P4HA3) in adipocytes and adiposetissue and to explore its effect on obesity and type 2 diabetes mellitus (T2DM).We initially demonstrated that P4HA3 was significantlyupregulated in the subcutaneous adipose tissue of obesity and T2DM patients, andits functional roles in adipocyte differentiation and insulin resistance wereinvestigated using in vitro and in vivomodels. The knockdown of P4HA3 inhibited adipocytedifferentiation and improved insulin resistance in 3T3-L1 cells. In C57BL/6Jdb/db mice fed with a high fat diet (HFD), silencing P4HA3significantly decreased fasting blood glucose and triglycerides (TG) levels,with concomitant decrease of body weight and adipose tissue weight. Furtheranalysis showed that P4HA3 knockdown was correlated with theaugmented IRS-1/PI3K/Akt/FoxO1 signaling pathway in the adipose and hepatictissues of obese mice, which could improve hepatic glucose homeostasis andsteatosis of mice. Together, our study suggested that the dysregulation ofP4HA3 may contribute to the development of obesity andT2DM.The aims of the present study were to evaluate the expression of prolyl4-hydroxylase subunit alpha 3 ( Type 2 diabetes mellitus (T2DM) is one of the most prevalent metabolic disorders,which is characterized by insulin resistance . ObesityAs the main component of adipose tissue, adipocytes are considered to be an importantlink between obesity and T2DM development due to their secretory function . Many cyP4HA3 gene is highly expressed in breast cancer tissue andelevated P4HA3 expression is correlated with poor survival outcomes(Prolyl 4-hydroxylase (P4H) is a key enzyme in collagen biosynthesis, which containsthree catalytic subunits  . Recentloutcomes. HoweverP4HA3 in adipocytes and adipose tissue and exploredits effect on obesity and T2DM.In this study, we analyzed the transcriptomic profiles of adipocytes from leanindividuals, non-diabetic obese individuals, and obese individuals with T2DM from apreviously published microarray dataset (GSE133099). Then, we evaluated theexpression of 2 in Chinese adults wasdefined as obesity. This study was approved by the Institutional Ethics ReviewBoard of the First Affiliated Hospital of Bengbu Medical College.Subcutaneous adipose tissue samples were collected from 30 lean patients, 30 obese non-diabetics patients  and30 obese with T2DM patients  at the First AffiliatedHospital of Bengbu Medical College between June 2019 and July 2020. Thespecimens were immediately frozen with liquid nitrogen and stored in a −80°Cfreezer. The diagnosis of obesity was based on the China National Nutrition andHealth Survey (CNNHS) data: a BMI of ≥28 kg/mC57BL/6J db/db mice are mice in “C57 black 6 genetic background” with db/dbmutation (genetic mutation with defective leptin receptor), which developobesity and T2DM. C57BL/6J db/db female mice (2 months old) were purchased fromthe Animal Research Center of Nanjing University (China) and kept in apathogen-free facility and maintained under a 12-h light/dark cycle at 22°C.Mice were fed with a high-fat diet (HFD) . At the 10th week of feeding, mice wererandomly divided into si-NC group and si-P4HA3 group. sh-P4HA3 group mice wereintraperitoneally injected with adeno-associated virus carrying P4HA3 shRNA(AAV-shRNA-P4HA3), while sh-NC group mice were injected with AAV-scrambledshRNA. At the 20th week of feeding, the mice were sacrificed after 4-h fasting.Animal care and experimental procedures were approved by the Ethics Committee inAnimal Experimentation of the First Affiliated Hospital of Bengbu MedicalCollege (China).12 vg/mL) and injections wereadministered intraperitoneally using an insulin syringe. Primers binding withinthe AAV inverse terminal repeats (ITRs) were used to measure the virus titerwith quantitative polymerase chain reaction (qPCR) as in a previous study (AAV9 virus containing shRNA targeting P4HA3 was produced by SunBio (China), withscramble non-targeting shRNA used as control. For virus injection, mice wereanesthetized with isoflurane (1-4%), and placed in a prone position. The viruswas diluted in sterile PBS (1×10us study .4 cells/well) were seeded into 6-wellplates and cultured at 37°C in a humidified incubator with 5% CO2.Then, the cells were transfected with 100 nM siRNA targeting P4HA3  using Lipofectamine® 3000  according to the manufacturer's protocol.The cells transfected with scramble siRNA (si-NC) were used as control.The 3T3-L1 adipocytes  or insulin in mice that had been fasted for 6 h. Bloodglucose levels were measured at 0, 20, 40, 80, and 120 min using an Infinityglucose meter .Mice were weighed on an electronic scale every 1 week. Body composition wasdetermined by time domain-nuclear magnetic resonance (TD-NMR) on a MinispecAnalyst AD lean/fat analyzer .g for 15 min at 4°C. The remaining cellswere incubated in erythrocyte lysis buffer for 10 min at room temperature toeliminate red blood cells. Cell debris was removed by filtering the cellsuspension through a 70-μm nylon filter. After centrifugation, the pelletedpreadipocytes were plated in basal medium consisting of DMEM/F-12 supplemented with 10% fetal calf serum (FCS) and incubated for 16-18 h. Afterincubation, cells were detached by trypsin and counted. Cells were seeded in a6-well plate at a density of 1×105 cells per cm2 andcultured in complete medium until 70% confluence. To initiate differentiation,the culture medium was replaced with 2 mL MDI induction medium per well . Onday 3 of differentiation, MDI induction medium was replaced with 2 mL insulinmedium (DMEM containing 10 µg/mL insulin). On Day 6, insulin medium was replacedwith fresh DMEM. On day 10, fully differentiated adipocyte-like cells wereobtained for subsequent analysis.Preadipocytes from human abdominal subcutaneous adipose tissue (SAT) wereisolated and cultured following standard protocols. In brief, to obtain stromalcells, SAT was digested with collagenase and separated from mature adipocytes bycentrifugation at 500 5 cells/well. After 24 h, the culture medium wasreplaced with 3 mL MDI induction medium per well. The medium was changed every 2days. On Day 7, the differentiated adipocyte-like cells were subject to furtheranalysis.For 3T3-L1 adipocyte differentiation, cells were seeded in a 6-well plate at adensity of 5×10Mature adipocytes were determined by Oil Red O (ORO) staining. After washing withPBS, the cells were fixed with ice-cold acetone for 30 min. Fixed cells werewashed with PBS and stained with 30% ORO in isopropanol for 60 min to visualizeintracellular lipid deposits. For quantification, ORO-stained particles wereeluted with 100% isopropanol and analyzed using Thermo Scientific VarioskanFlash (USA) for spectrophotometry readings at 514 nm.Radioimmunoprecipitation assay buffer (RIPA)  was used to extractproteins from cultured cells and the concentration was determined using abicinchoninic acid kit . Total protein (10 µg) wasresolved by 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE). The proteins were transferred to polyvinylidene difluoride (PVDF)membranes. Non-fat milk (10%) in PBS buffer (90%) was used to block themembranes at room temperature for 1 h. Then, the membranes were incubated withprimary antibody at 4°C overnight and secondary antibody at room temperature for2 h. The immunoreactive bands were developed using an enhanced chemiluminescencekit  and photographed on a gel imager system . The densitometry analysis was performed with ImageJ software .The antibodies used in this study were as follows : GAPDH; C/EBP-α ; PPAR-γ ;C/EBP-α ; IRS ; p-IRS ;AKT ; p-AKT ; FoxO1 ; p-FoxO1; PI3K ; p-PI3K ; β-actin. In addition, HRP-linked secondary antibody was used .-Δ ΔCT method using GAPDH as the referencegene. All quantitative qPCR reactions were performed in triplicate. The primersused for real-time qPCR are shown in Supplementary Table S1.Trizol reagent  was used to extract total RNAaccording to the instructions. The purified total RNA was dissolved in DEPCwater and its concentration was measured with NanoDorp. One microgram of totalRNA was used for reverse-transcription using the cDNA PrimeScript™ RT reagentKit . Quantitative expression analysis was performed using SYBRpremix EX TAQ II kit  on the Roche LightCycler 480 qPCR system. The PCR cycling condition used was: 95°C for 5 min, 40 cyclesof 95°C for 30 s, 60°C for 30 s, and 72°C for 60 s. Relative gene expressionlevel was calculated by 2Glucose uptake in differentiated 3T3-L1 adipocytes was assessed by analysis of2--DOG uptake. The cells were incubated with 100 nM insulin for 30min prior to glucose uptake assay. Assays were performed in Krebs-Ringerphosphate buffer supplemented with 0.2% bovine serum albumin (downstream media).Briefly, cells were washed in downstream media and incubated in downstream mediacontaining 5 mM glucose for 2 h. After washing with downstream media withoutglucose, 2--DOG (0.125 μCi/well) was added to cells for 10 min.Cells were then immediately washed 3 times with chilled PBS and lysed in PBSwith 0.1% Triton-X100. An aliquot (50 μL) from the lysate was taken for proteinmeasurement and the remainder was transferred to scintillation fluid forradioactive counting. The recorded counts per minute values were normalized tothe total cellular protein level for each sample.g for 10 minat 4°C, a 30-μL aliquot was taken from each sample for measurement. TG weremeasured using Triglyceride Quantification Colorimetric/Fluorometric Kit , according to the manufacturer's instructions.Lipids were extracted and dissolved in chloroform. An aliquot (30 μL) was removedfrom each sample for quantification. Cultured cells were directly lysed in 1%Triton X-100 in PBS. After centrifugation at 500 H&E staining was performed using H&E Stain Kit .Deparaffinized/hydrated sections were placed in adequate Mayer's hematoxylin(Lillie's Modification) to completely cover the tissue section and incubated for5 min. The section was rinsed twice with distilled water to remove excess stain.Then, adequate Bluing Reagent was applied to completely cover the tissue sectionand was then incubated for 30 s. After washing with distilled water, the sectionwas dehydrated in absolute alcohol, followed by staining with Eosin Y Solutionto completely cover the tissue for 2-3 min. The section was rinsed three timesusing absolute ethanol and then mounted on a slide. The images were collectedunder an inverse microscope  at 200× magnification.2FC (fold change)| ≥1.The published microarray dataset (GSE133099) was retrieved from Gene ExpressionOmnibus (GEO). Differentially expressed genes were identified by the edgeRpackage in R software. Deferentially expressed (DE) genes were identified basedon adjusted P value (<0.05) and the |logt-test and comparisons among multiplegroups were analyzed using one-way analysis of variance (ANOVA) with