Search Results (750)

The expression of the BNYVV silencing suppressor (VSR) p14 protein, together with the production of a viral non-coding RNA (ncRNA3), affects the long-distance movement on natural host Beta species. Through immunoprecipitation of Flag-p14 and expression of TurboID-p14 coupled to mass spectrometry, we identified several potential p14 cellular partners. These include proteins related to RNA metabolism, proteasome activation, and notably, SGS3—a key player in the siRNA transitivity pathway—and DRB4, the DCL4 cofactor described in A. thaliana silencing pathways. The interaction between p14 and DRB4 was specifically retrieved with the hypomorphic p14BA2 VSR mutant in nucleoli and was disrupted in the presence of ncRNA3, a condition that allows the viral mutant to move long distances. Subcellular fractionation of infected tissues revealed that ncRNA3 accumulates in the nucleus through its interaction with p14, while genomic RNA3 remains cytoplasmic. Our results suggest that a specific viral RNA is targeted to the nucleus to participate in silencing suppression function by destabilizing the p14-DRB4 complex. Moreover, the interaction between p14 and SGS3 provides a mechanistic explanation of the role of this VSR in silencing transitivity. Full article

The herpes simplex virus 1 (HSV-1) US3 is a multifunctional serine/threonine kinase that promotes HSV-1 replication and spread. But its role and the mechanisms by which US3 regulates actin cytoskeletal remodeling remain poorly defined. We combined flow cytometry, confocal microscopy, immunoprecipitation-mass spectrometry (IP-MS), protein complex mapping, and machine learning to characterize US3-mediated F-actin dynamics. Flow cytometry and confocal microscopy showed that wild-type HSV-1 induces significant F-actin remodeling, while the ΔUS3 mutant displays F-actin levels comparable to uninfected cells, identifying US3 as a key regulator. IP-MS identified 47 high-confidence US3 interactors enriched in conserved actin regulatory complexes, including Arp2/3 nucleation machinery, formin-associated assemblies, cofilin severing complexes, and Rho-family GTPase modules. Mapping interactors to the CORUM database revealed clustering within actin nucleation, polymerization, and severing complexes, indicating that US3 operates through organized cellular machines. Machine-learning classifiers trained on label-independent mass-spectrometry features were used to prioritize interactors resembling known actin regulators; under protein-group-aware cross-validation, logistic regression performed best (average precision 0.24; ROC-AUC 0.66), and the analysis was interpreted as prioritization rather than de novo discovery. Pharmacological inhibition of Arp2/3 and formin pathways significantly attenuated US3-dependent F-actin remodeling, supporting the functional involvement of these pathways. Together, these findings are consistent with an inferred hierarchical axis in which US3 modulates Rho GTPase signaling and cofilin activation to promote F-actin disassembly, coordinating cytoskeletal remodeling required for efficient viral egress and spread. Full article

Letermovir (LMV), a cytomegalovirus (CMV) terminase inhibitor, has a potentially low genetic barrier to the emergence of resistance, with single mutations in the terminase subunits (UL51, UL56, UL89) being associated with LMV resistance (LMV-R). We determined the fitness of LMV-R viruses (UL56 C325F/W/Y or M236V mutants) with different DNA polymerase (pol) (UL54) mutants in the presence and absence of anti-CMV drugs in dual-infection competition assays. After 7 days of growth, viral variants were quantified by targeted sequencing of the UL54 or UL56 genes by next-generation sequencing. Without antivirals, the UL56 C325F mutant was equally fitted as the wild-type virus, in contrast to C325W/Y mutants that had a reduced replication capacity. The UL56 C325F mutant showed the highest replication capacity among all terminase mutants tested when grown in competition with wild-type virus under LMV. The UL56 terminase mutants gained replication capacity when grown in competition with DNA pol mutant viruses under LMV pressure, with the terminase mutant UL56 C325F being able to overgrow various DNA pol mutants. Furthermore, the UL56 C325F mutant showed the highest replication capacity when grown in competition with another UL56 terminase mutant. Our results are in line with the C325F UL56 mutant being the most frequent LMV-R mutation identified in the clinic. Full article

