Nuclei Isolation from Ocular Tissues of the Embryonic Chicken for Single-Nucleus Profiling.

The generation of quality data from a single-nucleus profiling experiment requires nuclei to be isolated from tissues in a gentle and efficient manner. Nuclei isolation must be carefully optimized across tissue types to preserve nuclear architecture, prevent nucleic acid degradation, and remove unwanted contaminants. Here, we present an optimized workflow for generating a single-nucleus suspension from ocular tissues of the embryonic chicken that is compatible with various downstream workflows. The described protocol enables the rapid isolation of a high yield of aggregate-free nuclei from the embryonic chicken eye without compromising nucleic acid integrity, and the nuclei suspension is compatible with single-nucleus RNA and ATAC sequencing. We detail several stopping points, either via cryopreservation or fixation, to enhance workflow adaptability. Further, we provide a guide through multiple QC points and demonstrate proof-of-principle using two commercially available kits. Finally, we demonstrate that existing in silico genotyping methods can be adopted to computationally derive biological replicates from a single pool of chicken nuclei, greatly reducing the cost of biological replication and allowing researchers to consider sex as a variable during analysis. Together, this tutorial represents a cost-effective, simple, and effective approach to single-nucleus profiling of embryonic chicken eye tissues and is likely to be easily modified to be compatible with similar tissue types.

The people behind the papers - Alexandre Dumoulin and Esther Stoeckli.

The absence or dysfunction of primary cilia, which are non-motile protrusions on cells, leads to a group of neurodevelopment disorders called ciliopathies. In a new study, Esther Stoeckli and colleagues identify the role of primary cilium-mediated sonic hedgehog (Shh) signaling in commissural axon guidance in mice and chick embryos. We caught up with first author, Alexandre Dumoulin, and corresponding author, Esther Stoeckli, Professor at the University of Zurich, to find out more about the work.

Evaluation of chicken embryo extract and egg yolk extract as alternatives to basic cell culture medium supplement.

BACKGROUND: Fetal calf serum (FCS), an existing cell culture supplement, is effective but has several drawbacks, including being expensive, requiring a lengthy process of production, and requiring a hard currency. With this in mind, we planned to evaluate chick embryo extract and egg yolk extracts in cell culture as alternatives to fetal calf serum (FCS). METHODS: Specific pathogen-free eggs were purchased from the National Veterinary Institute, Bishoftu, Ethiopia, and incubated in a humidified incubator at 37 °C for 11 days. Egg yolk extract (EYE) and chick embryo extract (CEE) were collected after the egg was opened with caution not to destroy the yolk sack or the chick embryo itself. Chick fibroblasts and Vero cells were cultured in minimum essential medium (MEM) supplemented with egg yolk extract or chick embryo extract at ratios of 0:10, 1:9, 2.5:7.5, and 5:5% fetal calf serum. RESULTS: Fibroblast cell attachment was better in media supplemented with 5% CEE and 5% FCS. The confluency was also greater than 50% at this concentration. Vero cells cultured with 5% CEE and 5% FCS also exhibited very good cell attachment and a confluency of up to 70%. Viability and confluency were also observed at 5%:5% ratios of 50 and 70%, respectively. CONCLUSION: This investigation evaluated these two extracts as cell culture supplements and revealed promising results as alternatives to fetal calf serum. The limitation of this study is that it only used two cell types and additional cell lines, and different ratios should be tested. With the above findings, further research using different cell lines, ratios and conditions is warranted.

Number of Blastodisc Cells During the Laying Period for Two Successive Generations of Layer and Broiler Breeder.

