BRAF V600E mutation. This study employed the CRISPR/Cas9 technology to generate three isogenic A375 melanoma cell lines with point mutations of NRAS Q61K, KRAS G13D and MEK1 Q56P, respectively. They recapitulated the resistance to BRAF inhibitors in vitro as such mutations have been found in patients with acquired resistance to BRAF inhibitors during treatment. Hence, these novel isogenic cell lines become extremely useful tools for upcoming research in this field. Additionally, we determined that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. The KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy.BRAF inhibitor drug resistance has been a long-time challenge in the treatment of melanoma with BRAF V600E mutation drives uncontrolled cell growth in most melanomas. While BRAF V600E tumors are initially responsive to BRAF inhibitors, prolonged treatment results in inhibitor resistance and tumor regrowth. Clinical data have linked the NRAS Q61K, KRAS G13D and MEK1 Q56P mutations to the BRAF inhibitor resistance. However, development of novel therapeutics is hindered by the lack of relevant isogeneic cell models. We employed CRISPR/Cas9 genome engineering to introduce NRAS Q61K, KRAS G13D and MEK1 Q56P mutations into the A375 melanoma cell line with endogenously high expression of BRAF V600E. The resulting isogenic cell lines are resistant to BRAF inhibitors. The A375 MEK1 Q56P isogenic cells are additionally resistant to MEK inhibitors as single agent, but interestingly, these cells become sensitive to MEK/BRAF inhibitor combo. Our results suggest that resistance in the NRAS and MEK isogenic lines is driven by constitutive MEK/ERK signaling, while the resistance in the KRAS isogenic line is driven by EGFR overexpression. Interestingly, the KRAS G13D isogenic line displays elevated PD-L1 expression suggesting the KRAS G13D mutation could be a potential indication for immunotherapy. Overall, these three novel isogenic cell models with endogenous level RAS and MEK1 point mutations provide direct bio-functional evidence demonstrating that acquiring a drug-resistant gene drives tumor cell survival and may simultaneously introduce new indications for combo therapy or immunotherapy in the clinic. BRAF V600E [BRAF V600E oncogene functions by increasing signaling through the Ras/RAF/MEK/ERK pathway controlling cell survival and proliferation. Small molecule BRAF inhibitors such as dabrafenib and vemurafenib are highly effective for initial treatment of BRAF V600E melanomas. However, continued use of BRAF inhibitors leads to the accumulation of secondary activating mutations in Ras/RAF/MEK/ERK pathway genes that bypass BRAF inhibition, resulting in regrowth of BRAF-inhibitor resistant tumors [BRAF V600E tumor cells, or if BRAF inhibitor treatment itself drives the accumulation of additional mutations. Currently available cell-based models of drug-resistant melanoma are derived either from BRAF V600E melanomas that have been subjected to prolonged small molecule BRAF inhibition, or from cells overexpressing exogenous copies of the gene in question from a non-native promoter [BRAF V600E melanomas have yet to be defined, and this lack of mechanistic understanding hinders the development of novel chemotherapeutics and combination therapies effective for the treatment of tumors that have become resistant to BRAF inhibitor treatment regimens.More than half of all clinical melanomas carry an activating BRAF mutation, 90% of which are AF V600E ,2,3. Thet tumors ,5,6,7,8.promoter ,11. No sBRAF V600E mutation. Use of CRISPR/Cas9 to directly engineer BRAF-inhibitor resistant melanoma cells provides a unique opportunity to study mutations associated with BRAF-inhibitor resistance in the absence of any selective drug pressure or exogenous protein expression and enable such mutant alleles to be expressed from the native promoter at physiologically relevant levels. Furthermore, because these mutations have been precisely engineered using CRISPR/Cas9 rather than by selective drug pressure, each new engineered cell line is genetically identical to the cell line from which it was derived, except for the introduced point mutation. Thus, each engineered model cell line can be used together with its parental line as an isogenic pair, and any observed difference in drug-resistance phenotype can be directly attributed to the introduced point mutation.In this study, we use CRISPR/Cas9 gene editing to introduce three point mutations associated with acquired BRAF inhibitor resistance directly into A375 human malignant melanoma cells, which carry the KRAS G13D, NRAS Q61K, and MEK1 Q56P mutations confer resistance to the BRAF inhibitors dabrafenib and vemurafenib in BRAF V600E A375 cells. The MEK1 Q56P mutation also renders the cells resistant to MEK inhibitors such as trametinib and binimetinib. Interestingly, MEK1 Q56P A375 cells are more sensitive to treatment with a combination of MEK and BRAF inhibitors than to either inhibitor in isolation, at equivalent doses. Additionally, the NRAS Q61K and KRAS G13D mutations bypass BRAF inhibition via distinct mechanisms: resistance in KRAS G13D cells appears to be primarily mediated by an overexpression of EGFR, while resistance in NRAS Q61K cells is caused by a failure of inhibition of downstream MEK-ERK signaling. Finally, PD-L1 expression is constitutively elevated in KRAS G13D but not in NRAS Q61K or MEK1 Q56P A375 cells, pointing to a possible role for immune checkpoint inhibition in melanomas carrying this specific biomarker.We find that the BRAF V600E melanoma represent an important step forward for both the study of acquired BRAF inhibitor resistance and for the screening and development of novel chemotherapeutics and treatment regimens for patients with inhibitor resistant melanoma.These new CRISPR/Cas9 engineered isogenic models of acquired BRAF inhibitor resistance in NRAS Q61K upstream intron 5′-TAGGGCCAGGTTATAATTGGTGG-3′ downstream intron 5′-ACATAAGTGAAGCTTGCTACTGG-3′, KRAS G13D upstream intron 5′-GCATTTTTCTTAAGCGTCGATGG-3′ downstream intron 5′-GTATTTCAGAGTTTCGTGAGAGG-3′, MEK1 Q56P upstream intron 5′-CATGTTGGTGATAGTCATCCCGG-3′ downstream intron 5′-AAAATTCAAACAGCACGGCTGGG-3′.Point mutations were introduced into the NRAS, KRAS, and MEK1 genes by lipid-based co-transfection of two Cas9/gRNA all-in-one plasmid constructs targeting the introns to either side of the relevant coding exon . Cas9/gRNA all-in-one plasmids were derived from pcDNA3 and expressed tracrRNA and gRNA from a U6 promoter, and human codon optimized Cas9 and PuroR from a CMV promoter. Cells were also transfected with a pUC19-derived homologous sequence donor plasmid containing the desired point mutation and flanking exonic and intronic sequence. The following guide sequences were used: KRAS G13D , A375 NRAS Q61K  and A375 MEK1 Q56P  were grown and maintained in ATCC-formulated Dulbecco’s Modified Eagle’s Medium  with 10% fetal bovine serum . Cells were maintained at 37 °C and 5% CO2 in a humidified incubator.The melanoma cell line A375  and the derived isogenic lines A375 5 cells per well into 12-well culture plate and transfected with Cas9-sgRNA all-in-one plasmid and donor plasmids using TransfeX reagent. The transfected cell cultures were then enriched by puromycin selection. Single cells were isolated by automated cell sorting (Sony SH800) into 96-well plates and expanded for approximately 10–14 days. Once the single clones reached over 70% confluence per well, clones were then sub-cultured.For the establishment of isogenic cell lines, A375 cells were seeded at 1 × 10NRAS Q61K 5′- TGCATGTTTGTCTTCCTGAGA-3′ and 5′-ATCTTTTCCATTACATACTGAAAGATG-3′, KRAS G13D 5′-CCCCATGACACAATCCAGCTT-3′ and 5′-TCTGCTGCTGGTCTTTACTTTGG-3′, MEK1 Q56P 5′- GAGGATTTCTACTGTTGTGATTCAG-3′ and 5′-GGTATCCTGGCATTCCTTCCACAG-3′. The following nested sequencing primers were also used: NRAS Q61K 5′-GCAGGCATATAGAATTTGGTGGGTTTTCTTTTATGTAGGGTG-3′ and 5′- CTCTGGTTCCAAGTCATTCCCAGTAGCAAGC-3′, KRAS G13D 5′-AAGCGTCGATGGAGGAGTTTGTAAATGAAGTACAG-3′ and 