Hepatitis B virus (HBV) infection remains a major global health burden, with chronic infection leading to severe liver diseases including cirrhosis and hepatocellular carcinoma (HCC). HBV-encoded X protein (HBx) plays a critical role in viral replication and pathogenesis by modulating host cellular processes, including autophagy and lysosomal function. However, the molecular mechanisms by which HBx disrupts lysosomal biogenesis and autophagic degradation remain elusive. In this study, we show that HBx downregulates the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, which leading to impaired lysosomal acidification and autophagosome–lysosome fusion. Mechanistically, HBx-mediated TFEB downregulation involves the CUL4A (Cullin 4A)/CUL4B (Cullin 4B)-DDB1 (DNA damage-binding protein 1) E3 ubiquitin ligase complex and is dependent on the DDB1-interacting motif in HBx. HBx mutants defective in DDB1 binding (HBx^R96E and HBx^ΔDBD) fail to downregulate TFEB or impair lysosomal function. Collectively, our findings identify a pathway by which HBx disrupts lysosomal function via CUL4A/CUL4B–DDB1-dependent TFEB downregulation, providing insights into HBV-associated liver pathogenesis and highlighting potential targets for therapeutic intervention. Full article

A single vaccine cannot prevent or treat all cancers; however, recurrent tumor-specific epitopes may facilitate the development of pre-manufactured, HLA-matched mRNA vaccines tailored for specific molecular subgroups. We define the shared tumor-specific epitope as a recurring peptide derived from a viral oncoprotein, a driver mutation, a frameshift, an altered protein C-terminus, or a fusion junction, and we employ a rigorous cancer-cell-only criterion: a target must be recurrent within a defined subgroup, absent from essential normal tissues at the peptide–HLA level, naturally presented on tumor cells, and sufficiently clonal to minimize immune escape. Under this criterion, we present fifteen sequence-anchored reference designs alongside one conceptual placeholder across thirteen candidates divided into four superclasses: viral oncoproteins (such as HPV16/18 E6 and E7 as attenuated antigenic reference designs; Merkel cell polyomavirus serving as a design-specific placeholder), recurrent driver neoepitopes (including KRAS G12/G13, IDH1 R132H, and H3 K27M), hematologic neoantigens (such as NPM1 Type A C-terminus; and a single CALR exon 9 construct encoding the shared novel C-terminus of types 1 and 2 mutations), and fusion junctions (notably EWS-FLI1 and BCR-ABL). Each open reading frame is anchored to a canonical accession with its documented event; representative ORFs are provided as reference designs, with the intended residue-level verification records. These sequence designs are intended as reference constructs and are not suitable as clinical-grade or manufacturing-ready products; they require independent residue-level validation and comprehensive safety assessments prior to laboratory or clinical application. The historical record of non-personalized vaccination—including HPV and hepatitis B prophylaxis, intravesical BCG, and unsuccessful tumor-associated antigen trials—frames both the potential and limitations of such approaches. The practical product is not a universal vaccine but rather a governed library aligned with specific genotype, viral etiology, HLA context, and clinical setting. Currently, none of these designs have established proof-of-benefit-tier evidence. Full article

The amide functional group remains a cornerstone of medicinal chemistry, serving as an indispensable scaffold in the design of modern therapeutics. This review presents an analysis of FDA-approved drugs (small molecules and peptides with MW < 1300 Da) containing amide functionality between 2021 and 2025, highlighting its continued and evolving role in addressing contemporary medical challenges. An analysis of these novel therapeutics reveals the remarkable functional versatility of the amide bond. In antiviral agents like nirmatrelvir (Paxlovid^®), amides form the structural backbone of peptidomimetics, enabling high-affinity binding to a viral protease. In precision oncology, as seen with adagrasib (Krazati^®), the amide acts as a critical, metabolically stable linker that positions a covalent warhead for selective inhibition of a mutant kinase. This analysis underscores that amide’s unique combination of planarity, resonance stabilization, and capacity for robust hydrogen bonding continues to make it an essential element in the medicinal chemist’s toolkit, underpinning the development of next-generation therapeutics across oncology, infectious diseases, and neurology. To provide a practical framework for drug discovery, the synthetic routes for each drug are detailed, with particular emphasis placed on the key amide-forming strategies employed. Full article