Tukey'spost hoc test for pairwise comparison. P value of <0.05was considered statistically significant and all tests were two-sided.All statistical analyses were performed with R (version 3.6.3) and SPSS v26.0. The statistical difference between two groups was comparedusing unpaired Student's P4HA3 in adipocytes of obese individuals was much higherthan that of lean individuals, which was further elevated in adipocytes of theobese individuals with T2DM.Based on the published microarray dataset (GSE133099), we analyzed the expressionprofiles in mature adipocytes. In non-diabetic individuals, we identified 795significantly differentially expressed (DE) genes between obese and leansubjects. In obese individuals, we identified 134 DE genes between diabetic andnon-diabetic individuals. A total of 37 DE genes were shared between these twocomparison groups . NotablyP4HA3 in obese and T2DM, wecollected subcutaneous fat tissue samples from 30 obese patients, 30 obese withT2DM patients, and 30 lean people. As shown in To validate the upregulation of P4HA3 from obese patients was higher than lean people, andobese patients with T2DM showed an even higher level of P4HA3than obese non-diabetic patients  level and impaired the glucose uptake in thedifferentiated adipocytes  were fed with HFD for 10 weeks to induceobesity and diabetes. The mice were then randomly divided into sh-P4HA3 groupand sh-NC (negative control) group (n=6/group). The mice were intraperitoneallyinjected with AAV9 vectors encoding an AAV-shRNA-P4HA3 sequence (shRNA targetingP4HA3) or AAV-scramble shRNA sequence. The mice in thesh-P4HA3 group did not show body weight increase after AAV injection . In addition, after AAV9 vectors wasadministered (24 h after P4HA3 knockdown), food intake in miceof the sh-P4HA3 group was significantly reduced compared with the sh-NC group, which suggested that silencing P4HA3was beneficial for reducing food intake in mice. We also verified the knockdownof P4HA3 in liver, epidermal adipose tissue, inguinal adiposetissue, skeletal muscle, islet, and hypothalamus in mice injected withAAV-shRNA-P4HA3 . To examine the metabolic effect ofP4HA3 knockdown, FBG, insulin, free fatty acid (FFA), andTG levels were measured, and the results showed that P4HA3knockdown significantly reduced FGB, insulin, FFA, and TG levels in blood after6-h fasting -6, and IL-1 weresignificantly downregulated in the sh-P4HA3 group and the glycogenolytic gene (PYGL) were significantlydownregulated in the sh-P4HA3 group  remodeling and worse prognosis in breastcancer (P4HA3 in obesity and its association with T2DM are not wellcharacterized. Our study revealed the upregulation of P4HA3 inmature adipocytes from obese patients with T2DM. Further functional studydemonstrated that the knockdown of P4HA3 could reduce the mass ofepididymal and inguinal adipose tissues and decrease the levels of fasting bloodglucose, insulin, free fatty acid, and triglycerides in the obese mouse model. Theseresults indicated that P4HA3 upregulation may account for thedysregulation of adipocyte and insulin response in obesity combined with T2DM.Adipose tissue is a heterogeneous tissue responsible for systemic energy homeostasis(It was reported that the dysregulation of P4H is associated with tumor initiation andprogression, and P4H upregulation could augment the invasiveness potential of cancercells and enhance the metastasis to lymph nodes and lungs . High extcancer . Howevereostasis. The suseostasis. The exceostasis.P4HA3 silencing could tune down the expression of PPAR-γ andCEBP-α. Our results also demonstrated that P4HA3 knockdown couldsignificantly decrease lipid accumulation and impair adipocyte differentiation in3T3-L1 cells, suggesting that P4HA3 was implicated in theregulation of adipocyte differentiation.Through regulating temperature and calorie consumption, adipocytes play a pivotalrole in maintaining homeostasis and energy balance. However, imbalanced dietarypatterns or lack of exercise can lead to the fatty acid accumulation in adipocytes, which lin vivo model, our work showed that P4HA3knockdown significantly affected systemic metabolism, including diet-inducedobesity, insulin resistance, and liver steatosis. These changes can be attributed tothe reduced food intake and enhanced glucose tolerance and homeostasis regulation.At the molecular level, P4HA3 knockdown not only tunes downadipocyte differentiation, but it also downregulates the expression of inflammatoryfactors. Increased levels of inflammatory cytokines such as IL-1, IL-6, MCP-1, andleptin, and a decreased level of adiponectin are proposed to promote the developmentof insulin resistance and T2DM (P3HA4 silencing, which may be relatedto the improvement of insulin resistance induced by P3HA4silencing. It is also important to investigate the change of leptin and adiponectinin the future to get a full picture of how P3HA4 silencingorchestrates adipocyte cytokine profiles.In the and T2DM . IL-1,Mand T2DM . We obseP4HA3 can suppress the growth andmetastasis of pituitary adenoma via blocking PI3K-Akt pathway (P3HA3knockdown can improve insulin resistance by activating IRS-1/PI3K/Akt/FoxO1signaling pathway.IRS-1/PI3K/AKT signaling pathway is crucial in cell proliferation, differentiation,and adaptation, which also modulate the signal transduction initiated by insulinreceptor . FoxO1,  pathway . ConsistPYGL, PCK1, andG6PC in the liver of P4HA3-silenced obesemice. Since glycogenolysis and gluconeogenesis are primary drivers of hepaticglucose level, these data suggest that P4HA3 could regulate hepaticglucose level by modulating genes involved in gluconeogenesis.Liver is a central organ for carbohydrate metabolism via glycogenolysis andgluconeogenesis during fasting. The metabolic capacity is manifested as theexpression level and activity of rate-limiting enzymes in glycogenolysis andgluconeogenesis . The incin vivo P4HA3 silencing by intraperitonealinjection with AAV counteracts HFD-induced obesity and improves insulin resistance,we could not conclude whether the effects come directly from its influence inadipose tissues or liver tissues. Since we observed the downregulation ofP4HA3 expression in multiple tissues including liver andadipose tissues after AAV injection, we speculated that the protective effects mayresult from a systemic influence on multiple organs. Future work using tissuespecific knockout mice will be needed to pinpoint the key tissues implicated in theP4HA3-dependent regulation. In addition, future studies willalso need to fully investigate the expression and functional role ofP4HA3 between obese diabetic mice and lean mice.However, although the P4HA3 expression wasincreased in obese individuals with T2DM. We also provided evidence thatP4HA3 knockdown could ameliorate obesity and insulin resistancein C57BL/6J db/db mice with HFD feeding. P4HA3 knockdown could alsosuppress adipocyte differentiation, alleviate the inflammatory cytokine expression,and improve the regulation of hepatic glucose homeostasis and steatosis. Our datasuggested that targeting P4HA3 may serve as a potential therapeuticstrategy to ameliorate obesity-associated diabetes. Future work will be needed todelineate the mechanism by which P4HA3 becomes upregulated in obeseand diabetic individuals.In summary, this study demonstrated that


Previous research has indicated that various metabolites belonging to phenolic acids (PAs), produced by gut microflora through the breakdown of polyphenols, help in promoting bone development and protecting bone from degeneration. Results have also suggested that G-protein-coupled receptor 109A (GPR109A) functions as a receptor for those specific PAs such as hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA). Indeed, HA has a molecular structural similarity with nicotinic acid (niacin) which has been shown previously to bind to GPR109A receptor and to mediate antilipolytic effects; however, the binding pocket and the structural nature of the interaction remain to be recognized. In the present study, we employed a computational strategy to elucidate the molecular structural determinants of HA binding to GPR109A and GPR109B homology models in understanding the regulation of osteoclastogenesis. Based on the docking and molecular dynamics simulation studies, HA binds to GPR109A similarly to niacin. Specifically, the transmembrane helices 3, 4 and 6  and Extracellular loop 1 and 2 (ECL1 and ECL2) residues of GRP109A; R111 (TMH3), K166 (TMH4), ECL2 residues; S178 and S179, and R251 (TMH6), and residues of GPR109B; Y87, Y86, S91 (ECL1) and C177 (ECL2) contribute for HA binding. Simulations and Molecular Mechanics Poisson-Boltzmann solvent accessible area (MM-PBSA) calculations reveal that HA has higher affinity for GPR109A than for GPR109B. Additionally, in silico mutation analysis of key residues have disrupted the binding and HA exited out from the GPR109A protein. Furthermore, measurements of time-resolved circular dichroism spectra revealed that there are no major conformational changes in the protein secondary structure on HA binding. Taken together, our findings suggest a mechanism of interaction of HA with both GPR109A and GPR109B receptors.  