Search Results (158)

Here we discuss three veterinary alphaherpesviruses—pseudorabies virus, equid alphaherpesvirus 1, and bovine alphaherpesvirus 1—that were instrumental in uncovering the true extent of transcriptome complexity through long-read RNA sequencing, which earlier short-read approaches could not resolve. We focus on three major transcriptomic features whose discovery and characterization relied heavily on these viral models: (i) widespread transcriptional overlaps that complicate read assignment and may drive transcriptional interference; (ii) diverse transcript isoforms arising from alternative 5′ and 3′ transcript termini, as well as splicing; and (iii) non-coding RNAs clustered near replication origins that illuminate potential replication–transcription interactions on a shared nuclear template. Long-read viromics in these veterinary systems has additionally served as a stringent benchmark for transcript callers and annotation pipelines, because the extreme density of overlaps and co-terminal transcript families exposes errors that often go unnoticed in typical mammalian transcriptomes. This has made veterinary herpesvirus datasets disproportionately influential in shaping best practices for full-length isoform calling, transcript end mapping, and artifact-robust cDNA library handling. We also discuss animal gammaherpesviruses as proxies for human gammaherpesviruses, allowing experimental investigation of viral programs difficult to study in human infection. Finally, we describe pseudorabies virus applications as a retrograde transneuronal tracer. Full article

This study systematically investigated the genomic alterations in Saccharomyces cerevisiae driven by Replication Factor A (RFA) dosage insufficiency using a promoter-replacement strategy combined with mutation accumulation and whole-genome sequencing. Our findings reveal that transcriptional suppression of RFA2 or RFA3 leads to severe growth inhibition. RFA deficiency induces a distinct mutational spectrum characterized by a high frequency of monosomy and terminal deletions, indicative of severe replication stress. Furthermore, loss of heterozygosity is significantly enriched at centromeres and high-GC regions, underscoring the role of RFA in stabilizing intrinsic genomic barriers. Utilizing an APOBEC3B-induced mutagenesis assay, we demonstrate that RFA insufficiency leads to the extensive accumulation of exposed ssDNA with a distinct bias towards the lagging strand template. Notably, we observed that cells spontaneously inactivate Mismatch Repair (MMR) genes, such as MSH2 and PMS1, to survive RFA-induced stress. This hypermutant phenotype grants a certain degree of growth recovery on Low Galactose (LG) medium. Overall, these findings demonstrate that RFA dosage is a key determinant of genomic integrity and elucidate how repair pathway modulation drives adaptive evolution under replication stress. Full article

DNMT1 variants are linked to complex neurodegenerative syndromes, yet their variant-specific functional and epigenomic consequences remain poorly defined. DNMT1 variants were identified in eight patients using gene-panel or whole-exome sequencing. Functional effects were assessed by site-directed mutagenesis and transient expression in HEK293T cells. Genome-wide methylation profiling of peripheral blood leukocyte DNA was performed using Nanopore sequencing, enabling direct quantification of 5-methylcytosine (5mC). CpG island-level differential methylation and gene set enrichment analysis (GSEA) were conducted. Variants in the replication foci targeting sequence (RFTS) domain (p.Y511H, p.Y540C, p.H569R) exhibited reduced DNMT1 protein expression, decreased enzymatic activity, and cytosolic aggregation. Variants in the C-terminal catalytic domain (p.A1334V and p.P1546S) showed reduced protein expression with relatively mild enzymatic impairment. Patients carrying the p.Y511H variant demonstrated a significant reduction in global 5mC levels compared with controls. Principal component analysis revealed distinct methylomic profiles separating most patients from controls, with marked intra- and inter-familial heterogeneity. CpG island-level analysis identified a single significantly hypomethylated region in p.Y511H carriers, and GSEA revealed differential enrichment of multiple Gene Ontology biological pathways. This study defines domain-dependent functional effects of DNMT1 variants and provides the first nanopore-based methylome analysis, revealing variant-specific and heterogeneous epigenomic alterations. Full article