Background: While vaccines are designed to elicit targeted immune responses, in some cases, the immunogenic molecules employed can inherently interact with broader host cellular pathways as a secondary consequence. This phenomenon can be exemplified by COVID-19 vaccines. COVID-19 vaccines, including mRNA platforms, use the SARS-CoV-2 spike protein as an immunogen to induce the production of neutralizing antibodies. The spike protein binds the ACE2 (angiotensin-converting enzyme 2) receptor on human cells, mediating viral entry and infection. ACE2 is widely expressed across multiple tissues and is a key component of the renin–angiotensin–aldosterone system (RAAS) that acts as a homeostatic regulator of systemic and local blood flow, blood pressure, cardiac function, fluid balance and immunity. Some studies have proposed the interaction between the spike protein and ACE2 as a possible contributing factor to rare adverse effects observed following COVID-19 vaccination, including myocarditis, pericarditis, thrombosis, and reported alterations in blood pressure, though these mechanisms remain to be fully elucidated. Objectives: As a proof-of-concept approach in vaccine antigen development, we engineered SARS-CoV-2 spike mutants with impaired binding to the host receptor ACE2. Methods: By rational design, we produced and validated in vitro and in vivo spike point mutants that do not effectively bind ACE2. Results: The engineered spike mutants do not effectively bind the human entry receptor ACE2 while retaining the immunogenic properties equal to or better than the wild type spike and thus generate a protective response in animals when used as a vaccination agent. Conclusions: By establishing a straightforward molecular strategy for rational vaccine design, this work demonstrates the feasibility of limiting specific antigen–host receptor interactions while maintaining immunogenicity. This approach may be applicable to future vaccination strategies where antigen interaction with host cells could potentially interfere with physiological pathways. Full article

RPE65, an isomerohydrolase expressed in retinal pigment epithelium (RPE), is critical for the visual cycle. More than 115 missense variants of the RPE65 gene have been associated with Leber’s congenital amaurosis (LCA), a severe childhood retinal dystrophy. Due to high genetic heterogeneity, the variant-specific pathogenic mechanisms remain largely uncharacterized. In this study we focus on an LCA patient carrying compound heterozygous RPE65 variants (c.200T > G, c.430T > C), aiming to dissect the mechanistic/functional basis of mutated protein-driven retinal degeneration and evaluate gene therapy-mediated restoration using patient-specific hiPSCs-RPE (iRPE). Transient overexpression of wild-type/mutant RPE65 in HEK293T cells showed both variants markedly destabilize the RPE65 protein through the autophagosome–lysosome degradation pathway and its isomerohydrolase activity required for the retinoid visual cycle. We further established a patient-specific iRPE platform suitable for enzymatic activity analysis. Characterization of patient-specific iRPE cells revealed those compound heterozygous variants did not compromise iRPE morphology, most gene expression, or core canonical physiological features of iRPE. However, they significantly downregulate endogenous RPE65 protein abundance and dampen enzymatic function. Subsequently, we delivered RPE65 via adeno-associated viral (AAV) vectors driven by either the ubiquitous CMV promoter or RPE-specific VMD2 promoter into patient iRPE to validate therapeutic potency, and verified that exogenous RPE65 supplementation effectively restores deficient isomerohydrolase activity in this disease model. Collectively, this work elucidates the variant-specific pathogenesis of RPE65-associated LCA and preliminarily assesses the efficacy of gene augmentation, providing preclinical experimental evidence to support the referral of this patient for clinical RPE65 gene replacement therapy. Full article