5′-CATCATGGACCCTGACATACTCCCAAGGA-3′, MEK1 Q56P 5′-CAGACCTGGAGCTTTCTTTCCATGA-3′ and 5′-CTGGTCCCCAGGCTTCTAAGTAC-3′. Thermocycler parameters to amplify the 550 bp KRAS, 600 bp NRAS and 369 bp MEK1 on-target regions were as follows: step one, 98 °C for 1 min; step two, 98 °C 30 s; step three, 72 °C for 30 s, step four, 72 °C 30 s, repeat steps 2–4 for 29 times, step five, 72 °C for 4 min. PCR products were analyzed on 1.2% agarose double tier Lonza FlashGel. Positive KI mutant clones were identified by Sanger sequencing of PCR products.Genomic DNA was extracted from CRISPR/Cas9 edited A375 clones using QuickExtract DNA extraction solution (Epicentre/Lucigen) according to manufacturer instructions. The CRISPR/Cas9 targeted genomic regions for each cell line were then PCR amplified and analyzed by Sanger sequencing for the presence of the desired point mutation. The following PCR primers were used: Cells were lysed in 1% SDS in phosphate buffered saline (PBS) followed by sonication for 3 × 5 s at 30% amplitude (QSonica CL-18). Lysates were then clarified by centrifugation at 13,000 RPM for 10 min in a table-top microcentrifuge and protein concentration was measured by bicinchoninic acid assay. Protein was loaded at 20 μg per lane on 4–12% gradient gels and migrated according to standard protocols. Protein was transferred to a nitrocellulose membrane using a quick dry blotting system , which was then blocked with 5% nonfat milk protein in TBS-T. Blots were then probed with the following primary antibodies: rabbit monoclonal anti-EGF receptor , rabbit monoclonal anti-p44/42 MAPK (ERK1/2) , rabbit monoclonal anti-phospho-p44/42 (Thr202/Tyr294) , rabbit monoclonal anti-MEK1/2 , rabbit monoclonal anti-phospho-MEK1/2 (Ser217/221) , rabbit monoclonal anti-AKT , rabbit monoclonal anti-phospho-Akt (Ser473) , rabbit monoclonal anti-PD-L1 , and rabbit polyclonal anti-GAPDH . Blots were then probed with goat anti-rabbit IgG (H + L)-HRP conjugate  and bands were visualized using a Gel Doc XR+ gel documentation system (Bio-Rad).Cells were seeded in triplicate in 96-well culture plates at 2000 cells per well and grown overnight in the absence of any drug treatment. The following day, the indicated concentration of drug or an equivalent volume of DMSO vehicle control was added and the cells were grown for an additional three days. After three days of treatment, cell viability was assessed using CellTiter-Glo luminescent cell viability agent (Promega) according to manufacturer instructions. Luminescence was measured with a SpectraMax i3x multi-mode plate reader. The cell survival rate of drugged cells was normalized to the DMSO control group. The dose-response curves and IC50 values were generated using Prism (GraphPad). The BRAF inhibitors dabrafenib and vemurafenib, the MEK inhibitors trametinib and binimetinib, and the non-specific chemotherapeutic doxorubicin were obtained from ApexBio and Selleckchem.Cells were seeded in 96-well ultra-low attachment microplates (Corning) at 500 cells per well and grown for 72 h. After 72 h the indicated concentration of drug was added, and the spheroids were grown for an additional 72 h. At the end of the 6-day time course spheroids were co-stained with NucBlue Live ReadyProbes reagent  and 2.0 μM Calcein AM Green for two hours and then imaged by confocal microscopy on a Cellinsight CX7 high content analysis platform. For each spheroid, 15 confocal 20-micron Z-stacks were imaged in both the blue and green channels to visualize NucBlue-stained nuclei and Calcein AM Green-stained cytosol, respectively. The resulting 30 images were then assembled into a single image projection and analyzed using HCS Studio cell analysis software (Thermo Fisher). Total spheroid visible area was calculated by delineating the boundary of the spheroid in the green channel.6 cells per mL. Then 200 μL of each cell type was stained with 20 μL R-phycoerythrin (PE) conjugated mouse anti-human CD274 (PD-L1) antibody  for 30 min. Mouse IgG1k-PE  was as an isotype control. Stained cells were analyzed by flow cytometry. Where indicated, cells were stimulated overnight with 200 ng/mL recombinant human interferon gamma  before they were harvested for flow cytometry analysis.A375 parental cells and A375 isogenic cells were harvested with non-enzymatic cell dissociation solution, washed once in ice cold PBS, and re-suspended in flow cytometry staining buffer at 1 × 10BRAF V600E A375 cells using CRISPR/Cas9 genome engineering. The three mutations, NRAS Q61K, KRAS G13D, and MEK1 Q56P, were selected based on the strength of the clinical data supporting their association with BRAF inhibitor resistance, and because all three mutations affect the Ras/RAF/MEK/ERK signaling pathway [To generate inhibitor resistant model cell lines, three specific point mutations associated with BRAF inhibitor resistance in clinical samples were introduced into  pathway ,11. CRIS pathway  and ensuKRAS G13D c.41 G > A, heterozygous NRAS Q61K c.184 C > A, and homozygous MEK1 Q56P c.170 A > G point mutations were obtained from this screen. Total cellular RNA was then harvested from each candidate clone and the cDNA sequences of expressed KRAS, NRAS, and MEK1 genes, respectively, were obtained  and MEK1 Q56P mutations yielded dabrafenib resistance on the order of 10−7 and 10−8 representing a 100- and 10-fold decrease in sensitivity, respectively. A similar trend in vemurafenib resistance was obtained with A375, RAS and MEK1 Q56P mutation cell lines yielding IC50 values in the order of 10−8, 10−6 and 10−7, respectively. In order to determine whether the observed BRAF inhibitor resistance phenotypes were specific to BRAF inhibition, rather than to a general insensitivity to small molecule compounds, all four cell lines were tested for resistance to the BRAF non-specific chemotherapeutic doxorubicin. Doxorubicin is a DNA intercalator that induces apoptosis selectively in rapidly dividing cell populations and should thus be unaffected by the presence of engineered RAS and MEK point mutations. As expected, no significant difference in doxorubicin sensitivity was detected between WT and RAS or MEK mutant A375 cells, with all four lines having doxorubicin IC50 values on the order of 10−8 , ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), AKT, and phosphor-AKT (Ser473)  D. Basal RK1/2 Thr2/Tyr204,BRAF V600E melanoma [MEK1 Q56P A375 melanoma model for resistance to the FDA-approved MEK inhibitor compounds trametinib and binimetinib. MEK1 Q56P A375 cells were found to have a ~10-fold lower sensitivity to trametinib and a ~30-fold lower sensitivity to binimetinib relative to unmodified A375 cells . Spheroid sizes were analyzed on a high-content imaging platform.Following characterization of the drug resistance phenotypes and MEK/ERK/MAPK signaling perturbations in A375 RAS and MEK1 mutant melanoma models in 2D tissue culture, we next evaluated the drug responses of the parental cell line and the three isogenic cell lines in a 3D culture environment. WT, NRAS Q61K, KRAS G13D, and MEK1 Q56P cells were significantly more resistant to the BRAF inhibitors dabrafenib and vemurafenib than the WT A375 cells from which they were derived (MEK1 Q56P A375 spheroids, like MEK1 Q56P cells grown in 2D tissue culture, were also more resistant to the MEK inhibitors trametinib and binimetinib than WT A375 cells.As was observed in 2D tissue culture,  derived A,C. SimiNRAS Q61K spheroids subjected to treatment with 50 nM vemurafenib were reduced to ~60% of the size of an untreated NRAS Q61K spheroid (NRAS Q61K cells in 2D tissue culture with up to 1 μM vemurafenib for the same amount of time yielded less than a 10% decrease in cell viability (Interestingly, all four A375 melanoma model lines were more sensitive to the MEK and BRAF-specific inhibitors in 3D tissue culture than in 2D tissue culture, requiring lower concentrations of inhibitor to achieve a reduction in spheroid size comparable to the loss of cell viability observed in 2D tissue culture. For example, A375 WT spheroid