GPCRs are cell surface proteins involved in mediating and regulating wide range of biological processes including immune system, odor, vision, homeostasis, etc. ,2. They l trials ,7. Humanl trials . As of 2tistics) ,8. OwingInterestingly, the G-Protein Coupled Receptor (GPCR)—GPR109A, a class A GPCR,  is expressed in variety of cells and tissue types, more robustly in osteoclastic precursor macrophages ,12. Niac−/−) in mice, conducted by Chen et al., have revealed significantly higher bone mass and strength in tibia and spine of mice (weaned 4-week-old and 6-month-old) using densitometric, bone histologic, and molecular signaling analytic methods [−/− mice. Additionally, in GPR109A−/− mice compared with their respective untreated control mice, HA considerably inhibited bone resorption and increased bone mass in wild type mice but had no additional effects on GPR109A−/− mice [Although, importance in bone formation and resorption has mostly been given to micronutrients such as calcium, vitamin D, phosphate and macronutrients comprising fats and proteins , recent  methods . It was −/− mice . −/−) knockout studies, site-directed mutagenesis and structural similarity, it can be predicted that the actions of PAs on bone cells are mediated through a mechanism involving specific activation of GPR109A receptor. Other studies involving ligand binding assay (35S-GTPγS) using membrane prepared from mouse fetal calvarial cells have shown that HA stimulates 35S-GTPγS binding to membranes transfected with GPR109A but not to GPR109B. It has been hypothesized that HA mediates it action on bone cell through binding to GPR109A [Studies on the structural determinants of GPR109A receptor binding to niacin have been investigated using site-directed mutagenesis by Tunaru et al., revealed putative ligand binding residues in GPR109A receptor, and did not show any interaction of niacin to its close homolog with 95% amino acid sequence identity, GPR109B  . Based o GPR109A . Further GPR109A . In the present study, we implemented a computational pipeline involving molecular docking studies, molecular dynamics simulations and Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) calculations  combinedpose-0) is the AutoDock predicted top ranked pose with largest population size for both GPR109A and GPR109B structures, whereas the other run (pose-1) is the conformation from second largest AutoDock scored cluster. For easy interpretation in the naming pattern, pose-A0 and pose-A1 refers to GPR109A-HA complex whereas pose-B0 and pose-B1 refers to GPR109B-HA complex. All these complexes were subjected to 200 ns MD simulation followed by trajectory analysis; RMSD based structural clustering of GPR109A/B-HA interaction , and −14.96 (±4.87) kcal/mol, respectively (numbers in the parenthesis are standard deviations) , and hydrophobic interactions with F255 (TMH6) and F276 (TMH7) during MD simulation. However, in pose-A1, HA is stabilized by the H-bond formation with R111, R251 and K166. Compared to pose-A0, the H-bond occupancy for HA is less in pose-A1 and has weaker binding energy as demonstrated by MM/PBSA energy. GPR109A-HA complexes: The calculated binding energies of GPR109A-HA complexes; iations) . The negpose-B0 and pose-B1 the calculated binding energies are −13.52 (±7.07) and −1.48 (±5.78) kcal/mol, respectively (numbers in the parenthesis are standard deviations)  . In bothAccording to calculated binding energy values, all the complexes of GPR109B-HA are stable. Compared to GPR109A-HA complexes, GPR109B-HA complexes are energetically much less favorable. This is well supported by the finding of the weak binding of HA with GRP109B as observed by Chen et al., . CompariK166 (TMH4), S178 (ECL2) and S179 (ECL2) while residues in TMH2  and ECL1 (S91) play a major role in binding HA to GPR109B. To understand the importance of the specific residues of GPR109A and GPR109B for binding of HA, we generated two in silico chimeras of GPR109A and GPR109B similar to Tunaru et al., study [The results from H-bond analysis and MM/PBSA calculation have shown that HA has higher binding affinity with GPR109A than that of GPR109B. The major residues that are responsible for binding of HA to GPR109A are present on TMH4 and ECL2—., study . Chimera., study . pose-A0, carboxylic group of HA has H-bond interaction with R111 in chimera 3A4B. Absence of residue—S178 in the ECL2 region of chimera 3A4B might have paved a path for HA to move deep inside down the transmembrane helix to interact with R111. The cluster representative structure from MD simulation shows carboxyl group of HA interacting with the side chain of basic residue K166  and −23.54 kcal/mol (±7.17), respectively . MM/PBSAdue K166 E. pose-A0. Representative structure forms the most dominant cluster for chimera 3B4A shows that HA occupies a binding site surrounded by TMH3 and TMH4 with hydrophobic interaction mainly from F255, H189, F186 and L258  value greater than 100 µM for acifran [50 value ~2 µM) and weak binding with niacin . HA, which is quite larger than niacin in size, but similar to acifran (in terms of size and structure), shows strong interaction with chimera 3B4A similar to that of acifran. This indicates that residues in ECL2  and TMH3/TMH4/TMH6  regions are the major contributors in strong binding of HA to GPR109A. In a mutagenesis study by Tunaru et al., for similar chimeric structures, it was shown that chimera 3A4B was inactive with niacin and had very low binding of EC acifran . HoweverFree energy landscape (FEL) analysis was performed using cpptraj module of AMBER16  to exploTime resolved circular dichroism spectra revealed that HA interaction with GPR109A and GPR109B led to small conformational changes in the secondary structure content  of the protein . A smallpose-A0 and pose-A1 and then subjected the identified critical residues to Alanine mutation. The mutated complex (GPR109A-HA) was energy minimized followed by 50 ns MD simulations for further analysis. Since GPR109A-HA complexes are energetically more stable over GPR109B-HA, we considered GPR109A-HA complexes for further in silico mutation analysis to compare the importance of the residues of GPR109A that are interacting with HA for stable binding. Through interaction analysis of GPR109A with HA in MD simulations, we concluded that residues K166 (TMH4), S179 (ECL2), S178 (ECL2) and positively charged residues R111 (TMH3) and R251 (TMH6) are important for HA binding to GPR109A. Additionally, aromatic residues F255 (TMH6) and F276 (TMH7) are also concluded as important residues as they are found to be in the surrounding of HA during MD simulations. Based on these observations, we took the representative structures of the most dominant cluster from MD trajectory of complexes K166-S178-S179 Alanine triple mutant: According to H-bond analysis of pose-A0 complex, residues; K166 (TMH4), ECL2 residues; S178 and S179 are involved in H-bonding with HA during MD simulation. So based on these results, we generated Ala triple mutants for these residues taking representative structure form the most dominant cluster as the starting structure. Visualization of 50 ns MD trajectory and H-bond analysis both show that due to the Ala mutation HA moved away from TMH4 residue K166A, and ECL2 residues: S178A and S179A during MD simulation. Instead, HA’s carboxyl group made a H-bond with N-terminal residue N17 with occupancy of 20% of the whole MD trajectory. Aromatic ring was surrounded by TMH6 hydrophobic residues; I254, F255, and L258, while in the wild type it is stacked between residues; F255 (TMH6) and F276 (TMH7) (6 (TMH7) . Thus, aR111-R251 Alanine double mutant: As HA is energetically stable and makes H-bonds with R111 and R251 in pose-A1, we generated a double Ala mutant. In the alanine double mutant, HA moved towards ECL2 residues; S178 and S179 during MD simulation and made H-bonds with these residues with an occupancy time of 12% and 19 %, respectively. The HA aromatic ring was surrounded by TMH6 hydrophobic residues: I254, F255, L258, and TMH7 residue F276. Compared to K166A-S178A-S179A triple mutant where HA was unable to reach the arginine binding site, in this specific double mutant, HA was unable to stay near the R111A (TMH3) and R251A (TMH6) due to the absence of any salt bridges. As a result, HA moved towards the ECL2 residues; S178 and S179  and F276 (TMH7) were present near the HA aromatic ring during MD simulations, thus, we considered analyzing the importance of these residues in HA binding to GPR109A. We took the cluster representative structure of two complexes, pose-A0 and pose-A1, to generate the alanine mutant complexes: F255A-F276A-A0 and F255A-F276A-A1 In pose-A0, HA makes H-bonds with K166 and ECL2 residues in the wildtype receptor; S178, and S179, and the aromatic ring is stacked between F255 (TMH6) and F276 (TMH7). The H-bond calculation for mutant complex, F255A-F276A-A0, shows that H-bonds of HA with K166, S178 and S179 that were present in wild type complex are also present in the mutant complex with an occupancy time of 17%, 62% and 47%, respectively. Due to alanine mutation of residues F255 (TMH6) and F276 (TMH7), the HA aromatic ring no longer remains in this position during MD simulation and instead shifts to a new site surrounded with ECL2 residues; F186, W188, and H189 , R251 (TMH6), and the aromatic ring is surrounded by hydrophobic residues F255 (TMH6) and F276 (TMH7). In contrast, according to H-bond analysis, HA makes interactions with R111 (TMH3), K166 (TMH4), and R251 (TMH6) with an occupancy time of 57%, 24%, and 52%, respectively, in the F255A-F276A-A1 mutant complex. The HA aromatic ring is surrounded with residues; I254, L258, Y269 throughout the MD simulation. Thus, according to both the mutant complexes (F255-F276-A0 and F255-F276-A1), the hydrophobic residues F255 (TMH6) and F276 (TMH7) in wild type GPR109A are essential to strengthen HA binding , and the aromatic ring is surrounded by F255 (TMH6) and F276 (TMH7). Visualization of MD trajectory shows that HA moves away from the initially bound position after 4 ns of the MD simulation run, and by the time the simulation reaches the time of 6 ns, HA completely moved away from the initial binding site and exited out from the GRP109A protein ( protein . This sphttps://pubchem.ncbi.nlm.nih.gov/compound/464). AutoDock 4.2 was used to dock HA against GPR109A/B structures and attain initial conformations of the protein-ligand complexes [The homology model structures for both GPR109A and GPR109B were taken from our previous study of niacin interaction with the GPR109A receptor . Brieflyomplexes . To explomplexes . Kollmanomplexes . As HA complexes . We applomplexes . Based o+ and 70 Cl- ions were added to GPR109A system and 57 K+ and 60 Cl− ions were added to GPR109B system to make up to equivalent of 150 mM salt concentration. During the simulation, Langevin Dynamics was used to maintain constant pressure (1 atm) and temperature (310 K). Periodic boundary settings with a flexible cell were maintained with the cutoff distance of 12 Å for non-bonded interaction and particle mesh Ewald (PME) method was used to treat long-range electrostatic interactions with the switching distance of 10 Å. The coordinates of each system were saved every 1 ps during the entire simulation run. Based on RMSD of GPR109A/B—HA complex throughout the MD simulation, trajectories were extracted and further subjected to cluster analysis (as performed in our earlier study for interaction of niacin with GPR109A) [To get the insights on the interaction and dynamics of GPR109A/B on HA, MD simulations was performed using NAMD starting with the AutoDock predicted protein-ligand conformations . VMD v1.GPR109A) . DBScan GPR109A) . RepreseGPR109A) , NAMD [2GPR109A) , VMD [28perature 0 K. Peripose-0 to explore the HA binding site similar to the experimental studies conducted by Tunaru et al. for niacin binding [Apart from the two AutoDock predicted models, we have generated two in silico chimeras  of GPR10 binding . For theSecondary structural changes in the proteins (GPR109A and GPR109B) after binding to HA was studies by CD spectropolarimeter . The recombinant protein (GPR109A and GPR109B) was purchased from MyBioSource . The lyophilized protein from 20 mM Tris-HCl, 0.15 M NaCl, 0.05% Brij-78 and 6% Treahlose was reconstituted in distilled water to a final concentration of 0.01 µM. Equimolar concentration of HA (dissolved in distilled water) was mixed with the protein. CD spectra were recorded at 37 °C between wavelengths of 190–260 nm in a quartz cuvette of path length 1 mm. Spectra were recorded for protein (only) and for the protein-ligand complex to determine the changes in the secondary structure content. All the analysis were performed using Jasco application software version 1.11.06 .Structural basis of HA binding to GPR109A based on computational analysis  clearly revealed that HA has stronger binding with GPR109A, while the binding of HA with GPR109B is comparatively weaker. Our computational results are in agreement with our experimental findings on HA interaction with GPR109A and GPR109B . Further


Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the  Culicoides biting midge and therefore the global and seasonal distribution of AHSV is dependent on climate conditions of this insect vector [African horse sickness (AHS) is a severe and fatal disease of equids that is caused by African horse sickness virus (AHSV). AHS has been classified as a notifiable OIE-listed disease, resulting in the implementation of significant constraints on international trade and increased disease surveillance . AHSV hat vector ,6.Orbivirus genus and part of the Reoviridae family, African horse sickness virus (AHSV) is a complex non-enveloped virus with a characteristic double-stranded (ds) RNA genome consisting of ten segments contained within an icosahedral core particle that is encapsidated by an outer protein layer [Belonging to the in layer ,8,9,10. in layer ,12. The in layer ,14,15.Orbivirus genus and their functional significance, if any, remains to be discovered and is the focus of this study. AHSV VP7 is highly conserved among all nine serotypes [Culicoides-derived insect cell line (KC cells) infected with AHSV [During replication, certain hallmarks of infection start to appear such as the formation of structures and protein inclusions within the cytoplasm. NS1 self-assembles into tubule structures of unknown function within the cytoplasm of AHSV-infected cells ,17. Theserotypes . To dateerotypes ,22,23. Tith AHSV . The finith AHSV . Anotherith AHSV ,26,27. MOnce assembled, the core particles are released from VIBs and join the host exocytosis pathway with the aid of NS3/NS3A. It is during this virus egress stage that the outer capsid proteins VP2 and VP5 are incorporated onto the core particle to form a mature virion which then exits the cell via further NS3/NS3A facilitation ,29,30,31Currently, the only method of protection against the virus is a polyvalent live-attenuated vaccine that confers broad protection against all nine AHSV serotypes. With the recent developments in reverse genetics technology, it has become possible to produce recombinant viruses containing defined mutations and/or markers in their viral genomes resulting in the rapid production of safe attenuated and “Differentiation of Infected from Vaccinated Animals” (DIVA)-compliant vaccines, respectively ,36,37. TAHS can cause up to 95% mortality in susceptible horses . The outSeveral viruses have been shown to cause neurodegeneration in humans due to protein aggregation in the brain ,48,49. FGiven that AHSV pathogenicity and disease is more severe than that of BTV , it is pIn this study, we set out to use this soluble version of AHSV VP7 to study the impact of VP7 crystalline particle formation on virus replication by using a plasmid-based reverse genetics system to generate recombinant AHSV that does not form VP7 crystalline particles. Using the backbones of virulent (AHSV5) and attenuated (AHSV4) serotypes, we replaced wild-type segment 7 with a gene encoding our soluble mutant VP7, and used these recombinant viruses to study the effect of crystalline particle formation on virus yield and release in mammalian and insect cells. The interaction of soluble VP7 with other AHSV proteins during the replication cycle was also examined. The results obtained here allowed us to further understand the role of AHSV VP7 on replication, and possibly cellular pathogenesis. This study sheds light on the potential role of viral protein inclusions in disease.®, Merck, Darmstadt, Germany), 5% foetal bovine serum  and 1% penicillin and streptomycin . Geneticin  was added every third passage at 1 mg/mL. Cells were grown at 37 °C in the presence of 5% CO2. KC cells (derived from Culicoides variipennis) were obtained from the Onderstepoort Veterinary Institute  and cultured at 28 °C in TC-100 insect medium  with nonessential amino acids supplemented with 10% FBS , 1% penicillin and streptomycin , and antifungals .BSR-T7/5 cells are a clone of baby hamster kidney-21 cells that constitutively express T7 polymerase, and were used with permission from Ulla Buchholz, Department of Clinical Virology, Federal Research Centre for Virus Diseases of Animals, Tubingen, Germany . The celA plasmid-based reverse genetics system described previously  was usedACACTTGGCACGCATTACGCGCTGTCATTTTTCAGCAGATGAATATGCAGCCTATTAATCCGCCGATTTTTCCACCGACTGAAAGGAATGAAATTGTTGCGTATCTATTAGTAGCTTCTTTAGCTGATGTGTATGCGGCTTTGAGACCAGATTTCGCGATGAATGGTGTTAATCCGATGCCAGGGCCGATTAACAGAGCTCT-3′). This dsDNA fragment was inserted into the pJAD-S7 backbone using the In-Fusion® HD Cloning kit  cloning strategy. The dsDNA fragment was designed to include 15 bp homologous ends to the VP7 sequence and inserted into the VP7 plasmid backbone linearized by inverse PCR using primers that contained the same 15 bp homologous ends in their 5′ regions by In-Fusion® reaction according to manufacturer’s instructions. The sequence of the resulting plasmid, pJAD-S7-276, was confirmed by Sanger sequencing.To construct reverse genetics plasmids that encode the mutant soluble VP7 (sVP7), the In-Fusion cloning strategy  was usedACACCTGGCACGCCCTACGCGCAGCCATCTTTCAACAGATGAATATGCAGCCAATTAATCCGCCAATTTTTCCACCAACAGAAAGAAATGAGATTGTTGCATATTTGCTAGTAGCTTCTTTGGCAGACGTGTATGCGGCTTTAAGACCAGACTTCGCGATGAATGGTGTTAATCCGATGCCAGGGCCGATCAACAGAGCTCT-3′) and inserted into the S7 pSMART-T7 transcription vector backbone using In-Fusion® HD Cloning kit as described above. The sequence of the resulting plasmid, pSMART-T7-sVP7, was confirmed by Sanger sequencing.To construct the AHSV5-sVP7 transcription plasmid, the pSMART-T7 transcription vector containing a cDNA copy of AHSV5 S7 (GenBank KM886350) was used as a template. A dsDNA fragment containing the mutations that encode for sVP7 (underlined in sequence) in the AHSV5 backbone was synthesised by GenScript  (5′-ATGCGTATGTTTCGC50/mL (50% tissue infective dose per millilitre) [Recombinant viruses were rescued by plasmid-based reverse genetics system as described previously ,68. Monolilitre)  and convw/v) paraformaldehyde/PBS for 30 min at room temperature (RT). After fixation, cover slips were incubated for 15 min at RT in 0.5% Triton X-100/PBS to permeabilize the cells. BSR cells were blocked in 5% blocking solution (5% milk powder in PBS) directly after fixation followed by incubation with primary antibodies for 1 h. The cells were washed three times with wash buffer (0.5% (v/v) Tween20 in PBS) and incubated for 1 h with appropriate secondary antibody. Cells were washed and stained with 10 μg/mL DAPI  in 1% blocking solution for 10 min at RT. Coverslips were mounted onto glass slides using Vectashield Mounting Medium . Slides were then viewed using a Zeiss LSM 880 Laser Scanning Confocal Microscope (LSCM) coupled to an Airyscan detector. Images were processed and analysed for colocalisation by using Zeiss ZEN 3.3 Blue edition software package .Immunofluorescence microscopy was performed as previously described . Cell moFor the identification of AHSV VP7 and NS2 proteins, anti-VP7 guinea pig  and anti50/mL (50% tissue culture infective dose per millilitre) and converted to pfu/mL by multiplying by 0.7 [t-test was applied to statistical differences amongst the wild-type and mutant groups for each time point separately. In all the experiments, p ≤ 0.05 was considered statistically significant with the following denotations used: * (p ≤ 0.05) and ** (p ≤ 0.01).Virus growth kinetics was carried out in BSR and KC cells. Cells were grown to ~80% confluency in 24-well plates and each well was infected with appropriate virus at a MOI of 0.1, in triplicate, and incubated at 37 °C (BSR) or 27 °C (KC cells). After 1 h, BSR cells were washed and media replaced with 0.7 mL complete media to remove unbound virus. Cells were incubated for a further 12, 24, 36, 48 or 72 h. At each time point, both released and cell-associated virus were harvested separately. The supernatant of each well was carefully collected (representing released virus fraction) and the remaining cells harvested in 0.7 mL clean media by scraping cells and homogenizing through a 22G needle several times (representing cell-associated virus fraction). Debris was removed by centrifugation at 2500 rpm for 10 min and virus fractions stored at 4 °C. Virus titre was determined by endpoint dilution in BSR cells for each triplicate fraction, i.e., cell-associated virus, released virus, or total virus. Briefly, cells were infected with 10-fold dilutions of each sample (6 repeats), plates were sealed, incubated for 72 h and wells were scored according to presence or absence of visible CPE observed with an inverted light microscope. Titres were expressed as TCIDg by 0.7 . All expTo understand the role of AHSV VP7 crystal formation during AHSV replication, we aimed to generate recombinant ‘synthetic’ AHSV that contains a modified segment 7 that encodes a soluble VP7 protein (sVP7), by reverse genetics. To also determine whether AHSV VP7 crystalline particles could play a role in virulence or cellular pathogenesis, this study set out to generate both recombinant AHSV4 (attenuated) and AHSV5 (virulent) viruses that do not form VP7 crystalline particles, i.e., rAHSV4-sVP7 and rAHSV5-sVP7, to compare their properties with their wild-type counterparts. Therefore, four recombinant viruses  were generated by reverse genetics technology.A plasmid-based reverse genetics system was used to recover wild-type and mutant viruses. Here, recombinant AHSV4 (rAHSV4), representing wild-type AHSV serotype 4 virus, was derived using segments 1–10 representing the live attenuated AHSV4 strain known as “LP” . This avirulent AHSV4 LP strain is used as a vaccine in South Africa and its origin has been described previously by Baltus Erasmus . RecombiThe AHSV4 LP strain is an attenuated virus; it is avirulent in horses and has reduced CPE in mammalian cells. Therefore, we included analysis of a highly virulent OIE reference strain of AHSV5 (HS 30/62), also known as “Fourie” or FR , to alloSuccessful virus rescue from transfection was confirmed by the presence of