In the poultry industry, genetic selection for growth performance is associated with poor reproductive efficiency and an increase in embryo mortality. The identification of new biomarkers is essential to improve these parameters. The blastodisc, composed of blastodermal cells, undergoes cellular events to achieve embryo development. Factors such as hen's age, temperature and time of egg storage could influence the number of blastodermal cells and impair embryo development. In this study, we investigated the variability of the number of viable cells of blastodisc (NVCB) that could be dependent on the stage of laying and on the breed and potentially associated with reproductive parameters. In experimental breeds, eggs were collected during the whole cycle of laying. Then, the protocol was repeated on industrial breeds (breeder hens) during five successive days at three stages of laying (before, after laying peak and at the end of laying period) for two generations (mothers and offsprings). For each egg, the blastodisc was dissected in order to count viable cells. For both experiments, the NVCB increased during the laying cycle. The NVCB was higher in broiler blastodisc compared to layer blastodisc for both generations. For layer breed, the NVCB were negatively correlated with laying rate for the first generation while positively associated for offsprings. However, the NVCB was positively correlated with laying rates in both generations for broiler hens and with fertility and hatchability rates. The NVCB from fresh oviposited fertilised eggs could be a potential tool in predicting on reproductive performances in poultry.

Embryonic Thyroid Hormone Insufficiency Causes Structural Anomalies in the Embryo of Domestic Chick, Gallus domesticus.

Thyroid hormone (TH) is essential for growth and development, yet its specific role during embryogenesis remains incompletely understood. This study investigates the impact of TH deficiency, induced by thiourea, a known inhibitor of thyroid peroxidase (TPO), on the development of domestic chicks. Thiourea was administered before thyroid gland formation, and its presence in treated embryos was confirmed through liquid chromatography-mass spectrometry. In silico docking revealed a strong interaction between thiourea and the CCP-like domain of TPO, which was corroborated by TPO activity assays showing reduced enzyme function. This reduction in enzyme activity led to lower embryonic TH levels and increased thyroid-stimulating hormone (TSH) secretion. Morphological analysis of newly hatched chicks revealed significant structural anomalies, particularly in lateral plate mesoderm-derived structures, including omphalocele, limb deformities, anophthalmia and craniofacial defects. Alcian blue and Alizarin red staining demonstrated reduced ossification in ribs and forelimbs, while histological analysis showed incomplete abdominal wall closure and abnormal vertebral column development. Haematological profiling of TH-deficient newly hatched chicks revealed significantly lower blood cell counts, highlighting TH's critical role in haematopoiesis. These findings emphasise the multifaceted role of TH in embryonic development, with potential implications for understanding congenital hypothyroidism and its developmental impacts, especially in regions with limited healthcare access.

Research of recombinant influenza A virus as a vector for Mycoplasma pneumoniae P1a and P30a.

BACKGROUND: Mycoplasma pneumoniae (MP) is a common respiratory pathogen affecting the longevity of the elderly and the health of children. However, the human vaccine against MP has not been successfully developed till now due to the poor immunogenicity and side effects of MP inactivated or attenuated vaccine. Therefore, it is necessary to develop a MP genetic engineering vaccine with influenza virus strain as vector. METHODS: In this study, the major antigen genes P1a of MP adhesion factor P1(3862-4554 bases) and P30a of P30(49-822 bases) were inserted into the nonstructural protein (NS) gene of Influenza A virus strain A/Puerto Rio/8/34(H1N1), PR8 for short, to construct the recombinant vectors NS-P1a or NS-P30a. The recombinant pHW2000 plasmids containing NS-P1a or NS-P30a were cotransfected with the rest 7 fragments of PR8 into HEK293T cells. After inoculating chicken embryos, the recombinant influenza viruses rFLU-P1a and rFLU-P30a were rescued. RT-PCR and sequencing were used to identify the recombinant viruses. The hemagglutination titers of rFLU-P1a and rFLU-P30a were determined after five successive generations in chicken embryos so as to indicate the genetic stability of the recombinant viruses. The morphology of recombinant influenza viruses was observed under electron microscopy. RESULTS: P1a or P30a was designed to be inserted into the modified NS gene sequence separately and synthesized successfully. RT-PCR identification of the recombinant viruses rFLU-P1a and rFLU-P30a showed that P1a (693 bp), P30a (774 bp), NS-P1a (1992bp) and NS-P30a (2073 bp) bands were found, and the sequencing results were correct. After five successive generations, each virus generation has a certain hemagglutination titer (from 1:32 to 1:64), and the band of P1a or P30a can be seen in the corresponding positions. The virus particles under the electron microscope appeared as spheres or long strips connected by several particles, revealing a complete viral membrane structure composed of virus lipid bilayer, hemagglutinin, neuraminidase, and matrix proteins. CONCLUSION: The recombinant viruses rFLU-P1a and rFLU-P30a which carried the advantaged immune regions of the P1 and P30 genes in MP were successfully constructed and identified. And the genetic stability of rFLU-P1a or rFLU-P30a was relatively high. The typical and complete morphology of influenza virus was observed under the electron microscope. Our research provided a foundation for the further development of MP vaccines for human.