size was reduced to ~10% of the size of an untreated control A375 WT spheroid after three days of treatment with 25 nM dabrafenib A,B, but spheroid A,B, whiliability . ConversThese findings, in addition to demonstrating the resistance of engineered melanoma model cell lines to MEK and BRAF inhibitors in a 3D tissue culture system, clearly highlight the value of the 3D tissue culture format for the screening and optimization of novel chemotherapeutics and combination drug therapies for the treatment of drug-resistant melanomas.NRAS Q61K and KRAS G13D cells to combination drug treatment was intermediate to the sensitivity to each drug in isolation (MEK1 Q56P cells were more sensitive to a half-dose of dabrafenib in combination with a half-dose of trametinib than to a full dose of either drug alone, across a wide range of drug concentrations (After assessing the MEK and BRAF inhibitor resistance phenotypes of A375 melanoma model cell lines in both 2D and 3D tissue culture, we next evaluated each line for sensitivity to combination MEK/BRAF inhibitor treatment. Each cell line was treated in 2D tissue culture with either the BRAF inhibitor dabrafenib, the MEK inhibitor trametinib, or an equivalent combination drug treatment consisting of a half-dose of dabrafenib and a half-dose of trametinib A–C. A375solation B,C, MEK1trations D.MEK1 Q56P melanoma cells to combination MEK/BRAF inhibitor treatment, MEK1 Q56P cells were treated with either dabrafenib, vemurafenib, trametinib, binimetinib, doxorubicin, or a combination drug treatment consisting of a half-dose of dabrafenib plus a half-dose of trametinib. Total cellular protein was then analyzed by immunoblot for the basal levels of EGFR, MEK1/2, ERK1/2, and AKT expression, as well as the level of MEK1/2, ERK1/2 and AKT phosphorylation, which correlated with an increase in signaling through the Ras/RAF/MEK/ERK signaling pathway. As expected, MEK1 Q56P cells maintained phosphorylation of both MEK and ERK in the presence of BRAF and MEK inhibitors, while WT A375 cells had diminished MEK or ERK phosphorylation in the presence of these compounds (MEK1 Q56P cells are resistant to MEK and BRAF inhibitor treatment because, unlike WT A375 cells, MEK1 Q56P cells maintain signaling for cell survival and proliferation via phosphorylation of MEK and ERK proteins even in the presence of inhibitors that completely abrogate MEK/ERK signaling in unmodified A375 cells. However, when MEK1 Q56P cells were treated with an equivalent dose of dabrafenib and trametinib in combination, phosphorylation of MEK and ERK decreased dramatically, to levels of Ras/RAF/MEK/ERK pathway signaling that more closely resemble the response of WT A375 cells to BRAF or MEK inhibitor treatment alone (In order to examine the specific perturbations in Ras/RAF/MEK/ERK signaling underlying the sensitivity of ompounds E. This rMEK1 Q56P melanoma cells carry the activating BRAF V600E mutation as well as an activating MEK1 mutation, inhibition of BRAF alone fails to abrogate aberrant proliferation signaling through downstream MEK proteins, while inhibition of MEK alone cannot effectively compensate for the increase in upstream BRAF signaling. Instead, by inhibiting Ras/RAF/MEK/ERK signaling at multiple points along the signaling cascade (Because A375  cascade F, synergMEK1 Q56P melanoma cells grown in 3D tissue culture to combination MEK/BRAF inhibitor treatment. Spheroids were treated for three days with either 12 nM dabrafenib, 4 nM trametinib, 6 nM dabrafenib together with 2 nM trametinib, or vehicle control, then spheroids were then stained and imaged (MEK1 Q56P spheroids were more sensitive to the combination drug treatment condition than to an equivalent dose of dabrafenib or trametinib in isolation (MEK1 Q56P model of acquired BRAF inhibitor resistance, when used together with the isogenic WT A375 cells from which they were derived, is a valuable new tool for the screening and development of new combination therapies in both 2D and 3D tissue culture.Finally, we evaluated the response of d imaged G. As wassolation G,H. ThesBRAF V600E melanoma is associated with an immunosuppressed tumor microenvironment and consequent tumor immune evasion [KRAS G13D, but not in NRAS Q61K or MEK1 Q56P A375 cells relative to WT. Furthermore, pre-treatment of cells with interferon gamma, which typically increases the cell surface expression of PD-L1, had no effect on PD-L1 expression in KRAS G13D cells, but increased PD-L1 expression as expected in WT, NRAS Q61K, and MEK1 Q56P A375 cells. This finding indicates that PD-L1 expression in A375 KRAS G13D is constitutively elevated in a way that is unaffected by immune signaling, and suggests that combination therapies involving anti-PD-L1 immune blockade inhibition together with inhibitors targeting the Ras/RAF/MEK/ERK signaling pathway could be effective for the treatment of a subset of BRAF-inhibitor resistant melanomas.Because BRAF inhibitor resistance in  evasion  we testeKRAS G13D is due to an upregulation of trafficking of PD-L1 to the cell surface or to a general increase of cellular expression of PD-L1 protein, lysates from WT, NRAS Q61K, and KRAS G13D A375 cells grown in both 2D and 3D tissue culture were probed with anti-PD-L1 antibody (KRAS G13D cells, and that the level of PD-L1 expression was unaffected by tissue culture format.Next, in order to determine if the observed phenotype of constitutive cell-surface expression of PD-L1 in A375 antibody B, and itBRAF V600E melanoma. By using precision gene editing rather than selective drug pressure or exogenous overexpression of mutant alleles to generate drug-resistant model cell lines, we have conclusively demonstrated that three specific mutations associated with acquired BRAF-inhibitor resistance, NRAS Q61K, KRAS G13D, and MEK1 Q56P, are the direct cause of the observed drug-resistance phenotype. We have also confirmed that the molecular mechanism underlying BRAF-inhibitor resistance in these three melanoma models is the aberrant re-activation of Ras/RAF/MEK/ERK signaling, which is consistent with clinical data [NRAS Q61K and KRAS G13D drive MEK/ERK signaling in the presence of BRAF inhibitor, phosphorylation of MEK/ERK is much greater in NRAS Q61K than in KRAS G13D A375 cells after BRAF inhibitor treatment. However, both mutations confer a comparable degree of BRAF inhibitor resistance, suggesting that reactivation of MEK/ERK signaling predominantly accounts for the BRAF inhibitor resistance of NRAS Q61K cells, while it is likely that factors other than persistent MEK/ERK phosphorylation contribute to BRAF inhibitor resistance in KRAS G13D cells. Notably, we observe a significant increase in the basal levels of both EGFR and PD-L1 expression in KRAS G13D A375 melanoma cells but not in NRAS Q61K cells. This finding is consistent with patient data demonstrating that resistance to BRAF inhibitors in patients with secondary KRAS G13D mutations leads to an increase in basal EGFR expression by negative feedback through the Ras/RAF/MEK/ERK signaling pathway [KRAS G13D but not in the NRAS Q61K melanoma model is further consistent with findings of elevated PD-L1 levels in tumors with activating KRAS mutations and with studies demonstrating elevated PD-L1 expression in non-small-cell lung cancers with activating EGFR mutations and/or overexpression of EGFR [In this study, we have demonstrated how CRISPR/Cas9 genome engineering can be used to create novel, physiologically relevant cell-based models of acquired BRAF inhibitor resistance in cal data . However pathway ,27. The  of EGFR ,30,31,32MEK1 Q56P melanoma model to both MEK and BRAF inhibitors confirms the prior discovery of BRAF inhibitor cross-resistance with stable expression of exogenous MEK1 Q56 and MEK1 P124 mutant alleles in A375 cells [MEK1 Q56P mutation falls outside allosteric drug binding pocket [MEK1 Q56P A375 cells, but not in the KRAS G13D or NRAS Q61K melanoma models is consistent with clinical data demonstrating the effectiveness of combined MEK/BRAF