CPE, positive labelling of AHSV proteins and titration determination of passaged viruses. Furthermore, growth curves of rescued rAHSV and rAHSV5 and their respective wild-type reference strains AHSV4 (LP)  and AHSVThe detection of virus proteins following rescue and after multiple passage events confirmed the ability of the mutant rAHSV4-sVP7 and rAHSV5-sVP7 viruses to replicate, indicating that VP7 crystalline particle formation is not vital for virus fitness.As observed previously, when expressed alone, sVP7 has a homogenous intracellular distribution . TherefoThe small VP7 foci observed to be present for wild-type and mutant viruses in To compare the replication properties of wild-type and mutant rAHSV, next we examined the association of VP7 with VIBs. VIBs are virus factories which act as a scaffold for viral replication and assembly. VP7 foci have previously been shown to associate alongside VIBs . To compResults thus far indicated the presence of unique sVP7 tubule-like structures in rAHSV4-sVP7 LSCM data. To determine if the tubule-like distribution was due to association with characteristic AHSV NS1 tubules, dual labelling of VP7 and the tubule-forming NS1 protein was performed and analysed. Results showed that the tubule-like structures formed by sVP7 fully colocalised with the NS1 signal , indicatTaken together, results thus far show that soluble VP7 associates with VP2 and NS2 proteins similarly to wild-type VP7. The notable difference observed can be seen for the unique colocalisation and potential interaction of soluble VP7 with NS1 tubules.To determine the impact of VP7 solubility on AHSV replication, in vitro time trials were performed in mammalian cells to determine the replication kinetics of each virus, i.e., attenuated (rAHSV4 versus rAHSV4-sVP7) and virulent (rAHSV5 versus rAHSV5-sVP7). To obtain a clear picture of the virus replication kinetics, released versus cell-associated virus was determined, in triplicate, at 12 hourly intervals over 3 days for each virus. Virus titre means were plotted and represented on a logarithmic scale to illustrate growth curves. Total virus was reported from the sum of titres from released and cell-associated fractions and illustrated to scale to show proportional differences between each virus at each time point.Released virus titre was determined from collecting media separately from cell monolayers prior to cell lysis. Results showed that rAHSV4-sVP7 released significantly less virus throughout the 3-day period, culminating with a decreased yield of released progeny virus at 72 hpi A. Cell-aLooking at the overall wild-type rAHSV4 replication kinetics C, total Percentage of released progeny virus, calculated as percentage of released fraction over total virus yield, indicated that rAHSV4 virus release increased rapidly from 36 to 72 hpi D, while Similarly to rAHSV4-sVP7, the released fraction of the virulent strain rAHSV5-sVP7 also had reduced virus titres over the 72 h period compared to rAHSV5 E. IntereComparing attenuated versus virulent serotypes overall revealed that both rAHSV4-sVP7 and rAHSV5-sVP7 had a general reduction in virus release compared to their wild-type virus counterparts D,H. InteSince AHSV is a vector-borne disease, it was decided to repeat this part of the study in the insect vector cell line (KC cells) to determine if VP7 solubility affects replication differently in these cells. Time trials were conducted as above, except the released and cell-associated virus fractions could not be collected separately as the cell integrity of the fragile KC cells could not be guaranteed at time of collection. Instead, at time of virus harvest, total virus was collected and titred as described in In KC cells, both rAHSV4 and rAHSV5 wild-type virus titres increased continuously from 12 to 72 hpi  with no Insoluble aggregates or inclusions that are formed during virus infection have been evidenced to have various functions within the cell . For exaThis study aimed to understand the nature of the implied effects of crystalline particle formation on cellular biology and AHSV replication. A reverse genetics approach was used to generate a recombinant AHSV that contains a mutated segment 7 that encodes the soluble VP7 in its genome and expresses soluble VP7 during replication. Successful rescue of rAHSV-sVP7 for both attenuated and virulent strains confirmed that VP7 crystalline particle formation is not required for successful virus replication. The impact of this genetic knock-in of a soluble VP7 was analysed by observing virus protein-protein interactions and replication kinetics compared to wild-type rAHSV.The soluble VP7 protein interacted similarly to wild-type VP7 with outer capsid protein VP2 and VIB mediator NS2 in infected cells, indicating that sVP7 forms cores as usual during replication. This served to confirm that wild-type VP7 is required in its soluble form to assemble into cores during replication as previously suggested ,70. IntePrevious studies have shown that increasing the solubility of AHSV VP7 increases the yield of CLPs —likely dReleased and cell-associated virus fractions were collected to obtain a more detailed view of intracellular progeny virus production versus released virus. Initial infection rates were also compared by examining virus yield at 12 hpi. It was revealed that both wild-type and mutant viruses (serotypes 4 and 5) had similar initial replication rates and, following initial infection, differences in virus production and release were observed.AHSV is typically released from the mammalian cell via disruption of the plasma membrane in a lytic manner mediated by NS3 ,32. WhenWhen comparing rAHSV4 with rAHSV4-sVP7 replication kinetics in BSR cells, it was found that rAHSV4-sVP7 replicates slower than rAHSV4 with significantly reduced virus yield overall. The decreased total virus yield over 72 h was due to a decrease in intracellular virus production. Interestingly, there was also a significant reduction in rAHSV4-sVP7 release throughout the 72 h period. It is likely that the delay in virus replication observed for rAHSV4-sVP7 is due to reduced released progeny virus particles available for reinfection. VP7 crystalline particles therefore play a role in AHSV4 release and intracellular virus production in mammalian cells.Replication kinetics of rAHSV5-sVP7 revealed that similarly to rAHSV4-sVP7, there was decreased virus release throughout the 72 h period. Therefore, VP7 crystalline particles also play a role in AHSV5 release in mammalian cells. Most noteworthy for serotype 5 was that although at early hours post-infection rAHSV5-sVP7 had lower yields than rAHSV5, instead of declining after 36 hpi the mutant virus continued to produce intracellular virus causing exponential growth from 48 hpi resulting in a significant increase in total virus yield at 72 hpi. The exponential growth of rAHSV5-sVP7 in BSR cells was similar to what is observed of AHSV growth in KC cells due to lack of cell death (CPE) as a result of non-lytic release mechanisms. It is therefore likely that, like KC cells, the infected BSR cells were not damaged by rAHSV5-sVP7 virus replication due to reduced CPE resulting in the increased total virus yield observed for rAHSV5-sVP7. For this virulent serotype, it is therefore possible that VP7 crystalline particles contribute toward cellular pathogenesis by disrupting the cellular membrane to aid virus release therefore causing CPE in mammalian cells. These results therefore implicate a potential role of AHSV VP7 crystalline particles in rAHSV5 cellular pathogenesis.Replication kinetics of recombinant viruses in KC cells revealed similar results for both serotype 4 and 5. Since virulence factors would typically only play a role in host mammalian cells, it is expected that no notable differences be revealed in KC cells. The decreased virus yield of mutant viruses in KC cells at 72 hpi further indicates a potential role of VP7 crystalline particles in efficient intracellular virus production as observed for mutant viruses in mammalian cells. Since no CPE is observed for insect cells, VP7 crystalline particles are not required for non-lytic release therefore resulting in similar replication kinetics of wild-type and mutant AHSV4 and AHSV5 in KC cells observed here. Furthermore, virus replication kinetics in mammalian cells revealed that virus release was significantly reduced for both mutant viruses rAHSV4-sVP7 and rAHSV5-sVP7, further indicating that AHSV VP7 crystalline particles play a role in mammalian, i.e., lytic, virus release mechanisms for these strains.Virus protein aggregates or structures that disrupt cellular membranes to promote pathogenesis and human disease have been previously reported. For instance, the accessory protein PB1-F2 of influenza A virus oligomerises into amyloid fibres, and has been shown to rupture cell membranes and cause cell death at late time points in human lung epithelial and monocyte cells ,80. As oProtein aggregates that mediate virus pathogenesis have also been shown for the nuclear export protein of a highly mutating H1N1 influenza A virus. This protein intrinsically forms aggregates which have been correlated with its role in virion budding ,81. HepaSince no exponential growth of rAHSV4-sVP7 was observed, it would be suggested that VP7 crystalline particles do not contribute toward rAHSV4 cellular pathogenesis as they may for rAHSV5. The cellular pathogenicity mechanisms of these viruses differ considerably since rAHSV4 is an attenuated virus with lowered CPE in mammalian cells while rAHSV5 is a virulent virus and unattenuated in mammalian cells. Therefore the role of VP7 crystalline particles in each virus may differ. A possible explanation could arise from our LSCM data that revealed the presence of sVP7-NS1 tubules in rAHSV4-sVP7 infected cells. NS1 has previously been linked to virus morphogenesis and cellular pathogenesis ,85. PerhThis study has revealed that AHSV VP7 crystalline particles play a role in virus release during AHSV replication. Furthermore, AHSV VP7 crystalline particles are required for efficient intracellular virus production in mammalian and insect cells. It was further revealed that VP7 crystalline particles may play a role in the lytic release of AHSV5 which may contribute to cellular pathogenesis in mammalian cells. To confirm the role of AHSV VP7 crystalline particles in pathogenesis, further cell viability and in vivo studies should be performed. To further elucidate the role of VP7 crystalline particles in virus release, localisation analysis should be carried out with exit mediator protein NS3, and the association of NS1 with VP7 should be further explored in the context of virus production. This study advances the understanding of AHSV replication.