The first embryonic landscape of G-quadruplexes related to myogenesis.

BACKGROUND: DNA G-quadruplexes (G4s) represent a distinctive class of non-canonical DNA secondary structures. Despite their recognition as potential therapeutic targets in some cancers, the developmental role of G4 structures remains enigmatic. Mammalian embryonic myogenesis studies are hindered by limitations, prompting the use of chicken embryo-derived myoblasts as a model to explore G4 dynamics. This study aims to reveal the embryonic G4s landscape and elucidate the underlying mechanisms for candidate G4s that influence embryonic myogenesis. RESULTS: This investigation unveils a significant reduction in G4s abundance during myogenesis. G4s stabilizer pyridostatin impedes embryonic myogenesis, emphasizing the regulatory role of G4s in this process. G4 Cut&Tag sequencing and RNA-seq analyses identify potential G4s and DEGs influencing embryonic myogenesis. Integration of G4 and DEG candidates identifies 32 G4s located in promoter regions capable of modulating gene transcription. WGBS elucidates DNA methylation dynamics during embryonic myogenesis. Coordinating transcriptome data with DNA G4s and DNA methylation profiles constructs a G4-DMR-DEG network, revealing nine interaction pairs. Notably, the NFATC2 promoter region sequence is confirmed to form a G4 structure, reducing promoter mCpG content and upregulating NFATC2 transcriptional activity. CONCLUSIONS: This comprehensive study unravels the first embryonic genomic G4s landscape, highlighting the regulatory role of NFATC2 G4 in orchestrating transcriptional activity through promoter DNA methylation during myogenesis.

Improved surrogate eggshell incubation system for assisted reproductive technologies such as cloning, genome modification, and de-extinction efforts.

A germinal disc located on the egg yolk surface drives genetic modification. Windowed and surrogate eggshell incubation methods have been developed, but these exhibit limited abilities to generate transgenic chickens. In the present study, we investigated the frequency of observing the germinal disc according to the preincubation positioning direction and time and found that it depended on those conditions, but only a few chicks (2.8-5.6%) hatched using the windowed method. Then, we attempted to improve surrogate method via one- or two-step procedures. All eggs, including surrogates that were 10 g heavier than the donor eggs, were obtained from a poultry flock of the same age. With the one-step surrogate method, where the donor egg was transferred only once through a 3.5 cm hole on the point end, at the first day of preincubation, into the surrogate egg, the survival rate at day 4 was 30.8%, and the hatching rate was 11.8%. With the two-step surrogate method (transfer was on the 1st and 4th day of incubation), the survival rate at day 4 was improved to 90.7%, and a hatching rate of 70.0% was achieved. Therefore, this method can be effective for in ovo artificial incubation.

Chick Embryo Chorioallantoic Membrane as a Platform for Assessing the In Vivo Efficacy of Chimeric Antigen Receptor T-cell Therapy in Solid Tumors.

The fertilized chicken egg chorioallantoic membrane (CAM), a highly vascularized membrane nourishing the developing embryo, also supports rapid growth of three-dimensional vascularized tumors from engrafted cells and tumor explants. Because murine xenograft models suffer limitations of time, cost, and scalability, we propose CAM tumors as a rapid, efficient screening tool for assessing anti-tumor efficacy of chimeric Ag receptor (CAR) T cells against solid tumors. We tested the efficacy of human epidermal growth factor receptor 2 (HER2)-specific CAR T cells against luminescent, HER2-expressing (FaDu, SCC-47) or HER2-negative (MDA-MB-468) CAM-engrafted tumors. Three days after tumor engraftment, HER2-specific CAR T cells were applied to tumors grown on the CAM. Four days post-CAR T cell treatment, HER2-expressing FaDu and SCC-47 tumors treated with CAR T showed reduced viable cancer cells as assessed by luciferase activity. This reduction in viable tumor cells was confirmed by histology, with lower Ki-67 staining observed in CAR T cell-treated tumors relative to T cell-treated controls. Persistence of CAR T in CAM and tumor tissue 4 days post-treatment was confirmed by CD3 staining. Altogether, our findings support further development of the chick CAM as an in vivo system for rapid, scalable screening of CAR T cell efficacy against human solid tumors.