Background: Viral attachment mediated by host cell surface receptors is the first step in viral infection. As a key cell surface receptor, heparan sulfate (HS) mediates the attachment and entry of numerous non-enveloped viruses in livestock, thereby serving as a crucial molecular target for studying virus–host interactions. Methods: Based on the structural scaffold of a nanobody (Nb; PDB: 7TJC), we rationally designed and constructed a mutant Nb targeting HS, designated HS-Mut-Nb1, using molecular docking, site-directed mutagenesis, molecular dynamics (MD) simulations, and experimental characterization. Results: Molecular docking indicated that the active site of wild-type Nb for HS binding was located within the cavity jointly formed by the complementarity-determining region 3 (CDR3) and the framework regions (FRs) of the wild-type Nb. A comprehensive analysis integrating virtual alanine scanning, site-directed mutagenesis, and MD simulations revealed that the combination of three point mutations (Phe47Arg, Asp99Tyr, and Tyr108Pro) significantly enhanced the binding affinity of Mut-Nb1 for HS, with a calculated binding free energy (ΔG) of −83.26 ± 3.06 kcal/mol. Enzyme-linked immunosorbent assay (ELISA) results further confirmed that Mut-Nb1 exhibited high affinity for HS (K_D = 65.87 nM) and specificity (positive/negative ratio, P/N = 3.84; cross-reactivity, CR < 6.60%). Conclusions: This study not only provides novel candidate molecules for elucidating the mechanism of HS–virus interactions and developing related inhibitors but also offers a reference for the rapid construction of mutant Nbs. Full article

Semaphorin 3A (Sema3A), a known axon chemorepulsive protein during development, is localised in perineuronal nets (PNNs) in the adult brain. PNNs are condensed aggregates of extracellular matrix molecules surrounding specific types of neurons, which regulate neuroplasticity and memory. However, the role of PNN-associated Sema3A in these processes remains unclear. To address this topic, we investigated the contribution of Sema3A to cerebellum-dependent learning and memory in adult mice using the eyeblink conditioning (EBC) paradigm. We interfered with Sema3A signalling by employing: (i) a molecular approach, in which secreted Sema3A receptors (neuropilin-1 bodies) were expressed in the anterior interposed nuclei (AIN) via viral vector injection; and (ii) a genetic approach, using mutant mice with impaired Sema3A signalling (K108N mice). Mice expressing neuropilin-1 bodies showed reduced EBC performance at the beginning of the memory retention phase. However, increased inflammation was found in the AIN of these mice, challenging the interpretation of these findings. K108N mice showed enhanced EBC performance at the beginning of the memory retention phase. No synaptic structural changes were detected in the AIN of K108N mice at the end of the EBC paradigm. Based on our findings in K108N mice, constitutively altered Sema3A signalling is associated with subtle improvement in cerebellar memory. Full article

Enterovirus B (EVB) is the most prevalent species of human enteroviruses, responsible for a wide range of diseases, including hand, foot, and mouth disease, viral meningitis, myocarditis, and neonatal sepsis, imposing a significant disease burden primarily on children. Coxsackievirus B (CVB1-6) and various echovirus (E) serotypes are the major serotypes of EVB. Since no antiviral drug or vaccine is available, it is important to strengthen monitoring, risk assessment, and early warning of genomic variations for EVB. CVB1, CVB3, E6, and E30 were selected as representative EVB serotypes for this study due to the availability of three-dimensional structures and their global prevalence. To evaluate the mutation effects of structural proteins on structural stability and receptor-binding affinity, computational saturation mutagenesis of EVB serotypes was performed using FoldX. Furthermore, based on data from deep mutational scanning for CVB3, a risk prediction model for EVB fitness was constructed by machine learning algorithms and applied to other EVB serotypes. Finally, we integrated three phenotypes—structural stability, receptor-binding affinity and fitness—to evaluate genomic variation risk of EVB and tracked the prevalence of high-risk mutants in natural viral sequences through molecular evolution analysis and mutation profiles. We identified the N-terminus and C-terminus of VP1 and the EF loop of VP2 as the EVB regions of highest genomic variation risk, and high-risk mutations had played significant roles in viral evolutionary history. These findings provide a framework for multi-phenotypic and multi-data approaches to viral risk assessment and offer insights to support the development of antiviral drugs and vaccines. Full article

Metagenomics analysis is a critical tool in identifying and typing viral samples to aid surveillance, clinical, epidemiological, and other workflows. Despite advances in sequencing technology and analysis pipelines, there are still limitations that lead to reduced taxonomic resolution or false positives from highly recombinant or challenging samples. Here we describe MGtree, a novel metagenomics pipeline that utilizes a combination of full-length read alignments and phylogenetic analysis to classify samples of interest. We demonstrate that MGtree accurately genotypes viral samples from challenging norovirus and HPV datasets. MGtree outperforms the popular metagenomics programs Kraken2 and Centrifuge, and it succeeds with low-input samples where de novo assembly fails. MGtree’s correct assignments across highly mutant and coinfected samples highlights its ability to resolve viral genotypes and its potential to improve classification precision in complex samples. Full article