Tamoxifen resistance in estrogen receptor positive (ER+) breast cancer therapy increases, which is the leading cause of cancer treatment failure, as it can impair patients’ prognoses, cause cancer recurrence, metastasis, and death. Combination therapy with compounds is needed to overcome tamoxifen resistance. Oleanolic acid (OA) was known to increase tamoxifen sensitivity in tamoxifen-resistant breast cancer; however, the molecular mechanism of OA and its involvement in overcoming tamoxifen resistance remain unknown and need further investigation. This study was conducted to identify the potential gene targets and molecular mechanisms of OA in overcoming tamoxifen resistance.CD44, FGFR2, PIK3R1, and MDM2 were designated as potential target genes (PTGs), and PIK3R1 was suspected as the potential gene for OA to overcome tamoxifen resistance. Molecular docking confirms that OA can inhibit p85 activation. PIK3R1 is suggested to be the potential gene for OA in overcoming tamoxifen resistance in breast cancer therapy.A bioinformatic approach for functional network analysis was used in silico by utilizing secondary data in the Gene Expression Omnibus (GEO) database and analyzing them with GEO2R to obtain data on differentially expressed genes (DEGs). The DEG data were further examined with Database for Annotation, Visualization, and Integrated Discovery (DAVID), STRING, cBioPortal website, and Cytoscape with its plugin CytoHubba. Molecular docking was performed to predict the binding properties of OA on the protein encoded by the potential gene. The predicted molecular mechanism of OA in overcoming tamoxifen resistance involves inhibiting p85 activation, leading to the inhibition of the downstream activity of the PI3K signaling pathway, causing breast cancer to respond to tamoxifen therapy once again. Results of this study need to be validated by further studies, including in vitro and in vivo.The online version contains supplementary material available at 10.1186/s43141-022-00341-4. Breast cancer is one of the leading causes of mortality among women worldwide . Over 70Previous studies have shown the molecular mechanisms of tamoxifen resistance including alterations in ER signaling, activation of PI3K/AKT/mTOR (PI3K) signaling, and dan activation of NFκB signaling . TamoxifOlea europaea) furopyrimidin-2-yl]phenol). The docking score of the interaction of OA with p85 was higher than that of its native ligands, indicating a lower binding affinity to each receptor. This result is consistent with the involved amino acid on OA for each receptor. The native ligand of p85 has three amino acids, which interacted through Arene-H (Met772) and side chain donor , as seen in Table OA can alleviate the symptom of renal ischemia–reperfusion injury through the PI3K/AKT signaling pathway . OA and PIK3R1, which encodes the p85α regulatory subunit of PI3K [Through functional network analysis, a candidate gene, namely  of PI3K , was ide of PI3K . A clini of PI3K . Those f of PI3K , 48. Thi of PI3K . Figure When activated by RTK, PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 accumulates in the plasma membrane and attracts plasma membrane-localized phosphoinositide-dependent kinase 1 (PDK1) and protein kinase B/AKT, a critical mediator of the PI3K signaling pathway. AKT is phosphorylated by PDK1 and PDK2 and becomes active, thereby stimulating downstream pathways such as the mammalian target of rapamycin (mTOR) via tuberous sclerosis complex 1/2 (TSC1/2), NFκB, and MDM2 among others. The tumor suppressor protein phosphatase and tensin (PTEN) homolog catalyzes the dephosphorylation of PIP3 to PIP2, so it is also referred to as a negative regulator of PI3K signaling .PIK3CA, which encodes the p110α catalytic subunit of the PI3K, is the most prevalent in HR+ breast cancer. Mutations in PIK3R1, which encodes the p85α regulatory subunit of the PI3K, have also been identified in HR+ breast cancer [PIK3CA mutations (33.0%), 10 samples had PIK3R1 mutations (2.2%), and 283 samples had PIK3R1 underexpression (61.8%).The PI3K signaling pathway is one of the possible targets for overcoming breast cancer therapy resistance . Aberratt cancer . In a stt cancer  on 458 sPI3KR1 acts as a tumor suppressor [PIK3R1 can impair the function of the p85α regulatory subunit, resulting in an increased PI3K pathway activity [ppressor  and encoppressor . Its prippressor . In otheppressor . The motppressor . The undactivity , 42. Furactivity . This fiactivity , which dactivity .Numerous studies have demonstrated that OA can inhibit the PI3K signaling pathway. Li et al.  administA recently published review article discussed the development of OA and its derivate for anticancer therapy via several mechanism, including induction of apoptosis, regulation of cell cycle, inhibition of angiogenesis, inhibition of stem cells, reversal multidrug resistance, and increase immune system . OA beloPIK3R1 to overcome tamoxifen resistance in breast cancer therapy. Molecular docking shows that OA can inhibit p85 activation. OA acts molecularly by inhibiting p85 activation, leading to the inhibition of the downstream activity of the PI3K signaling pathway, causing breast cancer patients to respond to tamoxifen therapy once again. However, these findings should be confirmed through further research, including specific in vitro and in vivo studies.OA likely targets Additional file 1: Supplementary Figure 1. Distribution of the value obtained from the GEO database. The distribution of data for data series GSE67916 (A) and GSE86871 (B) is quite good.Additional file 2: Supplementary Table 1. DEGs of tamoxifen-resistant MCF-7 cell line (GSE67916). Supplementary Table 2. DEGs of the MCF-7 cell line treated with OA (GSE85871). Supplementary Table 3. GO enrichment analysis of the OTGs : Biological Process. Supplementary Table 4. GO enrichment analysis of the OTGs : Cellular component. Supplementary Table 5. GO enrichment analysis of the OTGs : Molecular Function. Supplementary Table 6. KEGG pathway enrichment analysis of the OTGs.