Malaria remains a major global health burden, and the emergence of resistance to blood stage antimalarials underscores the need for new interventions targeting earlier stages of the parasite’s life cycle. The pre-erythrocytic liver stage represents a critical bottleneck and an attractive target for chemotherapeutic and prophylactic interventions. In this study, we functionally characterized the putative E3 ubiquitin ligase Trophozoite Exported Protein 1 (TEX1; PBANKA_0102200) in Plasmodium berghei using gene knockout, tagging, and imaging approaches across the mosquito and liver stages. TEX1 knockout parasites (PbTEX1-KO) showed impaired development during mosquito-stage transitions, with significant reductions in ookinete formation, oocyst numbers, and sporozoites reaching the salivary glands. In hepatic stages, TEX1-KO parasites displayed reduced growth, abnormal nuclear division, and impaired liver stage maturation, ultimately leading to a dramatic decline in detached cell formation and blood stage infectivity. Endogenous C-terminal tagging of TEX1 with GFP and 3×HA revealed a discrete subnuclear localization pattern, indicating a critical role in DNA synthesis and/or mitotic regulation. Our findings reveal that TEX1 is required for nuclear replication and division and successful development in both the mosquito and liver stages of Plasmodium. Given its pivotal role and nuclear localization during hepatic schizogony, TEX1 represents a promising target for the development of liver stage antimalarial interventions. Full article

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed that WT Np formed discrete complexes on viral 5′ UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1–4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs despite binding more electrostatically. A major difference was observed in the binding kinetics; WT Np compacted and irreversibly bound single-stranded DNA, whereas phosphomimetic Np displayed reduced compaction and fast on/off binding kinetics. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants. Full article

Background/Objectives: BrdU (5′-bromo-2′-deoxyuridine), a synthetic thymidine (T) analog, is widely used to study cell proliferation and DNA synthesis. To precisely identify where and when DNA replication starts and terminates, it is essential to determine the BrdU incorporation rate and sites at a single-nucleotide resolution. Although several deep learning-based methods have been developed for detecting BrdU using Oxford nanopore sequencing data, there is a lack of accessible, easy-to-follow tutorials to guide researchers in preparing training data and implementing deep learning approaches as the nanopore sequencing technologies continue to evolve. Methods: Due to the lack of ground truth BrdU-positive data generated on the latest R10 flow cells, we prepared model training data from legacy R9 flow cells, consistent with existing tools. We processed publicly available synthetic and real nanopore DNA sequencing datasets, with and without BrdU incorporation, using a combination of open-source and custom software tools. Subsequently, we trained bidirectional gated recurrent unit (BiGRU)-based recurrent neural networks (RNNs) for BrdU detection using the TensorFlow library on the Google Colab platform. Results: We trained BiGRU-based RNNs for BrdU detection with a high specificity (>94%) but a moderate sensitivity due to limited BrdU-positive data. We detail the setup, training, testing, and fine-tuning of the model using both synthetic and real DNA sequencing data. Conclusions: Though the models were trained with data generated on legacy flow cells, we believe that this detailed protocol, covering both data preparation and model development, can be readily extended to R10 flow cells and basecallers for other base modifications. This work will facilitate the broader adoption of deep learning neural networks in biological research, particularly RNNs, which are well suited for modeling sequential and time-series data. Full article