inhibitor therapy for the treatment of melanomas with secondary activating RAS mutations and acquired BRAF inhibitor resistance [The resistance of our 75 cells . Howeverg pocket , indicatg pocket  or by anMEK1 Q56P isogenic A375 melanoma model to both MEK inhibitor and combination MEK/BRAF inhibitor when grown in 3D tissue culture highlight the utility of the 3D tissue culture format for high-throughput drug screening. The fact that all three melanoma models are also less sensitive to non-specific compounds such as doxorubicin in 3D than in 2D tissue culture further supports the idea that 3D tissue culture models are the more effective tool for the screening of targeted drugs than 2D cultures, and have the potential to significantly lower costs associated with expensive animal model. Furthermore, our results also show that there are significant differences in Ras/RAF/MEK/ERK pathway activation when the cells are grown in a 2D rather than a 3D culture format. Our finding of diminished basal AKT signaling in all four A375 melanoma models indicates that cells in 3D culture environment favor signaling for cell proliferation over signaling for cell survival in aggregate. It may also suggest that the 3D tissue culture format is particularly useful for screening inhibitors that affect the Ras/RAF/MEK/ERK signaling pathway.The increased sensitivity of the A375 melanoma model cell lines to BRAF inhibitor in 3D relative to 2D tissue culture, and the increased sensitivity of the KRAS G13D/BRAF V600E isogenic model of drug-resistant melanoma described here should focus on combination PD-L1 and BRAF/MEK inhibitor treatment. Initial studies could be performed in a standard 2D tissue culture format, but further refinement of such a treatment regimen can be accomplished via 3D tissue culture, or even 3D co-culture of A375 melanoma model cells with stromal cells, which would more closely mimic the physiological tumor micro-environment. With high expression levels of PDL-1 and EGFR on the cell surface plus both BRAF V600E and KRAS G13D existing within its genome, the A375 KRAS G13D mutant isogenic line is an ideal model suited for future studies focused on the tumor microenvironment and integrated combination therapy strategies.Acquired BRAF inhibitor resistance presents a significant clinical challenge as there are only few effective treatment options for melanomas that have developed secondary BRAF-inhibitor resistance. In this study, we have shown how CRISPR/Cas9 genome engineering can be used for the generation of novel cell-based models of acquired inhibitor resistance, which are ideally suited for the screening of new inhibitor compounds and combination therapies targeting treatment-resistant melanomas. Given the rapidly evolving combination treatment landscape, future studies using the KRAS G13D knock-in has dramatically increased both EGFR and PD-L1 expression levels, while isogenic cells carrying NRAS Q61K and MEK Q56P have constant activation of the MEK-ERK pathway. This indicates the expression of PD-L1 is directly linked to KRAS mutation or the downstream effects thereof, rather than to a general increase in Ras-Raf-MAPK signaling. It also suggests a potential utility of combining the targeted therapy with immunotherapy for clinical patients with acquired drug resistance.Melanoma remains the most lethal form of skin cancer exhibiting high mortality rates, due to a high likelihood of developing metastases and acquiring drug resistance. We used the CRISPR genome editing technology to target endogenous loci and knock in drug resistant mutation within the A375 melanoma cell line. The created isogenic cell lines represent a new type of drug resistance models that contains defined genetic resistance mechanisms, which provides an invaluable tool for developing next generation therapeutics that can overcome drug resistance in melanoma. Interestingly,


Vaccination with intact (146S) foot-and-mouth disease virus (FMDV) particles is used to control FMD. However, 146S particles easily dissociate into stable pentameric 12S particles which are less immunogenic. We earlier isolated several single-domain antibody fragments (VHHs) that specifically bind either 146S or 12S particles. These particle-specific VHHs are excellent tools for vaccine quality control. In this study we mapped the antigenic sites recognized by these VHHs by competition ELISAs, virus neutralization, and trypsin sensitivity of epitopes. We included two previously described monoclonal antibodies (mAbs) that are either 12S specific (mAb 13A6) or 146S specific (mAb 9). Although both are 12S specific, the VHH M3F and mAb 13A6 were found to bind independent antigenic sites. M3F recognized a non-neutralizing and trypsin insensitive site whereas mAb 13A6 recognized the trypsin sensitive VP2 N-terminus. The Asia1 146S-specific site was trypsin sensitive, neutralizing and also recognized by the VHH M8F, suggesting it involves the VP1 GH-loop. The type A 146S-specific VHHs recognized two independent antigenic sites that are both also neutralizing but trypsin insensitive. The major site was further mapped by cross-linking mass spectrometry (XL-MS) of two broadly strain reactive 146S-specific VHHs complexed to FMDV. The epitopes were located close to the 2-fold and 3-fold symmetry axes of the icosahedral virus 3D structure, mainly on VP2 and VP3, overlapping the earlier identified mAb 9 site. Since the epitopes were located on a single 12S pentamer, the 146S specificity cannot be explained by the epitope being split due to 12S pentamer dissociation. In an earlier study the cryo-EM structure of the 146S-specific VHH M170 complexed to type O FMDV was resolved. The 146S specificity was reported to be caused by an altered conformation of this epitope in 12S and 146S particles. This mechanism probably also explains the 146S-specific binding by the two type A VHHs mapped by XL-MS since their epitopes overlapped with the epitope recognized by M170. Surprisingly, residues internal in the 146S quaternary structure were also cross-linked to VHH. This probably reflects particle flexibility in solution. Molecular studies of virus-antibody interactions help to further optimize vaccines and improve their quality control. Aphthovirus within the Picornaviridae family. FMD is mostly controlled by vaccination. The 7 serotypes of FMDV  by definition lack cross-protection, but within serotypes cross-protection can also be limited. FMD vaccine is mainly produced as inactivated authentic virus capsids adjuvanted in an oil emulsion. However, many novel vaccines are being developed based on virus-like particles (VLPs) that lack the RNA genome, similar to natural empty capsids that sediment in sucrose gradients at 75S (Foot-and-mouth disease (FMD) affects cloven-hoofed animals, causing vesicular lesions at the feet, mouth and udders of lactating animals. The direct economic losses are high, due to loss of milk-production, growth and draft power. Indirect costs due to loss in trade and other restrictions also impact countries where the disease occurs . The caus at 75S , 5. A mos at 75S . This mAs at 75S . Two of The icosahedral FMDV capsid comprises 60 copies of four capsid proteins, viral protein (VP)1 to VP4. One copy of each VP associates into a protomer, five protomers associate to a pentameric (12S) structure arranged around a 5-fold symmetry axis, and 12 pentamers form the full capsid. The capsid outer surface is formed by VP1, VP2 and VP3 while VP4 is located internally. Upon capsid dissociation VP4 dissociates from 12S particles. The association of 12S pentamers in a 146S particle results in additional 2-fold and 3-fold symmetry axes. FMDV targets host cell receptors, including integrins and heparan sulfate. Protective antibodies can block such virus-receptor interaction to neutralize the virus but other mechanisms also exist , 10. BasA set of VHHs binding to serotype O, including VHHs M3F, M8F, M23F, and M220F were earlier mapped into four separate epitope bins indicated by roman