Mitochondrial dynamics regulate cell morphology in the developing cochlea.

In multicellular tissues, the size and shape of cells are intricately linked with their physiological functions. In the vertebrate auditory organ, the neurosensory epithelium develops as a mosaic of sensory hair cells (HCs), and their glial-like supporting cells, which have distinct morphologies and functional properties at different frequency positions along its tonotopic long axis. In the chick cochlea, the basilar papilla (BP), proximal (high-frequency) HCs, are larger than their distal (low-frequency) counterparts, a morphological feature essential for sound perception. Mitochondrial dynamics, which constitute the equilibrium between fusion and fission, regulate differentiation and functional refinement across a variety of cell types. We investigate this as a potential mechanism for regulating the shape of developing HCs. Using live imaging in intact BP explants, we identify distinct remodelling of mitochondrial networks in proximal compared with distal HCs. Manipulating mitochondrial dynamics in developing HCs alters their normal morphology along the proximal-distal (tonotopic) axis. Inhibition of the mitochondrial fusion machinery decreased proximal HC surface area, whereas promotion of fusion increased the distal HC surface area. We identify mitochondrial dynamics as a key regulator of HC morphology in developing inner ear epithelia.

Authorizing of Immunogenicity of Concentrated and Purified Newcastle Disease Virus V4 Strain using Downstream Processing.

Newcastle disease virus (NVD) from the Paramyxoviridae family is a single-stranded negative-sense RNA virus. This infection can affect both domestic poultry and almost all other bird species. It has been considered a very severe difficulty for the poultry industry all over the world. Even though it remains a potential threat to poultry industries, this virus is a powerful oncolytic virus as well. In this study, a process was accomplished to achieve concentrated and highly purified NDV V4 strain particles. Downstream processing of Newcastle virus strain V4 was characterized by amplifying virus in embryonated chicken eggs. Through a sequence of steps, harvesting allantoic fluid, clarification by centrifuge, concentration by ultrafiltration, and size exclusion separation, the reduced volume and pure virus particles were considered for the amount of ovalbumin, hemagglutinin activity, transmission electron microscopy (TEM), electrophoresis, and additionally immunogenicity of prepared antigens. The results presented a high recovery of HA activity in concentrated and purified virus with the removal of ovalbumin and the typical morphology based on TEM. Sepharose CL-4B was determined as the best media among all used resins to purify the virus. Prepared formulations as vaccines demonstrated positive hemagglutinin inhibition for 6 months and stability for 2 years. Strong evidence from organized studies supports the effectiveness of this method in concentrating and purifying intact NDV, which could be valuable in vaccine research, antiserum preparation, or even as an alternative oncotic agent to traditional methods. Despite further studies being conducted, this method can be utilized particularly on a semi-industrial scale to produce various vaccine components.

Rapid adaptive evolution of avian leukosis virus subgroup J in response to biotechnologically induced host resistance.