Hepatitis B Virus (HBV) continues to pose a significant global health challenge, with over 254 million chronic infections and current therapies being non-curative, necessitating lifelong treatment. The HBV ribonuclease H (RNase H) is essential during HBV reverse transcription by cleaving the viral pregenomic RNA after it has been copied into the (−) polarity DNA strand, enabling the viral polymerase to synthesize the (+) DNA strand. Although RNase H inhibition terminates viral replication and thus viral infectiveness, its targeting as an HBV treatment is unexploited. Its catalytic site contains four carboxylates that bind to two Mg²⁺ ions essential for RNA hydrolysis. As part of our ongoing research on RNase H inhibitors, we developed 23 novel N-hydroxypyridinedione (HPD) analogues. Specifically, 17 HPD imines, 4 HPD oximes, 1 2,6-diamino-4-((substituted)oxy)pyrimidine 1-oxide derivative, and 1 barbituric acid analogue were designed, synthesized, and tested for their anti-HBV activity. The HPD derivatives could be docked in the RNase H active site to coordinate the two Mg²⁺ ions and effectively inhibited viral replication in cellular assays. The 50% effective concentration (EC₅₀) values of these HPD compounds ranged from 0.5 to 73 μM, while the 50% cytotoxic concentration (CC₅₀) values ranged from 15 to 100 μM, resulting in selectivity indexes (SIs) up to 112. Furthermore, the novel HPD derivatives exhibited favourable pharmacokinetic-relevant characteristics, including high cellular permeability, good aqueous solubility, and overall drug-like properties. These findings indicate that HPD imines and oximes possess substantial antiviral potency and selectivity against HBV, underscoring the potential of the HPD scaffold as a promising framework for the development of next-generation anti-HBV agents. Full article

JC virus (JCV) replicates within the nuclei of glial cells in the human brain and causes progressive multifocal leukoencephalopathy. JCV possesses a small, circular, double-stranded DNA genome, divided into early and late protein-coding regions. The non-coding control region (NCCR) functions bidirectionally for both early and late genes, and the agnogene is located downstream of TCR and upstream of three capsid proteins in the late region. Previously, in cell culture systems, we demonstrated that these capsid proteins accumulate in intranuclear domains known as promyelocytic leukemia nuclear bodies (PML-NBs), where they assemble into virus-like particles (VLPs). To investigate the agnogene’s function, VLPs were formed in its presence or absence, and differential gene expression was analyzed using microarray technology. The results revealed altered expression of histone-modifying enzymes, including methyltransferases (EHMT1, PRMT7) and demethylases (KDM2B, KDM5C, KDM6B), as well as various kinases and phosphatases. Notably, CTDP1, which dephosphorylates the C-terminal domain of an RNA polymerase II subunit, was also differentially expressed. The changes were predominant in the presence of the agnogene. These findings indicate that the agnogene and/or its protein product likely influence epigenetic regulation associated with PML-NBs, which may influence cell cycle control. Consistently, in human brain tissue, JCV-infected glial cells displayed maintenance of a diploid chromosomal complement, likely through G2 arrest. The precise mechanism of this, however, remains to be elucidated. Full article

The genome of the herpesvirus is a linear double-stranded DNA. The viral genome replicates in the host cell to form a concatemeric DNA, which is then cleaved to produce a unit-length genome. This unit-length genome is packaged into procapsid to produce mature virus particles. The terminase large subunit, pUL15, mediates the cleavage and packaging of viral concatemeric genomes. Duck plague virus (DPV) is a member of the α herpesvirus subfamily. Previous studies have demonstrated that the C-terminal region of DPV pUL15 exhibits non-sequence-specific DNA cleavage activity in vitro, but the characteristics of DPV full-length pUL15 remain unclear. In this study, it was determined that the full-length pUL15 exerted non-sequence-specific nuclease activity. Additionally, full-length pUL15 was capable of binding to DNA and hydrolyzing ATP. To analyze the functional domain of DPV pUL15, pUL15 mutants were constructed, expressed, and purified. The results revealed that DNA-binding and ATPase functions of pUL15 were primarily mediated by its N-terminal region, and the nuclease activity was conducted by its C-terminus. The loss of the nuclease activity did not effect on the DNA-binding and ATPase activity. Taken together, this study’s findings demonstrated that DPV pUL15 is a multifunctional enzyme with ATPase, nuclease, and DNA-binding activities. These results will provide important clues for subsequent studies on the function of terminase and the process of viral genome packaging, and provide a foundational basis for the development of broad-spectrum anti-herpesviral drugs targeting the conserved terminase complex, with direct relevance to veterinary medicine. Full article

