high costs and logistical complexities of ex vivo manufacturing remain significant barriers to global patient access. In vivo cell therapy, which involves the direct injection of lentiviral vectors (LVVs) to engineer cells within the patient’s body, offers a promising, cost-effective alternative. However, transitioning from ex vivo to in vivo applications necessitates a fundamental shift in LVV biomanufacturing to ensure safety and efficacy. This paper examines the critical bottlenecks in the current LVV production landscape. In upstream processing, we explore LVV particle assembly and maturation mechanisms, the effect of transgene size on LVV functional titers and the formation of non-functional byproducts, including empty and partially formed LVV particles and extracellular vesicles (EVs). These impurities pose severe risks of immunotoxicity and insertional mutagenesis when delivered in vivo. In downstream processing, we highlight the challenges of purifying labile LVV particles, emphasizing the need for rapid, high-resolution separation techniques like continuous processing to maintain functional titers. Furthermore, we address the limitations of current analytical assays, which often fail to distinguish mature, functional LVVs from structurally similar but inactive contaminants. We conclude that the future of in vivo lentiviral therapy depends on developing novel purification strategies based on subtle biophysical differences—such as surface charge and capsid morphology—and implementing robust, high-throughput analytics to ensure delivery of high-purity, potent therapeutic viral vectors. Full article

Hepatocellular carcinoma (HCC) represents the second leading cause of cancer mortality worldwide and is mostly caused by hepatitis B virus (HBV) infection. HBV can induce HCC by an indirect mechanism of continuous necro-inflammation, contributing to hepatocyte damage and promoting cancer, as well as by viral intrinsic factors. Among them, the major contributors to the development of HBV-related HCC are represented by (i) HBV DNA integration in genes modulating cell proliferation, (ii) HBV pro-oncogenic proteins, such as HBx and HBs, and (iii) the accumulation of viral mutations, enhancing the tumorigenic features of HBV proteins. The currently available antiviral treatments, based on the usage of Nucleos(t)ide analogs (NUCs), substantially control HBV replication. However, even a successful NUC treatment does not completely abrogate HCC risk, since it rarely allows achievement of an HBV functional cure, the therapeutic end-point associated with HBsAg loss and more favorable liver outcomes. To date, novel therapeutic strategies based on innovative direct antivirals (nucleic acid polymers, small interfering RNAs, antisense oligonucleotides, covalently closed circular DNA (cccDNA) inhibitors, and capsid assembly modulators) and immune-therapeutics (therapeutic vaccines, checkpoint inhibitors, and Toll-like receptor agonists) are under evaluation in clinical trials. These approaches are showing promising data in terms of an HBV functional cure, thus representing novel strategies that could be beneficial for reducing the burden of HBV-related HCC. Lastly, further efforts in drug development are necessary to identify new compounds that could achieve a sterilizing HBV cure, implying the complete elimination of cccDNA and integrated HBV DNA, the only end-point that completely eradicates HBV and its related oncogenic risk. Full article

Epizootic hemorrhagic disease (EHD) is an important livestock disease caused by Epizootic hemorrhagic disease virus (EHDV). The recent incursion and wide distribution of EHDV in Europe have increased the need for effective vaccine candidates. Background/Objectives: The VP2 protein of EHDV forms the outer capsid layer of the virion and is essential for viral assembly and host cell entry. Owing to its antigenic properties, VP2 represents a major target for vaccine development. However, the recombinant production of VP2 is limited by low stability and poor yields, representing a significant barrier for the generation of safe and effective subunit vaccines. Methods: To overcome these limitations, the VP2 protein from EHDV serotype 8 (EHDV-8) was rationally engineered with targeted modifications at both the amino and carboxyl termini of its coding sequence. Recombinant expression was performed using a baculovirus vector-mediated system in Trichoplusia ni pupae (CrisBio^® technology), employed as living biofactories. Results: The engineering of VP2 resulted in up to a tenfold increase in protein yields compared with the wild-type sequence, while maintaining the trimeric structural integrity of the recombinant protein. Both wild-type and engineered VP2 protein variants were formulated and used to immunize IFNAR(−/−) mice, a model susceptible to EHDV infection. Both engineered and wild-type VP2 formulations elicited comparable neutralizing antibody responses in vaccinated animals. Furthermore, immunization with either formulation conferred full protection against lethal EHDV-8 challenge. Conclusions: In this work, we demonstrated that the rational engineering of the VP2 protein significantly improved recombinant expression yields in a baculovirus-based system without compromising structural integrity or immunogenicity. These findings additionally demonstrate the feasibility of producing high-quality VP2 antigens in T. ni pupae using CrisBio^® technology and support their potential application in the development of subunit vaccines against EHDV. Full article