Genetic editing of the germline using CRISPR/Cas9 technology has made it possible to alter livestock traits, including the creation of resistance to viral diseases. However, virus adaptability could present a major obstacle in this effort. Recently, chickens resistant to avian leukosis virus subgroup J (ALV-J) were developed by deleting a single amino acid, W38, within the ALV-J receptor NHE1 using CRISPR/Cas9 genome editing. This resistance was confirmed both in vitro and in vivo. In vitro resistance of W38-/- chicken embryonic fibroblasts to all tested ALV-J strains was shown. To investigate the capacity of ALV-J for further adaptation, we used a retrovirus reporter-based assay to select adapted ALV-J variants. We assumed that adaptive mutations overcoming the cellular resistance would occur within the envelope protein. In accordance with this assumption, we isolated and sequenced numerous adapted virus variants and found within their envelope genes eight independent single nucleotide substitutions. To confirm the adaptive capacity of these substitutions, we introduced them into the original retrovirus reporter. All eight variants replicated effectively in W38-/- chicken embryonic fibroblasts in vitro while in vivo, W38-/- chickens were sensitive to tumor induction by two of the variants. Importantly, receptor alleles with more extensive modifications have remained resistant to the virus. These results demonstrate an important strategy in livestock genome engineering towards antivirus resistance and illustrate that cellular resistance induced by minor receptor modifications can be overcome by adapted virus variants. We conclude that more complex editing will be necessary to attain robust resistance.

Region-selective and site-specific glycation of influenza proteins surrounding the viral envelope membrane.

Analysis of protein modifications is critical for quality control of therapeutic biologics. However, the identification and quantification of naturally occurring glycation of membrane proteins by mass spectrometry remain technically challenging. We used highly sensitive LC MS/MS analyses combined with multiple enzyme digestions to determine low abundance early-stage lysine glycation products of influenza vaccines derived from embryonated chicken eggs and cultured cells. Straightforward sequencing was enhanced by MS/MS fragmentation of small peptides. As a result, we determined a widespread distribution of lysine modifications attributed by the region-selectivity and site-specificity of glycation toward influenza matrix 1, hemagglutinin and neuraminidase. Topological analysis provides insights into the site-specific lysine glycation, localizing in the distinct structural regions of proteins surrounding the viral envelope membrane. Our finding highlights the proteome-wide discovery of lysine glycation of influenza membrane proteins and potential effects on the structural assembly, stability, receptor binding and enzyme activity, demonstrating that the impacts of accumulated glycation on the quality of products can be directly monitored by mass spectrometry-based structural proteomics analyses.

Establishment and Characterization of a Chicken Myoblast Cell Line.

Skeletal muscle, which is predominantly constituted by multinucleated muscle fibers, plays a pivotal role in sustaining bodily movements and energy metabolism. Myoblasts, which serve as precursor cells for differentiation and fusion into muscle fibers, are of critical importance in the exploration of the functional genes associated with embryonic muscle development. However, the in vitro proliferation of primary myoblasts is inherently constrained. In this study, we achieved a significant breakthrough by successfully establishing a chicken myoblast cell line through the introduction of the exogenous chicken telomerase reverse transcriptase (chTERT) gene, followed by rigorous G418-mediated pressure screening. This newly developed cell line, which was designated as chTERT-myoblasts, closely resembled primary myoblasts in terms of morphology and exhibited remarkable stability in culture for at least 20 generations of population doublings without undergoing malignant transformation. In addition, we conducted an exhaustive analysis that encompassed cellular proliferation, differentiation, and transfection characteristics. Our findings revealed that the chTERT-myoblasts had the ability to proliferate, differentiate, and transfect after multiple rounds of population doublings. This achievement not only furnished a valuable source of homogeneous avian cell material for investigating embryonic muscle development, but also provided valuable insights and methodologies for establishing primary cell lines.

Embryonic thermal challenge is associated with increased stressor resiliency later in life: Molecular and morphological mechanisms in the small intestine.

Developing chick embryos that are subjected to increased incubation temperature are more stressor-resilient later in life, but the underlying process is poorly understood. The potential mechanism may involve changes in small intestine function. In this study, we determined behavioral, morphological, and molecular effects of increased embryonic incubation temperatures and post-hatch heat challenge in order to understand how embryonic heat conditioning (EHC) affects gut function. At 4 days post-hatch, duodenum, jejunum, and ileum samples were collected at 0, 2, and 12 h relative to the start of heat challenge. In EHC chicks, we found that markers of heat and oxidative stress were generally lower while those of nutrient transport and antioxidants were higher. Temporally, gene expression changes in response to the heat challenge were similar in control and EHC chicks for markers of heat and oxidative stress. Crypt depth was greater in control than EHC chicks at 2 h post-challenge, and the villus height to crypt depth ratio increased from 2 to 12 h in both control and EHC chicks. Collectively, these results suggest that EHC chicks might be more energetically efficient at coping with thermal challenge, preferentially allocating nutrients to other tissues while protecting the mucosal layer from oxidative damage. These results provide targets for future studies aimed at understanding the molecular mechanisms underlying effects of embryonic heat exposure on intestinal function and stressor resiliency later in life.