Background/Objectives: Intrinsically disordered protein regions (IDRs) are difficult to study due to their flexible nature and transient interactions. Computational folding using AlphaFold may offer one way to explore potential folding of these regions under various conditions. Human DNA topoisomerase IIα (TOP2A) is an essential enzyme involved in regulating DNA topology during replication and cell division. TOP2A has an IDR at the C-terminal domain (CTD) that has been shown to be important for regulating TOP2A function, but little is known about potential conformations that it may undertake. Methods: Utilizing the AlphaFold 3 (AF3) model by way of AlphaFold Server, TOP2A was folded as a dimer first without and then with 29 literature-supported post-translational modifications (PTMs) and DNA to observe whether there is predicted folding. Results: TOP2A CTD does not fold in the absence of PTMs. With the addition of PTMs, however, the CTD is predicted to fold into a globular bundle of loops and α-helices. While DNA alone did not induce folding, in the presence of PTMs, DNA ligands increased helicity of the folded CTD and caused it to interact at different core domain interfaces. In addition, DNA is predicted to enable folding of the TOP2A CTD in the presence of fewer PTMs when compared to the absence of DNA. Conclusions: AF3 predicts the folding of TOP2A CTD in the presence of specific PTMs, and this folding appears to shift to allow binding to DNA in functionally relevant regions. These studies provide predicted folding patterns that can be tested by biochemical approaches. AF3 may support the development of testable hypotheses regarding IDRs and enables researchers to model protein-DNA interactions. Full article

Minute virus of canines (MVC), which is a member of the Bocaparvovirus genus, is a non-enveloped, single-stranded DNA virus that causes respiratory and gastrointestinal disease in canines, as well as causing infertility and fetal death in pregnant dogs. The non-structural small protein NP1 of bocaparvoviruses is a unique feature that distinguishes the bocaparvovirus subfamily from other parvovirus subfamilies. In the life cycle of the MVC, NP1 plays an indispensable role in viral DNA replication and pre-mRNA processing. Currently, there is a paucity of studies reporting the characterization of host cell proteins interacting with NP1 during MVC replication. In this study, we screened and identified host cell proteins interacting with MVC-NP1 through immunoprecipitation (IP) combined with liquid chromatography and tandem mass spectrometry (LC-MS/MS) analysis; MCM7 (Mini-chromosome Maintenance Protein 7) has been identified and confirmed to interact directly with NP1 through its N-terminal domain. Furthermore, functional studies reveal that MCM7 is essential in MVC replication. The knockdown of MCM7 decreased the expression of this MVC protein significantly, as well as suppressing MVC replication by arresting the cell cycle in the G0/G1 phase during infection. Conversely, up-regulating MCM7 can rehabilitate the expression of MVC proteins, as well as supporting MVC replication. In conclusion, this study elucidates the interaction between the NP1 protein of MVC and the host factor MCM7, demonstrating that MCM7 is a key factor in the replication process of MVC. These findings provide a potential target for future antiviral therapy. Full article

The papillomavirus (PV) life cycle is strictly controlled and can be divided into the following three distinct stages: initial infection, maintenance, and amplification. The papillomavirus E2 gene encodes a multifunctional protein responsible for regulating transcription and replication by recruiting viral and host factors to the viral DNA genome. Our lab has previously reported that tyrosine 102 may impact bovine (BPV) and human (HPV) viral replication in cell culture systems. This tyrosine is conserved in the E2 protein of the murine papillomavirus MmuPV1. To investigate how this amino acid impacts the MmuPV1 lifecycle in vivo, we generated potential phosphorylation mimetic (Y102E) and phosphorylation deficient (Y102F) mutants in the E2 open reading frame. The Y102F mutant protein supported both transcriptional activation and transient replication, while Y102E was defective. However, Y102E was capable of associating with E1 and the Brd4 C-terminal motif. When these E2-mutated MmuPV1 genomes were introduced into the skin of immunocompromised mice, only Y102F was capable of inducing papilloma development and production of infectious progeny virus. These findings demonstrate that while highly conserved, tyrosine at this position is not required by the virus. These data suggest that the chemical nature of the amino acid at this position can influence E2 activity and viral replication. Full article