numerals I to IV . Most ofIn this study we used different approaches to map the antigenic sites recognized by especially the serotype A 146S-specific VHHs earlier isolated, but also the 12S-specific VHH M3F and Asia1 146S-specific VHHs M332F and M658F. These VHHs were analyzed in competition ELISAs, virus neutralization tests and binding to trypsin-treated FMDV antigen. We further mapped the antigenic sites of two type A broadly strain reactive and 146S-specific VHHs, M691F, and M702F  was done as described earlier (Production of FMDV antigens of strains A/TUR/14/98 (A/TUR), A24/Cruzeiro/BRA/55 (A24Cru), A10/HOL/1/42, O1/BFS1860/UK/67 and Asia1/Shamir/ISR/89 (Asia1 Shamir) was done as described earlier ( earlier . A peptiThe 146S specific mAb 22.9 . We refe50) incubated with duplicate serial two-fold dilution series of VHHs for 1 h. The non-neutralized virus was then detected by adding IBRS-2 cells and 2 days later the plates were read macroscopically after staining the monolayers with amido black. VHH neutralization was calculated using the Spearman-Kärber method (50 of virus at the 50% end-point (VNT50). Most VHH dilution series started at 1 mg/ml. However, the M220F VHH dilution series started at 0.05 mg/ml since it was produced in baker's yeast at low level.VHH concentrations required for neutralization of FMDV A/TUR, A24Cru and Asia1 Shamir were determined as described previously  using 10r method , 29 and 3 buffer (pH 9.6) overnight at 4°C. Plates were incubated at room temperature (RT) with 100 μl/well of a 2-fold dilution series over 12 wells starting at 1 μg/ml 146S of suitable FMDV antigens. Bound FMDV antigens were detected by subsequent sequential incubation with 100 μl/well 0.25 μg/ml of biotinylated VHH and 0.5 μg/ml of a streptavidin horse radish peroxidase (HRP) conjugate . Bound HRP was detected by staining with 3,3',5,5' tetramethylbenzidine. The color reaction was stopped by addition of 0.5 M sulfuric acid (50 μl per well) and the absorbance at 450 nm (A450) was measured using a Multiskan Ascent spectrophotometer . A four-parameter logistic curve was fitted to absorbance and FMDV concentrations using the SOFTmax Pro 2.2.1 program (Molecular Devices) and was used to interpolate the Effective Concentration (EC) resulting in a specific A450 value for each VHH or antibody. VHHs or antibodies that did not reach the A450 value defining the EC value were given an EC value of the highest FMDV concentration used (1 μg/ml).The procedures for ELISAs have been described , 27. HigBinding to a BSA-conjugated VP2 peptide was analyzed by coating ELISA plates with 4 μg/ml VP2-22-BSA that were subsequently incubated with 0.5 μg/ml biotinylated VHH or mAb and streptavidin-HRP conjugate as described above. Controls included plates coated with 4 μg/ml BSA or 2.5 μg/ml of crude FMDV antigens of strains A/TUR, A24Cru or Asia1 Shamir.* ([A450 with competing VHH or mAb] – [A450 without Ag coating]) / ([A450 without competing VHH or mAb – A450 without Ag coating]).The ability of VHHs or mAbs to bind independent antigenic sites of FMDV was studied by blocking/competition double-antibody sandwich (DAS) ELISA using biotinylated VHHs and mAb 9 or mAb 32. ELISAs were performed using 0.5 μg/ml unlabeled VHH for coating and subsequent capture of crude FMDV antigens (1 μg/ml 146S), that contained about 20% 12S particles, in addition to 146S. The same VHH as used for coating was used in the next step as biotinylated VHH. For mAb 9 and mAb 32, plates were coated with unlabeled VHH M691F. Initially the optimal concentration of biotinylated VHH or mAb for competition was determined by titration of biotinylated VHH or mAb without competition, as described above for determining EC values. A biotinylated VHH or mAb concentration was selected that provided about 80% of the maximal absorbance value observed with the highest VHH or mAb concentration analyzed. In the final experiment, plates containing VHH-captured FMDV were first incubated with the unlabeled VHH or mAb (5 μg/ml) in 90 μl/well for 30 min (blocking step). Then, without washing plates, 10 μl biotinylated VHH or mAb in the predetermined concentration was added and incubated for another 30 min (competition step). A control without antigen and a control without biotinylated VHH or mAb were included. Bound biotinylated VHH was detected by incubation with 0.5 μg/ml streptavidin-HRP conjugate. Bound mAbs were detected with 2,000-fold diluted rabbit anti-mouse immunoglobulin HRP conjugate (RaM-HRP). The % inhibition of antigen binding due to a competing VHH or mAb was calculated as 100–100The Octet Red96 System  was used for affinity measurement based on Biolayer Interferometry. An assay temperature of 30 °C was used. PBS containing 0.05% Tween-20 was used as kinetics buffer in all steps of each assay. High precision streptavidin (SAX)-sensors (Sartorius) were hydrated and subsequently loaded with biotinylated M678F (2 μg/ml) for 300 s, with A/TUR 146S particles (2 μg/ml) for 900 s and kinetics buffer for 300 s (baseline step). The concentrations of FMDV particles and VHHs were optimized for affinity measurements prior to the experiments. Then association of serial dilutions of unlabeled VHHs was done for 300 s and finally dissociation for 1,800 s. A reference sensor without unlabeled VHH was included to correct for baseline drift.ka) and off-rate (kd) were determined by global fitting of the association and dissociation phases of a series of unlabeled VHH concentrations. The mathematical model used assumes a 1:1 stoichiometry, fitting only one VHH in solution binding to one binding site on the surface. The equilibrium dissociation constant (KD), a measure for affinity, was then calculated as the ratio of kd and ka. The Octet Analysis Studio v12.2 software (Sartorius) was used for data analysis.The on-rate  linker followed by digestion of the cross-linked complex with 5 proteases and identification of cross-linked peptides by MS analysis . The usem/z 350–1,700 were analyzed by LC-MS/MS using a Thermo LTQ Orbitrap XL mass spectrometer . MS data were analyzed using xQuest V2.0  using SDG purified A24Cru 146S particles complexed with either M691F or M702F VHH. Strain A24Cru was used for this purpose because it produces a high amount of 75S particles in addition to 146S particles and thus could also be used for analyzing VHH binding to 75S particles. Prior to XL-MS analysis, the A24Cru 146S particles were subjected to digestion with five proteases and MS analysis (see below) without complexing with VHH and cross-linking to determine whether sufficient peptides can be identified that cover the complete FMDV capsid sequence. Such peptide mass fingerprints were made for VP1, VP2, and VP3 but not for VP4, which is internal in the FMDV 3D structure and thus not accessible to antibodies. For both VHHs 10 μl 0.5 mg/ml 146S particles were mixed with 10 μl 0.5 mg/ml VHH in PBS and 2 μl N,N-dimethylformamide containing 2 mg/ml disuccinimidyl suberate (DSS) cross-linker. The DSS consisted of an equimolar mixture of two forms that contain either 12 hydrogen (H12) or 12 deuterium (D12) atoms but were otherwise chemically identical. After incubation for 3 h at room temperature (RT) cross-linking was quenched with 20 mM ammonium bicarbonate and proteins were reduced with 50 mM dithiothreitol for 1 h at 37°C and subsequently alkylated with 100 mM iodoacetamide for 1 h at RT. Then five different aliquots of the reduced/alkylated sample were separately incubated with five different proteases , overnight at 37°C , 25°C (chymotrypsin) or 70°C (thermolysin). Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis was then performed. Peptides were separated on a C18 PepMapRSLC column at a flow rate of 300 nl/min ramping a gradient from 2 to 40% mobile phase B  using an Ultimate 3000-RSLC system. Peptides in the range of est V2.0  and Stavest V2.0  using a The A24Cru strain was sequenced again using earlier described methods . The proWe mapped the antigenic sites of 30 VHHs binding to serotype A or Asia 1 FMDV by competition of biotinylated VHH with unlabeled VHH in DAS-ELISA. For competition ELISA we used FMDV strains A/TUR, A24Cru, A10/HOL/1/42 and Asia1 Shamir –D. We eaEpitope binning of strain A/TUR  was doneVHHs M3F, M8F, and M220F also recognize three independent antigenic sites  on strain Asia1 Shamir . The 146n = 15) recognition  was at least tenfold higher in trypsin-treated samples as compared to untreated samples. Using this criterium only VHHs M8F, M332F, and M658F showed trypsin sensitive binding  in ELISA . VHHs we binding , B, D. TSince only the Asia1 Shamir 146S-specific VHHs M332F and M658F fell into the same epitope bin as M8F, we titrated both untreated FMDV antigen consisting mostly of 146S and heated antigen (12S) in the M8F ELISA. M8F showed considerable 146S specificity for strain Asia1 Shamir  althoughNone of the VHHs bound in ELISA to a peptide representing the N-terminus of VP2 conjugated to BSA  althoughThe above classification of antigenic sites is summarized in KD values of 0.33 nM for M691F and 1.1 nM for M702F  GH-loop that could be resolved by X-ray crystallography due to reducing the disulfide bond involving C134 at the base of the GH loop . The putCross-linked residues VP2-H209 and VP3-K208 are surface accessible only at the inner surface of 12S pentamer  which isResidues VP3-E70 and K139 that were mutated in mAb 9 resistant mutants  are alsoThe epitope recognized by M170 on type O FMDV as resolved by cryo-EM of M170/FMDV complex  was visuKD= 0.33 nM) probably explains some of the non-reciprocal competition seen with M691F, which shows less than 50% inhibition by VHHs M659F, M676F, M677F, M678F, and mAb 9 while inhibiting the binding of these 5 VHHs/mAb for >98% (The antigenic sites of type A and Asia1 FMDV binding VHHs were characterized by competition ELISA, virus neutralization tests, trypsin sensitivity of the epitope, broadness of the serotype A recognition and, for 2 VHHs, XL-MS. In competition ELISAs some VHHs showed non-reciprocal competition, which complicates antigenic site mapping. In case of M8F this could be due to VHH binding causing structural changes in the FMDV capsid, since M8 binding is known to decrease the virion stability and stimulate viral uncoating . Such effor >98% , C. Takifor >98% . HoweverThe 12S-specific non-neutralizing VHH M3F was found to bind a different antigenic site than mAb 13A6. Furthermore, M3F did not bind to a trypsin sensitive epitope and did not bind a peptide representing the VP2 N-terminus. Freiberg et al.  were surMost type A 146S-specific VHHs bound site V whereas M686F and M688F bound a separate site, that was given roman numeral III, similar to the site identified in type O by M23F , since MThe 146S-specific VHHs M691F and M702F both recognized antigenic site V. Their epitopes were further mapped by XL-MS. Several VHH residues were cross-linked to different FMDV residues that are located at a distance that far exceeds the 11.4 Å length of the spacer arm of the DSS cross-linker used. This could be due to the VHH-FMDV complexes adopting different conformations. Such flexibility was earlier reported for complexes of FMDV with VHHs M8 and M170  or integThe site recognized by 146S-specific VHHs M170, M691F, and M702F overlaps with antigenic sites 2 and 4, which lie close together  and are M702F was cross-linked to VP3 residues T115, R120 and T191 at the 2-fold symmetry axis surface that is accessible in a 12S particle, but not in a 146S particle. Holes in the viral capsid have earlier been observed at the 2-fold axis of other picornaviruses, including poliovirus 135S particles  and acidWe have shown for type A FMDV that both 12S and 146S particle specificity relies on at least two independent antigenic sites. The major 146S specific antigenic site that is also recognized by the VHHs M691F and M702F was located close to the 2-fold and 3-fold symmetry axes on a similar position as the type O 146S specific VHH M170. Since this site was located on a single 12S particle the 146S-specific binding of M691F and M702F is probably also caused by different conformations of 12S and 146S particles, as earlier suggested for M170 . The croThe original contributions presented in the study are included in the article/AD, MH, and WP designed and coordinated the study. AD and SS performed grant acquisition and financial reporting. HL and MH performed the experiments. The XL-MS study done at Coval X was coordinated by AD and MH. HL, MH, SS, and AD completed data analysis. MH wrote the first version of the manuscript, that was subsequently edited by HL, AD, and WP. All authors contributed to the article and approved the submitted version.


Influenza A viruses are a major pathogen that causes significant clinical and economic harm to many animals. In Indonesia, the highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in poultry since 2003 and has caused sporadic deadly infections in humans. The genetic bases that determine host range have not yet been fully elucidated. We analyzed the whole-genome sequence of a recent H5 isolate to reveal the evolution toward its mammalian adaptation.We determined the whole-genome sequence of A/chicken/East Java/Av1955/2022  from a healthy chicken in April 2022 and conducted phylogenetic and mutational analysis.Phylogenetic analysis revealed that Av1955 belonged to the H5N1 clade 2.3.2.1c (Eurasian lineage). The six gene segments  out of the eight segments derived from viruses of H5N1 Eurasian lineage, one (PB2) from the H3N6 subtype and the remaining one (M) from the H5N1 clade 2.1.3.2b (Indonesian lineage). The donor of the PB2 segment was a reassortant among three viruses of H5N1 Eurasian and Indonesian lineages and the H3N6 subtype. The HA amino acid sequence contained multiple basic amino acids at the cleavage site. Mutation analysis revealed that Av1955 possessed the maximal number of mammalian adaptation marker mutations.Av1955 was a virus of H5N1 Eurasian lineage. The HA protein contains an HPAI H5N1-type cleavage site sequence, while the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. The virus has increased mammalian adaptation markers by mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among previously circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets. Influenza A viruses are a major pathogen that causes significant clinical and economic harm to various species, including poultry, pigs, horses, marine mammals, and humans . The surThe HPAI H5N1 subtype was first discovered in Indonesia in 2003 , spreadiAn incursion of a new HPAI H5N1 clade, clade 2.3.2.1c, from the mainland of Southeast Asia occurred in Indonesia in 2012 . The claClade 2.3.2.1c caused only one fatal infection in humans in Indonesia, the last case in 2017. The genetic bases that determine host range have not yet been fully elucidated. Influenza virus evolves by the mutation of the genes and by the reassortment of the gene segments. The Animal Care and Use Committee at the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia gave their approval for every step of this study .We performed genome analysis by next-generation sequencing as previously described . Total Rhttps://gisaid.org) on 10 August 2022 identified viruses with the highest identity to the nucleotide sequence of one or more gene segments of Av1955.BLAST analysis in the GISAID EpiFlu database (AF117241) or A/Brevig Mission/1/1918(H1N1) .The genetic information processing software Genetyx v14  generated the phylogenetic trees using the RAxML with 100 bootstrap replicates. The trees are rooted in the A/South Carolina/1/1918(H1N1)  : EPI_ISL_307026; A/chicken/East Java/Av240/2014 (H5N1 Indonesian lineage) (HA clade 2.1.3.2b) : EPI_ISL_307019; A/turkey/East Java/Av154/2013 (H5N1 Eurasian lineage) (HA clade 2.3.2.1c) : EPI_ISL_307002; A/eagle/Jakarta Timur/20616-206-III/2016 (H5N1) : EPI_ISL_266799; A/chicken/East Java/Av1210/2017 (H5N1) : EPI_ISL_401823; A/chicken/East Java/Spg119/2018 (H5N1) : EPI_ISL_365528; and A/chicken/East Java/Av1534/2019 (H5N1) : EPI_ISL_401824.We noted the accession number of each genome segment of the viruses in The BLAST analysis revealed that the HA sequence of Av1955 was most close to Spg119 of H5N1  and the NA to Av1210 of H5N1 (97.3%) among the viruses in the GISAD database . The HA The BLAST analysis revealed that the genome sequence of Av1955 was most close to Spg119 for the PB2 segment , HA (97.7%), NP (98.3%), and NS (98.5%); to Av1534 for the PB1 (97.9%) and PA (98.1%); to Av1210 for the NA (97.3%); and to Eg20616 for the M (98.9%) among the viruses in the GISAD database . These rAv1955 was most close to Av39 for the PB2 (93.2%); to Av154 for the PB1 (95.9%), PA (96.0%), HA (95.2%), NP (97.0%), NA (96.2%), and NS (97.2%); and Av240 for the M (98.1%) among the three isolates in East Java during 2013–2014. In the phylogenies, the PB2 of Av1955 closely clusters with Spg119 and Av39; all of the PB1, PA, HA, NP, and NA cluster with Eg20616, Av1210, Spg119, Av1534, and Av154; the M with Eg20616, Spg119, and Av154; and the NS with Av1210, Spg119, Av1534, and Av154.The PB2, M, and NS of Eg20616 closely cluster with Av240, while the other six segments with Av154, indicating that Eg206166 is a reassortant between Av240 and Av154; Eg20616 acquired the PB2, M and NS segments from an Av240-like virus, and the other six segments from an Av154-like virus.The PB2 and M of Spg119 closely cluster with Av39 and Av240, respectively, while the other six segments with Av154, indicating that Spg119 is a triple reassortant between Av39, Av240, and Av154; Spg119 acquired the PB2 segment from an Av39-like virus, the M segment from an Av240-like virus, and the other six segments from an Av154-like virus.From the described results, we proposed a model of the genesis of Av1955 by multi-steps of inter- and intra-subtype reassortment, as illustrated in There were deletions in NA at 49–68 and NS at 80–84 with Av1955 and all reference viruses except Av39. The Av1955 PB1-F2 was truncated at 25, while that of the reference viruses, except for Av39 and Av240, was truncated at 57. Av39 and Av240 possessed 87 open reading frames for PB1-F2, almost full of 90. The M2 of Av1955 was a resistant type to amantadine and rimantadine.The markers of the donor segments were the same number as the recipient, Av1955, with PB1 (eight), PA (three), HA (five), M1 (3), M2 (zero), and NS2 (one). The markers in the transferred proteins were the maximum among the four donor viruses except for the HA. The HA donor, Spg119, had five marker mutations in HA, while Av1210 and Av1534 had six and seven, respectively. Av1955 acquired 39 mammalian adaptation markers in its proteins, an 8% increase from 36 of Spg119 (the donor of three genome segments).Sequencing and phylogenetic analysis  identifiThere were no mammalian adaptation mutations in the receptor binding region of the HA protein among the seven reference viruses and Av1955. Their amino acid sequences were E-186, G-221, Q-222, and G-224 (H5 numbering), consistent with the avian type specifically binding to a 2,3-linked sialic acid receptor on the cell surface of avian hosts. If the mutations occur, viruses are unable to infect avian hosts. The HA protein of Av1955 contains an HPAI H5N1-type cleavage site sequence with multiple basic amino acid residues (PQRERRRKR). In contrast, the virus was isolated from a healthy chicken suggesting its low pathogenicity nature. All of the H5N1 isolates in East Java, Indonesia, since 2013 were from sick poultry except a few reassortants possessing a PB2 genome segment derived from avirulent subtypes like H3N6; Spg119, A/chicken/East Java/Spg119/2018 (H5N1), was one of the exceptions isolated from a healthy chicken . The virus obtained its PB2 genome from an H3N6 virus, as shown in Av1955 possesses 39 mammalian adaptation marker mutations out of 149 identified by Av1955 is a virus of the H5N1 Eurasian lineage (HA clade 2.3.2.1c). The virus has increased mammalian adaptation markers by substitution mutation and intra- and inter-subtype reassortment, gathering gene segments possessing the most abundant maker mutations among circulating viruses. The increasing mammalian adaptation mutation in avian hosts suggests that they might be adaptive to infection in mammalian and avian hosts. It highlights the importance of genomic surveillance and adequate control measures for H5N1 infection in live poultry markets.10.7717/peerj.14917/supp-1Supplemental Information 1Click here for additional data file.


Escherichia coli (ETEC) binds to specific receptors on small intestinal enterocytes, followed by secreting of toxins causing diarrhea in post-weaning piglets . However, the F18 subunit vaccine is not sufficient to stimulate an immune response that can protect weaning pigs from F18-positive ETEC (F18+ETEC). Recently, a body of evidence shows that flagellin protein (FliC) helps to increase the immunity of fused proteins. Therefore, in this study, we combined FliC with F18 to enhance the immune response of F18. The f18 gene was obtained from F18+ETEC, then was fused with the fliC gene. The expression of recombinant FliC-F18 protein was induced by Isopropyl-beta-D-Thiogalactopyranoside (IPTG). The purified protein was tested in vivo in mouse models to evaluate the immunostimulation. Results showed that the fusion of FliC and F18 protein increased the production of anti-F18 antibodies. Besides, the anti-F18 antibody in the collected antiserum specifically identified F18+ETEC. This result provides proof-of-concept for the development of subunit vaccine to prevent PWD using F18 antigen.F18 plays an important role in helping Enterotoxigenic  FliC-F18 was recombinantly expressed.FliC-F18 increased the production of anti-F18 antibodies without complete adjuvant.+ETEC.The isolated antibodies specifically identified F18Escherichia coli strains involved PWD express F4 (K88) or F18 fimbriae  is one of the most common threats to the swine industry worldwide  supE44 thi λ-recA1 gyrA96 ΔlacU169 (ϕ80 lacZ ΔM15)] and E. coli BL21(DE3) (F+ ompT hsdSB (rB− mB−) gal dcm(DE3) were used as host strains for cloning and protein expression, respectively. Both were cultured in Luria-Bertani (LB) medium containing 30 μg/mL of ampicillin at 37°C. F4 enterotoxigenic E. coli (F4+ETEC) and F18 enterotoxigenic E. coli (F18+ETEC) were kindly provided by Dr. Eric Cox (Ghent University). F18+ETEC and pET-22b/fliC plasmid  inducer (Biobasic). F4+ETEC was used in the cross-reaction experiment.f18 gene was obtained from the genome of F18+ETEC (GenBank: GQ325633.1)  to create sticky ends, followed by a recombinase-free cloning (RFC) protocol , then screened again by colony PCR with primers F18F/F18R and T7pro/T7ter. Plasmids collected from positive colonies were checked by the structure using digestion reaction with NdeI and XhoI, before sending for sequencing.The 25633.1) . The pETf18 gene was obtained from the genome of F18+ETEC by PCR with specific primers F18fF and F18R  were digested with BamHI and XhoI (Thermo Scientific). The digested result of the pET22b/fliCgfp plasmid was separated by agarose electrophoresis, followed by the collection of the gel band containing digested pET22b/fliC. The gel was treated immediately after collection, then was ligated with digested f18 gene by T4 DNA Ligase  to the C-terminal of FliC. The resulting mixture was transformed into competent E. coli DH5α. All the screening steps were the same with the construction of pET-22b/f18 recombinant plasmid, but replace primers F18F with F18fF, and replace NdeI with BamHI.The and F18R . The f18fliC-f18, pET22b/fliC and pET22b/f18 were transformed into competent E. coli BL21(DE3). Colonies that grew on LBAmp agar plates were re-screened by colony PCR with specific primers (F18F/F18R and F18fF/F18R). After screening steps, positive colonies were inoculated in 5 ml LBAmp media shaking tubes and allowed to grow at 37°C in 16 h, followed by sub-cultured at 1:10 (v/v). After OD600 reached 0.8–1.0, the cultures were induced with 0.1 mM of IPTG. The protein