Protein–protein interactions (PPIs) are fundamental to viral replication, regulating transcription, assembly, and genome packaging. Despite their biological importance, few FDA-approved therapeutics directly target these complexes. The split luciferase complementation assay (SLCA) is a quantitative bioluminescence system to measure protein–protein interactions in vitro after the proteins in question have been fused in-frame to N and C luciferase fragments. The SLCA can be performed both in vitro using purified protein components and in live cells, as the luciferase substrate luciferin is cell-permeable, allowing detection of protein interactions in intact cells. Assay performance, however, depends on the expression level and stability of the fusion proteins used. SLCA has been successfully applied to target Rev–Rev interactions in human immunodeficiency virus type 1 (HIV-1) for high-throughput small-molecule screening, establishing a proof-of-concept to target other parts of the viral life cycle. The system can be extended to other pathogens that currently do not have specific antiviral therapies such as HIV-1 Tat–cyclin T1, Capsid dimerization in Dengue virus, capsid interactions in equine encephalitis viruses, capsid assembly in Epstein–Barr virus, and nucleoprotein oligomerization in rabies virus. These applications demonstrate how the assay’s ability to quantify multimeric structural interactions is essential to viral replication, providing an avenue to identify small-molecule inhibitors that prevent viral replication and spread. Although there are challenges to protein stability and assay optimization, the sensitivity and adaptability of the SLCA has broader implications in virology to accelerate antiviral drug development. Full article

Human immunodeficiency virus type 2 (HIV-2) is a lentivirus closely related to HIV-1 but exhibits distinct molecular and clinical features that influence viral infectivity and efficacy of antiretroviral therapy. The HIV capsid is a critical structural component with multifaceted roles during infection and mediates some of the observed divergence between HIV-1 and HIV-2. Unlike HIV-1, study of the HIV-2 capsid is limited and standard protocols for the in vitro assembly of HIV-1 capsid protein (CA) lattice structures have not been successfully translated to the HIV-2 context. This work identifies effective approaches for the assembly of the HIV-2 CA lattice and leverages this to biochemically characterize HIV-2 CA assemblies and mutant phenotypes. Our findings elaborate on the sensitivity of HIV-2 CA to chemical conditions and reveal that it assembles into a more varied spectrum of particle morphologies compared to HIV-1. Utilizing these assemblies, we tested the hypothesis that HIV-1 and HIV-2 employ divergent mechanisms to stabilize CA oligomer forms and investigate the effects of non-conserved substitutions at the CA inter-protomer interfaces. This work advances our understanding of the key biochemical determinants of HIV-2 CA assembly that are distinct from HIV-1 and may contribute to their divergent virological properties. Full article

Virus-like particles (VLPs) formed as a result of self-assembly of viral capsid proteins are widely used as a platform for antigen presentation in vaccine development. However, since the inclusion of a foreign peptide into the capsid protein can alter its spatial structure and interfere with VLP assembly, such insertions are usually limited to short peptides. In this study, we have demonstrated the potential of capsid protein (CP) of single-stranded RNA phage PQ465 to present long peptides using green fluorescent protein (GFP) as a model. GFP was genetically linked to either the N- or C-terminus of PQ465 CP. Hybrid proteins were expressed in Escherichia coli and Nicotiana benthamiana plants. Spherical virus-like particles (~35 nm according to transmission electron microscopy) were successfully formed by both N- and C-terminal fusions expressed in E. coli, and by plant-produced CP with GFP fused to the C-terminus. ELISA revealed that GFP in VLPs was accessible for specific antibodies suggesting that it is exposed on the surface of PQ465-GFP particles. VLPs carrying GFP were recognized by anti-CP antibodies with less efficiency than VLPs formed by empty CP, which indicates shielding of the CP core in PQ465-GFP particles. Therefore, PQ465 CP can be used as a chimeric VLP platform for the display of relatively large protein antigens, which can operate in bacterial and plant expression systems. Full article

Plant viruses have evolved from being viewed exclusively as pathogens into versatile and powerful tools for modern biotechnology. Among them, Tomato bushy stunt virus (TBSV) holds a special place due to its well-studied molecular biology and unique structural properties. This review systematizes the knowledge on TBSV’s dual role as a multifunctional platform. On one hand, we cover its application as a viral vector for the highly efficient expression of recombinant proteins in plants, as well as a tool for functional genomics, including Virus-Induced Gene Silencing (VIGS) and the delivery of CRISPR/Cas9 gene-editing components. On the other hand, we provide a detailed analysis of the use of the stable and monodisperse TBSV virion in nanobiotechnology. Its capsid serves as an ideal scaffold for creating next-generation vaccine candidates, platforms for targeted drug delivery to tumor cells, and as a building block for the programmable self-assembly of complex nanoarchitectures. In conclusion, key challenges limiting the widespread adoption of the platform are discussed, including the genetic instability of vectors and difficulties in scalable purification, along with promising strategies to overcome them. Full article