Bibliometric study of the application of the chicken embryo chorioallantoic membrane model in cancer research: the top 100 most cited articles.

The chicken embryo chorioallantoic membrane (CAM) model has played a crucial role in various aspects of cancer research. The purpose of this study is to help researchers clarify the research direction and prospects of the CAM model. A bibliometric analysis was conducted on the top 100 most cited articles on use of the CAM model in tumour research, retrieved from the Web of Science Core Collection database. Tools such as Bibliometrix, VOSviewer, CiteSpace and Excel were utilized for the visualization network analysis. The 100 articles analysed were mainly from the USA, China and European countries such as Germany and France. Tumour research involving CAM model experiments demonstrated reliability and scientific rigor (average citation count = 156.2). The analysis of keywords, topics and subject areas revealed that the applications of this model ranged from the biological characteristics of tumours to molecular mechanisms and signaling pathways, to recent developments in nanotechnology and clinical applications. Additionally, nude mouse experiments have been more frequently performed in recent years. We conclude that the CAM model is efficient, simple and cost-effective, and has irreplaceable value in various aspects of cancer research. In the future, the CAM model can further contribute to nanotechnology research.

A pre-vertebrate endodermal origin of calcitonin-producing neuroendocrine cells.

Vertebrate calcitonin-producing cells (C-cells) are neuroendocrine cells that secrete the small peptide hormone calcitonin in response to elevated blood calcium levels. Whereas mouse C-cells reside within the thyroid gland and derive from pharyngeal endoderm, avian C-cells are located within ultimobranchial glands and have been reported to derive from the neural crest. We use a comparative cell lineage tracing approach in a range of vertebrate model systems to resolve the ancestral embryonic origin of vertebrate C-cells. We find, contrary to previous studies, that chick C-cells derive from pharyngeal endoderm, with neural crest-derived cells instead contributing to connective tissue intimately associated with C-cells in the ultimobranchial gland. This endodermal origin of C-cells is conserved in a ray-finned bony fish (zebrafish) and a cartilaginous fish (the little skate, Leucoraja erinacea). Furthermore, we discover putative C-cell homologs within the endodermally-derived pharyngeal epithelium of the ascidian Ciona intestinalis and the amphioxus Branchiostoma lanceolatum, two invertebrate chordates that lack neural crest cells. Our findings point to a conserved endodermal origin of C-cells across vertebrates and to a pre-vertebrate origin of this cell type along the chordate stem.

Synergistic activity of an RNA polymerase PA-PB1 interaction inhibitor with oseltamivir against human and avian influenza viruses in cell culture and in ovo.

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections.

A time series transcriptome profiling of host cell responses to Newcastle disease virus infection.

Newcastle disease virus (NDV), an avian paramyxovirus, causes major economic losses in the poultry industry worldwide. NDV strains are classified as avirulent, moderately virulent, or virulent according to the severity of the disease they cause. In order to gain a deeper understanding of the molecular mechanisms of virus-host interactions, we conducted Illumina HiSeq-based RNA-Seq analysis on chicken embryo fibroblast (DF1) cells during the first 24 hours of infection with NDV strain Komarov. Comparative analysis of uninfected DF1 cells versus NDV-infected DF1 cells at 6, 12, and 24 h postinfection identified 462, 459, and 410 differentially expressed genes, respectively. The findings revealed an increase in the expression of genes linked to the MAPK signalling pathway in the initial stages of NDV infection. This overexpression potentially aids viral multiplication while hindering pathogen detection and subsequent immune responses from the host. Our findings provide initial insights into the early responses of DF1 cells to NDV infection.

Arginyltransferase 1 (ATE1)-mediated proteasomal degradation of viral haemagglutinin protein: a unique host defence mechanism.

The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.