Andes virus (ANDV), a highly pathogenic orthohantavirus, enters host cells through low pH–triggered membrane fusion mediated by the Gc glycoprotein, a class II fusion protein containing a single C-terminal transmembrane domain (TMD). While the ectodomain has been extensively characterized, the role of the TMD in late-stage fusion remains unclear. Here, we investigated the minimal functional length and sequence requirements of the ANDV Gc TMD using site-directed mutagenesis. C-terminal deletion mutants and serine-to-alanine substitutions were evaluated for protein expression, virus-like particle production, cell–cell fusion, pseudotyped vector entry, and hemifusion activity. Deletion of the Gc cytoplasmic tail (CT) or a single C-terminal TMD residue was tolerated, whereas deletion of two or more residues impaired particle production and fusion, indicating that at least 21 of the 22 TMD residues are required for efficient membrane fusion and viral entry. Hemifusion assays showed that deletion of two or three residues, or substitution of the strictly conserved S1121, allowed lipid mixing but blocked progression to full fusion, while deletion of four residues also abolished hemifusion. In contrast, mutation of the less conserved S1126 had minimal effect. These results identify a precise TMD length and a conserved polar TMD residue as critical determinants of fusion pore formation in ANDV. Full article

Bovine herpesvirus type 1 (BoHV-1) causes severe respiratory and reproductive diseases in cattle, leading to significant economic losses worldwide. Current inactivated vaccines in China fail to induce robust cellular immunity and cannot differentiate infected from vaccinated animals (DIVA). To address these limitations, we constructed two triple-gene-deleted BoHV-1 vaccine candidates, ∆T3 (∆TK-∆gE/gI) and ∆g3 (∆gG-∆gE/gI), using Red/ET two-step recombination technology based on an infectious BAC clone. Their safety and immunogenicity were evaluated in a rabbit model. Both deletion mutants exhibited growth kinetics similar to the wild-type strain but with significantly reduced viral titers and plaque areas. The triple-gene deletion mutants showed superior safety compared with single-gene deletion mutants, with minimal clinical signs and low viral shedding. Immunization with ∆T3 and ∆g3 induced robust gB-specific and neutralizing antibody responses. Following challenge with the virulent BoHV-1 BC01 strain, vaccinated rabbits maintained normal body temperatures, showed significantly reduced viral shedding (approximately 100-fold), and exhibited milder pulmonary lesions. These findings demonstrate that ∆T3 high-dose and ∆g3 low-dose regimens are promising DIVA-compatible vaccine candidates, offering an effective strategy for IBR control and eradication programs. Full article

Equine infectious anemia virus (EIAV), with the simplest lentiviral genome, is a key model for studying fundamental lentiviral biology. Infectious viral particles are produced only when the Gag protein selectively encapsidates full-length genomic RNA via the packaging signal (Psi), yet the structural and functional features of EIAV Psi remain poorly characterized. Using computational prediction and dimethyl sulfate probing, we identified four stem-loops (SLs) within a ~120 nt region in the 5′ leader of the genome, spanning from downstream of the primer binding site through 20 nt into the gag coding sequence. In vitro dimerization assays demonstrated that a palindromic sequence (5′-CUGGCCAG-3′) within SL3 acts as a critical determinant of RNA dimerization. Functional screening using both an EIAV pseudovirus packaging system and the infectious clone EIAVuk revealed that deletion or mutation of the stem-loops significantly impairs viral packaging and replication, with SL2 deletion or its stem disruption causing the most severe defects. RNA-seq analysis of RNAs bound by wild-type Gag versus a zinc-finger mutant (H391K/H410K) identified two candidate Gag-associated sites: the SL2 stem and the SL2-SL3 junction. Targeting these regions with phosphorothioate-modified antisense oligonucleotides potently inhibited pseudovirus production and the replication of infectious EIAVuk. Our findings defined the secondary structure and functional organization of the EIAV core packaging region and established the SL2 stem and SL2-SL3 junction as candidate packaging determinants and promising targets for RNA-based antiviral intervention. Full article