Rationale: Whatever the mucosa primary infected, HPV-positive cancers are traditionally associated with a favorable outcome, attributable to a high sensitivity to radiation therapy. However, the direct impact of viral E6/E7 oncoproteins on the intrinsic cellular radiosensitivity  remains mostly speculative.Methods: Using several isogenic cell models expressing HPV16 E6 and/or E7, the effect of viral oncoproteins on global DNA damage response was first investigated by in vitro/in vivo approaches. The binary interactome of each individual HPV oncoprotein with factors involved in the various host DNA damage/repair mechanisms was then precisely mapped by Gaussia princeps luciferase complementation assay . The stability/half-life of protein targets for HPV E6 and/or E7 as well as their subcellular localizations were determined. At last, the host genome integrity following E6/E7 expression and the synergy between radiotherapy and compounds targeting DNA repair were analyzed.Results: We first showed that the sole expression of one viral oncoprotein from HPV16 was able to significantly increase the sensitivity to irradiation of cells without affecting their basal viability parameters. In total, 10 novel targets  for E6 and 11  for E7 were identified. Importantly, not degraded following their interaction with E6 or E7, these proteins have been shown to be less linked to host DNA and to colocalize with HPV replication foci, denoting their crucial implication in viral life cycle. Finally, we found that E6/E7 oncoproteins globally jeopardize host genome integrity, increase the cellular sensitivity to DNA repair inhibitors and enhance their synergy with radiotherapy.Conclusion: Taken together, our findings provide a molecular insight into the direct hijacking of host DNA damage/repair responses by HPV oncoproteins, demonstrate the significant impact of this phenomenon on both intrinsic cellular radiosensitivity and host DNA integrity and suggest novel connected therapeutic vulnerabilities. Although over 250 million doses of human papillomavirus (HPV) vaccine have already been distributed since its introduction 15 years ago, carcinogenic HPV genotypes (most notably HPV16) are still responsible for an estimated 690,000 cancers per year worldwide TP53ink4a (a surrogate biomarker for HPV infection) or the defect of TGFβ signaling were also reported to explain the enhanced radiosensitivity of HPV-positive tumors. While the down-regulation of E3 ubiquitin ligase TRIP12 has been demonstrated to be essential in p16ink4a-mediated repression of DNA damage/repair The main factors determining the success or failure of radiotherapy were first explained in the mid 70's by Prof. Rodney Withers in a seminal article entitled “The four R's of radiotherapy”. The repopulation of surviving normal and tumor cells between dose fractions, redistribution of malignant cells in the G2/M cell cycle phase, reoxygenation of the hypoxic areas and repair of DNA damage induced by ionizing radiation were originally proposed. A few months later, the intrinsic radiosensitivity of irradiated cells was presented as the fifth “R”. At last, a “6R” model has been recently suggested, adding the reactivation of immune responses to the five aforementioned R's in vitro/in vivo models as well as by mapping the interactome of HPV E6 and E7 oncoproteins with the different families of DNA repair proteins, the present study attempts to answer all these questions.Altogether, this context opens quite a few theoretical and translational questions. Could HPV-related “manipulation” of host DNA repair mechanisms represent the Achilles' heel of HPV-positive cancers? Are viral oncoproteins alone able to sensitize infected cells to irradiation-induced DNA single/double-strand breaks? Do E6 and E7 directly interact with key proteins involved in DNA damage repair pathways? Is the function, half-life and/or cellular localization of DNA repair proteins altered by viral oncoproteins? Could E6 and E7 oncoproteins enhance the sensitivity to DNA repair inhibitors and/or positively impact their synergy with radiotherapy? By using various ink4a) and HPV DNA genotyping [Abbott RealTime High-Risk HPV assay ]. The study was approved by the institutional review board at the University Hospital of Liege (#2021/25).A total of 59 oropharyngeal and 113 anal canal SCC were retrieved from pathology archives of the University Hospital of Liege (Belgium). Before selection, all cases were re-examined by experienced histopathologists. The paraffin-embedded specimens were processed and archived in the local Biobank throughout the project. For each specimen, the HPV status was determined by both immunohistochemistry (anti-p16Eleven HPV-negative and 7 HPV-positive cell lines were maintained in Dulbecco's modified Eagle's medium (DMEM), Minimum Essential medium (MEM) or Roswell Park Memorial Institute (RPMI) 1640 medium  supplemented with 10% fetal calf serum and various additives. Precise cell culture conditions are detailed in HPV-negative malignant vulvar (A431), floor of mouth (UPCI-SCC-111) and osteosarcoma (U-2OS) cells (previously used as a model for studying HPV-related carcinogenesis 5 in 150-300 µl of DMEM) were subcutaneously injected (left flank) in Nude mice aged 4-6 weeks (n = 13 per condition). Throughout the experiment, tumor size was monitored every 2-3 days with a digital caliper (Thermo Fisher Scientific). When tumor volume [π /6 x (length × width2)] reached 100-200 mm3, a unique dose of 6 Gy was administrated using a small animal irradiator . At day 9 after irradiation, the treated animals were anesthetized and 20 mg/kg luciferin (Promega) was injected intraperitoneally. Ten minutes later, mice were imaged using an IVIS® Lumina III in vivo Imaging System . For ethical reasons, mice were euthanized when the average tumor volume exceeded 1000 mm3 in the control group. All animals were purchased from Janvier Labs  and the authors strictly complied to the ethical recommendations established by the Federation of European Laboratory Animal Sciences Associations (FELASA). The procedures were initially reviewed and approved by the local ethics committee (#19-2109).A431 Luc or A431 E6E7 cells (7.5x102O2 in methanol for 5 min. Antigens were retrieved in 10 mM citrate buffer (pH6) for 23 min at 100 °C. Before the primary reaction (1 h at room temperature), non-specific antigens were blocked using serum-free protein block reagent  for 10 min. The following primary antibodies were used: anti-p16ink4a  and anti-γH2AX . For the immunoperoxidase staining, the rabbit EnVision detection kit (Dako) was used and positive cells were visualized using SignalStain DAB Substrate kit .Immunohistochemical experiments were performed as previously described ink4a staining was considered as positive when >75% cancer cells strongly expressed this surrogate biomarker for HPV infection. The number of γH2AX-positive tumor cells per mm2 was precisely determined using QuPath 0.2.0 software for digital pathology image analysis (computerized counting) All immunolabelled tissues were evaluated independently by two histopathologists. As previously described TP53 were amplified by classical PCR. The primer sequences were previously described Genomic DNA was extracted from cultured cells using the NucleoSpin Tissue kit . Exons 2 to 11 of ALKBH2, BRCA1, BRCA2, CHEK2, CLK2, DNA2, DUT, ENDOV, ERCC3, H2AX, MNAT1, PARP3, PER1, PMS1, PNKP, POLDIP2, RAD51, RBBP8, RMI1, RPA1, UVSSA, XRCC1, XRCC2, XRCC3, XRCC4, XRCC5 and XRCC6 in head and neck cancers were evaluated using The Cancer Genome Atlas (TCGA) public dataset through the cBioPortal interface The expression levels of Total RNA was extracted and purified from cultured cells using the NucleoSpin RNA isolation kit (Macherey-Nagel). One µg was then reverse transcribed using RevertAid Reverse Transcriptase and oligodT primers (Thermo Fischer Scientific). qPCR experiments  were performed using the FastStart Universal SYBR Green Master mix  and the following primers sequences: HPV16 E6 forward: 5'-TGG AAT CTT TGC TTT TTG TCC-3'; E6 reverse: 5'-CTG CGA CGT GAG GTG TAT TAA C-3'; HPV16 E7 forward: 5'-GGT TAC AAT ATT GTA ATG GGC TC-3'; E7 reverse: 5'-AGC TCA GAG GAGGAG GAT GAA-3'; GAPDH forward: 5'-ACC AGG TGG TCT CCT CTG AC-3'; GAPDH reverse: 5'-TGC TGT AGC CAA ATT GGT TG-3'. Each experiment was performed in triplicate and normalized to the amount of GAPDH mRNA from the same sample.Cells were seeded in order to reach ~10% confluence. After cell adhesion, 24-well plates were incubated for 7 days in the IncuCyte S3 Live-Cell Analysis System . Every 12 h, pictures were taken (4 fields per well). Collected data were finally analyzed using the IncuCyte S3 software.The percentage of apoptotic cells was determined by flow cytometry , using annexin V-FITC and propidium iodide, according to the manufacturer's recommendations (BD Biosciences).Cells were harvested using trypsin-EDTA solution, washed with PBS and fixed with iced cold 70% ethanol overnight at -20 °C. Fixed cells were then washed once with PBS and incubated for 30 min at room temperature with a solution containing RNase A  and propidium iodide . Cells were finally analyzed by flow cytometry .2EDTA and 0.5 mg/ml proteinase K (pH 8.0). Electrophoresis was conducted for 25 min at 0.6 V/cm in a solution containing 90 mM Tris, 90 mM boric acid and 2 mM Na2EDTA (pH 8.5). Following an incubation with Vista Green DNA dye for 20 min, comets were detected, photographed (6 fields per well) using a fluorescence microscope  and quantified (CometScore 2.0 software). Tail length was used to estimate the degree of DNA damage (double-strand breaks) in each condition.Cells were first irradiated with 40 Gy  at room temperature and incubated for the indicated times  at 37 °C in order to allow for DNA repair. Cells were then collected and processed for neutral comet assay as extensively described previously 4 cancer cells transduced with HPV16 E6 and/or E7 or firefly luciferase (control) were exposed to 0, 2 or 4 Gy irradiation. After a 4 h recovery, cytochalasin B , a potent cytokinesis inhibitor, was added to the media for 18 h. The cells were then quickly washed with PBS, fixed with 4% paraformaldehyde (15 min at room temperature) and stained with 4′,6-diamidino-2-phenylindole (DAPI). Micronucleus frequency was finally determined in once-divided binucleated cells according to previously described criteria Plated on a glass coverslip, 5 x 102 atmosphere. Plates were then washed in PBS, fixed and colored for 45 min using a solution containing 5% glutaraldehyde  and 0,5% Crystal violet . After a washing step with deionized water, plates were dried, scanned  and quantified using the ColonyArea ImageJ plugin . Instead of the traditional manual counting, this standardized/computerized approach allows to determine the percentage of area covered by cell colonies HPV-negative and -positive cells were seeded in 6-well plates at a density comprised between 250 and 2,000 cells per well, depending on their growth rate. After 24h, cells were irradiated with 0, 1, 2, 4 or 6 Gy using a Gammacell 40 Exactor (Best Theratronics) and incubated for 10 days at 37 °C in a humidified 5% COGaussia princeps luciferase are linked to the N-terminal ends of the proteins of interest through a 20-amino acid flexible hinge region. pSpica-N2 vectors expressing E6 or E7 from different high-risk (carcinogenic) HPV genotypes  were previously generated via Gateway cloning. In order to verify both the cloning step and the accuracy of all transferred sequences, resulting plasmids were sequenced  using the following forward primer: 5'-CAG CTC TTA AGG CTA GAG TAC-3'. A library of 200 pSpica-N1 plasmids coding for 179 DNA damage and repair proteins (and 21 isoforms) was generated . Cells were transfected with 100 ng pSpica-N2-HPV E6 or E7 and 100 ng pSpica-N1 (from the DNA repair library) using PEImax . Twenty-four hours