Human noroviruses (HuNoVs) stand as the primary cause of acute viral gastroenteritis outbreaks worldwide, particularly impacting children under the age of five. In Russia, reports of norovirus gastroenteritis have surged, especially in the post-COVID-19 era starting in 2022, with elevated infection rates reported into 2024. These viruses exhibit significant mutational variability, leading to the emergence of recombinant strains that can evade immune responses. A comprehensive examination of the complete genome is crucial for understanding the evolution of norovirus genes and for predicting potential outbreaks. This research focuses on analyzing the genotypic composition of HuNoVs circulating in the Sverdlovsk region during 2024, using Sanger sequencing and next-generation sequencing (NGS). Biological samples were collected (n = 384) from patients diagnosed with norovirus infection within the region. Bioinformatics analysis targeted the nucleotide sequences of the ORF1/ORF2 fragment and the assembly of complete genomes for the GII.4 and GII.7 genotypes. In total, 220 HuNoVs were characterized, representing 57.3% of the collected samples. The main capsid variants forming the predominant genotypic profile included GII.4 (n = 88, 40%), GII.7 (n = 86, 39%), and GII.17 (n = 14, 6%). Using NGS, we successfully assembled 8 out of 10 complete genomes for noroviruses GII.4[P16] and GII.7[P7]. Non-synonymous substitutions appeared at amino acid sites corresponding to the subdomains of VP1 in these strains. This molecular–genetic analysis provides contemporary insights into the genotypic composition, circulation patterns, and evolutionary dynamics associated with the dominant genovariants GII.4[P16] and GII.7[P7]. Full article

Aperiodic tessellations of polykite unitiles, such as hats and turtles, and the recently introduced hares, red squirrels, and gray squirrels, have attracted significant interest due to their structural and combinatorial properties. Our primary objective here is to learn how we could build a self-assembling polyhedron that would have an aperiodic tessellation of its surface using only a single type of polykite unitile. Such a structure would be analogous to some viral capsids that have been reported to have a quasicrystal configuration of capsomeres. We report on our use of a graph–theoretic approach to examine the adjacency and symmetry constraints of these unitiles in tessellations because by using graph theory rather than the usual geometric description of polykite unitiles, we are able (1) to identify which particular vertices and/or edges join one another in aperiodic tessellations; (2) to take advantage of being scale invariant; and (3) to use the deformability of shapes in moving from the plane to the sphere. We systematically classify their connectivity patterns and structural characteristics by utilizing Hamiltonian cycles of vertex degrees along the perimeters of the unitiles. In addition, we applied Blumeyer’s 2 × 2 classification framework to investigate the influence of chirality and periodicity, while Heesch numbers of corona structures provide further insights into tiling patterns. Furthermore, we analyzed the distribution of polykite unitiles with Voronoi tessellations and their Delaunay triangulations. The results of this study contribute to a better understanding of self-assembling structures with potential applications in biomimetic materials, nanotechnology, and synthetic biology. Full article

P23-77 is a thermophilic bacteriophage that infects Thermus thermophilus bacteria. The genome of the virus is enclosed in an icosahedral capsid. This capsid is made of the small major capsid protein (VP16), the large major capsid protein (VP17), and the minor capsid protein (VP11). In addition to these three structural proteins, membrane-associated proteins (VP15, VP19, VP20, VP22, and VP23) have been identified in the virus and may serve as scaffold proteins to help with viral assembly. Previous studies have expressed VP11, VP16, and VP17 in E. coli. A mixture of these proteins can lead to the formation of complexes. However, the potential to express membrane-associated proteins has never been explored. Here, we demonstrated, for the first time, the expression and co-expression of some membrane-associated proteins with capsid (coat) proteins, both in the natural host and in E. coli. Co-expression of these proteins did not result in the assembly of virus-like particles. We explored further strategies to express and purify some of the proteins for future studies. We observed that the insertion of a purification tag (Strep-II tag, but not a histidine tag) significantly reduced the expression levels of some of the proteins. Six of the eight structural proteins were successfully purified to homogeneity using different approaches. We showed that VP20 and VP22 migrated on SDS PAGE gel at sizes larger than their predicted molecular weights. Predicted 3D structures of the proteins show that most of them are helical in nature with disordered regions. The work presented here will help pave the way for the expression and purification of these proteins. This will help determine their 3D structures and may shed light on the requirements for viral assembly. Full article