The whitefly Bemisia tabaci (Hemiptera: Aleyrodoidea) causes direct feeding damage to crop plants and transmits pathogenic plant viruses, thereby threatening global food security. Although whitefly-infecting RNA viruses are known and proposed as biocontrol agents, no insect DNA virus has been found in any member of Aleyrodoidea. Using rolling circle amplification (RCA) of viral DNA from whiteflies collected from crop fields in Morocco, followed by Illumina sequencing of the RCA products, we found a novel insect single-stranded (ss) DNA parvovirus (family Parvoviridae) in addition to plant ssDNA geminiviruses transmitted by whiteflies. Based on its genome organization with inverted terminal repeats and evolutionarily conserved proteins mediating viral DNA replication (NS1/Rep) and encapsidation (VP), encoded on the forward and reverse strands, respectively, we named this virus Bemisia tabaci ambidensovirus (BtaDV) and classified it as a founding member of a new genus within the subfamily Densovirinae. This subfamily also contains three distinct genera of ambisense densoviruses of other hemipteran insects (Aphidoidea, Coccoidea, and Psylloidea). Furthermore, we provide evidence for the genetic variants of BtaDV circulating in whitefly populations and for its partial sequences integrated into the B. tabaci genome, with one integrant locus potentially expressing a fusion protein composed of viral Rep endonuclease and host DNA-binding domains. This suggests a long-term virus-host interaction and neofunctionalization of BtaDV-derived endogenous viral elements. Full article

Calpain-5/CAPN5 is a calcium-activated, non-lysosomal cysteine (thiol) protease. The substrate repertoire of CAPN5 is not known. Calpains catalyze limited proteolysis of their substrates, generating neo-N-termini that correspond to internal residues of their nascent substrate proteins. To identify such neo-N-termini generated by CAPN5, we employed an N-terminomics approach called TAILS (Terminal amine isotopic labeling of substrates) to quantitatively compare the N-terminal peptides detected in parental and CAPN5-deficient SH-SY5Y neuroblastoma cells. Thirty neo-N-termini corresponding to 29 protein groups and 24 unique proteins were detected to be depleted in the CAPN5^−/− cells. A subset of the identified putative substrates was further studied with CAPN5 co-immunoprecipitation, in vitro calcium-induced CAPN5 proteolysis assay, and their cellular fragmentation patterns were compared in parental and CAPN5-deficient SH-SY5Y cells. Here, we provide evidence for CAPN5-mediated proteolysis of the synaptic proteins DLGAP4, IQSEC1 and MPDZ, the neurodegeneration-related EWS, hnRNPU, TFG and UGP2, the DNA replication regulator MCM3, and the neuronal differentiation regulator LMTK1. Our data provide new relevance for neovascular inflammatory vitreoretinopathy (NIV), a progressive eye disease caused by pathogenic mutations in CAPN5. Data are available via ProteomeXchange with identifier PXD064313. Full article

The ability of a nucleic acid molecule to self-replicate is the driving force behind the evolution of cellular life and the transition from RNA to DNA as the genetic material. Thus, the physicochemical properties of genome replication, such as the requirement for a terminal hydroxyl group for de novo DNA synthesis, are conserved in all three domains of life: eukaryotes, bacteria, and archaea. Canonical DNA replication is initiated from specific chromosomal sequences termed origins. Early bacterial models of DNA replication proposed origins as regulatory points for spatiotemporal control, with replication factors acting on a single origin on the chromosome. In eukaryotes and archaea, however, replication initiation usually involves multiple origins, with complex spatiotemporal regulation in the former. An alternative replication initiation mechanism, recombination-dependent replication, is observed in every cellular domain (and viruses); DNA synthesis is initiated instead from the 3′ end of a recombination intermediate. In the domain archaea, species including Haloferax volcanii are not only capable of initiating DNA replication without origins but grow faster without them. This raises questions about the necessity and nature of origins. Why have archaea retained such an alternative DNA replication initiation mechanism? Might recombination-dependent replication be the ancestral mode of DNA synthesis that was used during evolution from the primordial RNA world? This review provides a historical overview of major advancements in the study of DNA replication, followed by a comparative analysis of replication initiation systems in the three domains of life. Our current knowledge of origin-dependent and recombination-dependent DNA replication in archaea is summarised. Full article