post-transfection, cells were washed and then incubated for 30 min at room temperature with the Renilla lysis buffer . After injection of 50 µl of luciferase substrate reagent in each well, the luciferase activity was measured during 10 s using a Centro LB960 microplate luminometer . As previously described Twenty-four hours before transfection, 3x105 HEK-293T cells per well of a 6-well plate were seeded. Cells were transfected with 1.5 µg pCineo-3xFLAG  and 1.5 µg pSpica-N1 from our library using PEImax (Polysciences). Twenty-four hours post-transfection, cells were harvested and incubated for 30 min on ice in 100 µl lysis buffer . After a centrifugation step , 96 µl collected supernatant was incubated overnight with 30 µl anti-Flag M2 Magnetic Beads (Sigma-Aldrich). The 4 remaining µl represent the total fraction of the extraction. Total and immunoprecipitated proteins were analyzed by western blotting. The nitrocellulose membranes were incubated overnight at 4 °C with anti-Gaussia luciferase antibody . After several washings, the membranes were incubated for 1 h at room temperature with a secondary goat anti-rabbit antibody . The protein bands were finally revealed using a chemiluminescence system .HPV16 E6 or E7 sequence was first transferred into the pCineo-3xFLAG vector, allowing the expression of a 3xFLAG tag at the N-terminal end of the viral oncoproteins. The day before transfection, 4x102 2 mM, EDTA 0.1 mM, NP-40 0.2%, DTT 1 mM and protease inhibitors) for 30 s. After centrifugation , the cytoplasmic fraction (supernatant) was collected. The pellet was then washed 5 times  before being lysed in buffer B  for 30 min. The fraction corresponding to the nucleic acid-free proteins was collected after centrifugation (320 g for 7 min at 4 °C). Finally, the proteins linked to the chromatin were isolated after sonication (30 s) in Laemmli buffer  at 4 °C. After quantification , 30 µg of proteins were separated by electrophoresis (SDS-PAGE) and transferred onto PVDF membranes (Roche), that were blocked with 5% nonfat milk in TBS-tween 0.1%, before being incubated overnight at 4 °C with the primary antibodies  were treated with 100 µM Cycloheximide (Sigma Aldrich). The proteins were isolated in SDS buffer  at different time points (up to 24 h) and then quantified . The level of given proteins was finally determined by western blot . The linear genome was then recircularized by incubating 10 µg of the digested plasmid with 400 U of T4 ligase, 10X ligation reaction buffer and RNase free water  overnight at 16 °C. Finally, DNA was precipitated (NaCl 5 M and isopropanol at -20 °C), centrifuged (16 000 g for 30 min at 4 °C) and resuspended in TE buffer .HPV-negative malignant vulvar (A431) cells were stably transduced with HA-tagged HPV16 E1 and 3xFlag-tagged HPV16 E2 or mCherry (negative control) , as described above (see Isogenic cell models expressing HPV16 E6 and/or E7). Given that the constitutive expression of E1 and E2 proteins is lethal for the cells, we used the Double-Floxed Inverted Open reading frame (DIO) technology to invert the Open Reading Frame (ORF). Therefore, the E1 and E2 genes are not expressed unless a Cre treatment is applied to the cell culture. Transduced cells were selected with 133 µg/ml hygromycin B or 0.33 µg/ml puromycin (InvivoGen).5 E1/E2-transduced cells were seeded per well. After 24 h, cells were transiently transfected with 0.5 µg of the recircularized HPV16 genome using FuGene 6 (Roche), according to the manufacturer's instructions. Six hours later, a non-integrative Cre lentivirus was added to the cell culture in order to induce the expression of HPV16 E1 and E2, by flipping the ORF to the right direction. Thirty-six hours after the Cre induction, the cells were quickly washed with PBS, fixed with 4% paraformaldehyde (15 min at room temperature), permeabilized with PBS-0.1% triton X-100 (15 min) and blocked with an animal-free blocking solution . The cells were then incubated with the primary antibodies overnight at 4 °C , library preparation (Illumina DNA PCR-Free Library Prep Kit using 25 ng DNA) and quantification/normalization [KAPA Library Quantification Kit ] were then performed at the GIGA-Genomics platform (University of Liege). Sequencing (30X coverage) was conducted on the Illumina NovaSeq6000 platform (PE150 mode). Sequencing reads were aligned to the reference human genome (GRCh38) using the Burrows-Wheeler Alignment tool, with “bwa mem” settings HPV16 E6E7 isogenic cell pairs generated from immortalized keratinocytes (HaCaT) were grown , 5,000 (HaCaT) or 8,000 (UPCI-SCC-111) cells per well in 96-well plates containing working dilution of each DNA repair inhibitor and incubated for 72 h at 37 °C. Cell viability was assessed by MTT proliferation assay (Roche). The individual IC50 values of each drug against the HPV E6E7 isogenic cell pair were calculated using nonlinear regression analysis .Potent/selective inhibitors of PARP1/2 (Niraparib and Veliparib), RAD51 (RI-1), homology-dependent DNA repair (YU238259) and DNA topoisomerase I (Camptothecin) were purchased from Selleckchem . For ICBoth control and E6E7-trasnduced HaCaT cells  were exposed to growing doses of irradiation  and/or to different concentrations (lower than IC50) of Niraparib, Veliparib, RI-1, Camptothecin or YU238259. After 72 h, cell viability was assessed by MTT proliferation assay (Roche). Synergy between radiotherapy and each DNA repair inhibitor was evaluated according to the Zero Interaction Potency (ZIP) method and using the open-source SynergyFinder 3.0 software 2 test. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.Statistical significance between 2 or more groups/conditions was assessed using GraphPad Prism 8 software . Normal distribution was determined by both the skewness score and a D'Agostino and Pearson normality test. The outlier identification was performed using the ROUT method, with a Q=5% By using the TCGA public dataset for head and neck SCC, both the overall survival and the mRNA levels of the most frequently used DNA damage/repair biomarkers  were first assessed. A pathway enrichment analysis (GSEA) was also performed. All collected data were separated according to the HPV status . As expected, [and reported in previous publications TP53 mutation statuses were transduced with HPV16 E6 and/or E7 , the expression of E6 and/or E7 viral oncoproteins was consistently associated with an increased cellular sensitivity to irradiation. Using this gold standard method for measuring long-term effects of radiotherapy, it is interesting to notice that a synergistic effect of E6 and E7 was only observed with wild-type TP53 cells (U-2OS) (3) were established. As shown in Figure in vitro data. Of note, in vivo experiments using malignant UPCI-SCC-111 and U-2OS cells were also tried. Different cell numbers and conditions  were tested but their weak tumorigenicity  did not enable their use in mouse models.In order to determine whether viral oncoproteins actively contribute to the elevated sensitivity of HPV-infected cells to radiation therapy, immortalized keratinocytes (HaCaT) and three cancer cell lines  from different origins and displaying different 7 Figure B-D. The 7 Figure . The ave7 Figure . Althoug (U-2OS) . Finallyhttps://www.mdanderson.org/documents/Labs/Wood-Laboratory/human-dna-repair-genes.html), 230 proteins, divided into 16 different families, are directly (or indirectly) implicated in DNA repair mechanisms. As mentioned by the authors (Wood RD and Lowery M), it is important to notice that some proteins act in several pathways but, for the sake of simplicity, each ORF is only listed once (in its main family) in the present study . According to the Human DNA Repair Gene Database  transduced or not with HPV16 E6E7 were treated with a translation inhibitor (cycloheximide) and the level of each individual protein was monitored over a 24h period. In parallel, subcellular protein fractionations were performed Figure A. Whereaversus 8.95, p = 0.0035) in the presence of HPV16 compared to the control condition lacking viral genome  was 2.5-fold higher , the number of SNPs and InDels was first estimated at different time intervals. In parallel, the COSMIC mutational signatures were extracted. As shown in Figures in vitro studies published in the past few years and using mainly oropharyngeal cancer cells In the last decades, multiple studies reported that HPV-positive cancers represent a distinct entity associated with a favorable prognosis. In addition, both the specific mutational signature and the elevated immune infiltration of HPV-driven neoplasms (compared to their HPV-unrelated counterparts) were described TP53 status. In parallel to clonogenic growth analyses, cytokinesis-block micronucleus assays demonstrated that both E6- and E7-transduced cells already displayed an increase of chromosome breakages compared to control cells at basal (non-irradiated) condition. These interesting observations (exacerbated following irradiation) are in agreement with recent studies analyzing the effect of viral oncoproteins from HPV16 as well as of E6 from cutaneous βHPV8 on genome instability Playing a major role in viral replication, the implication of HPV E1/E2 in host DNA damage/repair response manipulation has been thoroughly studied Given that neither E6 nor E7 possesses an intrinsic enzymatic activity, they act through their interactions with host proteins. Aiming at determining the interactome of HPV E6 and E7 oncoproteins with the different sub-families of DNA repair factors, a cDNA library covering about 80% of the entire DNA damage/repair responses was assembled and the GPCA was used as the high-throughput screening method. This luminescence-based technology represents a major improvement for the identification of binary protein-protein interaction. Indeed, compared to the currently more popular yeast two-hybrid which recovers less than 25% of total interactions This assumption was further supported by both the absence of degradation of these DNA repair proteins in presence of viral oncoproteins and their colocalization with E1/E2 foci. Of note, these protein recruitments to viral replication foci should no longer appear in invasive tumors displaying a pure integrated infection. However, given that the interactome of HPV E6/E7 with the DNA damage/repair system is not influenced by the viral status  of infected cells , the negative impact for host DNA should occur in all HPV-related cancers. In order to further characterize the revealed interacting pairs, truncated or mutated forms of the relevant viral oncoproteins were tested. All identified targets of E7 exhibited negative GPCA signals with the CR1+CR2 construct (lacking the last 62 amino acids of E7), confirming that most E7-related interactions required the C-terminal region of the oncoprotein mut breast, ovarian and prostate cancers In conclusion, viral E6 and E7 oncoproteins actively contribute to increase the intrinsic radiosensitivity of infected cells. Beside the high blood vessel and T-cell densities detected within tumor microenvironment, this latter parameter undoubtedly also contributes to the better response to radiation therapy of HPV-positive tumors. Even if all identified E6/E7-interacting DNA damage/repair proteins are not involved in signaling pathways directly activated in response to radiotherapy , a DNA repair global weakness takes place in HPV-infected cells. Indeed, without drastically modifying the extracted/detected mutational signatures, the expression of viral oncoproteins was clearly associated with a substantial increase of SNPs/InDels in host genome. Therefore, despite an upregulation of multiple factors involved in various DNA repair mechanisms , these gSupplementary figures, tables 1 and 3.Click here for additional data file.Supplementary table 2.Click here for additional data file.Supplementary table 4.Click here for additional data file.