Antigenicity and immunogenicity define a potent immunogen in vaccinology. Nowadays, there are simplified platforms to produce nanocarriers for small-peptide antigen delivery, derived from various infectious agents for the treatment of a variety of diseases, based on virus-like particles (VLPs). They have good cell-penetrating properties and protective action for target molecules from degradation. Human papillomavirus (HPV) causes anogenital warts and six types of cancer in infected women, men, or children, posing a challenge to global public health. The HPV capsid is composed of viral type-specific L1 and evolutionarily conserved L2 proteins. Produced in heterologous systems, the L1 protein can self-assemble into VLPs, nanoparticles sized around 50–60 nm, used as prophylactic vaccines. Devoid of the viral genome, they are safe for users, offering no risk of infection because VLPs do not replicate. The immune response induced by HPV VLPs is promoted by conformational viral epitopes, generating effective T- and B-cell responses. Produced in different cell systems, HPV16 L1 VLPs can be obtained on a large scale for use in mass immunization programs, which are well established nowadays. The expression of heterologous proteins was evaluated at various transfection times by transfecting cells with vectors encoding codon-optimized HPV16L1 and HPV16L2 genes. Immunological response induced by chimeric HPV16 L1/L2 VLP was evaluated through preclinical assays by antibody production, suggesting the potential of broad-spectrum protection against HPV as a prophylactic nanovaccine. These platforms can also offer promising therapeutic strategies, covering the various possibilities for complementary studies to develop potential preventive and therapeutic vaccines with broad-spectrum protection, using in silico new epitope selection and innovative nanotechnologies to obtain more effective immunobiologicals in combating HPV-associated cancers, influenza, hepatitis B and C, tuberculosis, human immunodeficiency virus (HIV), and many other illnesses. Full article

Sindbis virus (SINV), a prototype of the Alphavirus genus (family Togaviridae), is a globally distributed arbovirus causing febrile rash and debilitating arthritis in humans. Viral structural proteins—capsid (C), E1, and E2—are fundamental to the virion’s architecture, mediating all stages from assembly to host cell entry and pathogenesis, thus representing critical targets for study. This review consolidates the historical and current understanding of SINV structural biology, tracing progress from early microscopy to recent high-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography. We detail the virion’s precise T = 4 icosahedral architecture, composed of a nucleocapsid core and an outer glycoprotein shell. Key functional roles tied to protein structure are examined: the capsid’s dual capacity as a serine protease and an RNA-packaging scaffold that interacts with the E2 cytoplasmic tail; the E1 glycoprotein’s function as a class II fusion protein driving membrane fusion; and the E2 glycoprotein’s primary role in receptor binding, which dictates cellular tropism and serves as the main antigenic target. Furthermore, we connect these molecular structures to viral evolution and disease, analyzing how genetic variation among SINV genotypes, particularly in the E2 gene, influences host adaptation, immune evasion, and the clinical expression of arthritogenic and neurovirulent disease. In conclusion, the wealth of structural data on SINV offers a powerful paradigm for understanding alphavirus biology. However, critical gaps persist, including the high-resolution visualization of dynamic conformational states during viral entry and the specific molecular determinants of chronic disease. Addressing these challenges through integrative structural and functional studies is paramount. Such knowledge will be indispensable for the rational design of next-generation antiviral therapies and broadly protective vaccines against the ongoing threat posed by SINV and related pathogenic alphaviruses. Full article

Herpesviruses are prevalent infectious agents in humans, with complex structures and life cycles. The viability and detail of a model of capsid assembly and maturation can now be examined against the recently available mature herpesvirus capsids structures. The first large assembly product is the icosahedral procapsid with an outer shell composed of major capsid proteins (MCPs) connected by triplexes (heterotrimers composed of one Tri1 protein and two Tri2 proteins), and an inner shell of scaffold proteins. The asymmetric triplexes have specific and conserved orientations, suggesting a key role in assembly. In the mature capsid structures, triplexes bound to three MCPs may represent an assembly unit where, in most cases, the N-terminus of one MCP wraps around the E-loop of another MCP. The model accommodates the incorporation of a portal into capsid, required for genome encapsidation and viral viability. Cleavage of the scaffold triggers maturation of procapsid. Each of the MCPs rotates mostly as a rigid body, except for the flexible peripheral parts that remodel to close the capsid inner surface. Angularization of the capsid shifts the portal outward to a better contact with the capsid shell. Understanding these events in the herpesvirus life cycle to atomic detail could facilitate the development of drugs that uniquely target assembly and maturation. Full article