expressions were performed overnight at 16°C. The expression results were analysed by SDS-PAGE and Coomassie Brilliant Blue stained, followed by Western blot and probed with anti-His-tag antibody (Santa Cruz).The protein expression was conducted as described with some modifications . pET22b/SDS-PAGE and Western blotting were performed as described with some modifications . The proE. coli BL21(DE3)/pET22b/f18 (or pET22b/fliC-f18 or pET-22b/fliC) was sonicated on ice to get the supernatant containing soluble proteins that were used as raw material to purify F18  by affinity chromatography with HP Hitrap column . After being equilibrated with a binding buffer , the column was loaded with the soluble protein solution, then equilibrated and washed with the binding buffer. After that, an elution buffer  was added to elute the target protein.FliC-F18, F18 and FliC proteins were purified as described with some modification . The bioThe purity of F18  was tested by SDS-PAGE, followed by Coomassie Brilliant Blue stained and GelAnalyzer software. Finally, purified F18  was dialysed, concentrated and measured by the Bradford method.Mus musculus var. Albino), 3–5 weeks old, 18 g–20 g on average, were divided into four groups (3 mice/group). The blood of these mice was collected two days before the first dose injection. Groups 1, 2, 3 and 4 were injected with 25 ng FliC + 50 μL FCA , 25 ng F18 + 50 μL FCA, 25 ng F18 + 50 μL FIA , and 25 ng FLiC-F18 + 50 μL FIA, respectively. Booster doses were injected after one week, then repeated every one week with half the amount of antigen, but the same amounts of adjuvants. The amount of antigen protein used for injection was calculated based on the purity of that protein. Five days after each injection, 200 μL–300 μL of blood was obtained from the tail vein of each mouse; particularly for the last blood collection, 300 μL–400 μL of blood was collected from the orbital sinus of each mouse. The serum was collected and stored at −20°C for later analysis.Animals were maintained and performed in the experimental animal facility, and experiments by the Directive 2010/63/EU guideline approved by The Animal Care and Use Committee of University of Science, Vietnam. The total of 12 healthy male white mice ; dots 2 and 4 were loaded 50 μL of 1:200 diluted antisera which were collected from FLiC-F18 + FIA injected group (group 4); dots 3 and 5 were loaded PBS buffer . After incubating for 1 h at room temperature, the membranes were washed five times with PBST before accordingly adding antimouse IgGHRP , and goat antiswine IgGHRP . These secondary antibodies were incubated for 1 h at room temperature before washing five times with PBST. Finally, TMB substrate was added to observe the signal. Also, the crossreaction experiment was performed similarly but using F4+ETEC instead of F18+ETEC.Dot blot was performed according to the Dot Blotter 48 Clever  protocol with some modifications. The total of 0.5 μL of F18f18, there was one band between 400 bp and 500 bp on the gel . Whereas there was no band on the negative control (PCR reaction without F18+ETEC genome)  and T7ter primer (which bind to T7 terminator) to confirm the presence of pET22b/f18 plasmid. Three of five colonies contained pET22b/f18 plasmid which showed as the bands approximately 582 bp  incorporated with the length from T7 terminator to the recognition site of the XhoI enzyme on the plasmid (132 bp). In contrast, there was no amplicon band in the negative control  and gfp fragment (715 bp)  and T7ter primer (which bind to T7 terminator of plasmid pET22b/fliC-f18) to confirm the presence of pET22b/fliC-f18. Amplicons were about 582 bp  and the fragment from the T7 terminator to the restriction site of XhoI on the plasmid (132 bp). When PCR with T7pro and T7ter primers, the resulting band was approximately 2,190 bp , f18 gene (450 bp), the DNA fragment from T7 promoter of the plasmid to the starting point of fliC gene (100 bp), and the DNA fragment from the endpoint of f18 gene to T7 terminator of the plasmid (125 bp). Therefore, the recombinant pET22b/fliC-f18 was confirmed.The  lane 1, , which wlane +f, . Also, p lane 2, . pET22b/fliC-f18, pET22b/fliC and pET22b/f18 plasmids were transformed into E. coli BL21(DE3) competent cells. Positive clones were induced by IPTG to produce target proteins. After verifying by SDS-PAGE and Coomassie brilliant blue staining (E. coli BL21(DE3).The pET22b/staining , there wstaining . The resWith the fused His-tag, FLiC-F18, FliC and F18 were purified using HisTrap column. The protein purity was then evaluated by GelAnalyzer software. The results showed that the purity of FLiC-F18, FliC and F18 was 98%, 80% and 84.5%, respectively . Althoug450 nm of serum samples at the beginning of this experiment were 0.065 ± 0.005, which was similar to the background signal (0.055 ± 0.005) (control sample using PBST instead of antisera). This result meant that there was no anti-F18 antibody before F18 antigen injection. In the antisera of group 1 (FliC + FCA), there was no anti-F18 antibody as well (The F18 specific antibody titers in collected antisera from mice groups were determined via indirect ELISA to evaluate the ability of FliC in enhancing the immune response of the F18 antigen. The OD as well . The ant as well . This re+ETEC and tested the cross-reactivity of the anti-F18 antibody with F4+ETEC as well. In the case of F18+ETEC, the colourimetric signal generation was seen on dots 1 and 2 but did not appear on the others (+ETEC (+ETEC. In other words, antisera collected from the FliC-F18 + FIA injected group was specific to the F18 antigen.This experiment evaluated the reaction of the anti-F18 antibody in antisera collected from FliC-F18+FIA injected group with F18e others . In conts (+ETEC . This inet al. 2011; +CD11c+ cells, and T lymphocytes (Vaccines against F18 have not shown promising results due to poor immune response and difficulty producing specific antibodies (phocytes .+ETEC is an unsolved problem. All strategies include oral live and subunit vaccines as well as encapsulated vaccines, or parenteral vaccines stills had limited success until now (+ETEC infection. However, the cause of PWD is a non-invasive infection, thus mucosal immunity with F4/F18specific IgA plays an important role in preventing this disease. Further researches could focus on FliC-F18 based oral vaccines for post-weaning pigs because FliC had the potential to promote humoral immune response by oral immunisation (+ETEC.Vaccination against F18+ETEC. This study is a prototype of the F18 subunit vaccine development to prevent PWD associated with F18+ETEC. However, further research is needed to demonstrate the complete protection of FliC-F18 in the immunised mice and piglets.The immunostimulation of F18 was robustly enhanced by fusing with FliC protein. The anti-F18 antibodies in the immunised mice significantly increased and specifically recognised the F18


Plasmodium vivax and blocking transmission of Plasmodium falciparum, and thereby enhancing malaria elimination. However, there is a need to identify G6PD deficient individuals and administer the drug with caution due to its haemolytic side effects. The main objective of this study is to determine the prevalence of G6PD deficiency among malaria-suspected individuals.Glucose-6-phosphate dehydrogenase (G6PD) is cytosolic enzyme, which has a vital role for the integrity and functioning of red blood cells. Lower activity of this enzyme leads to the occurrence of acute haemolytic anaemia after exposure to oxidative stressors like primaquine. Primaquine is an important drug for the radical cure of A facility-based cross-sectional study was conducted from September 2020 to September 2021 in Metehara Health Centre, Eastern Ethiopia. A structured questionnaire was used to collect the socio-demographic and clinical information of the study participants. Capillary and venous blood samples were collected based on standard procedures for onsite screening, dried blood spot preparation, and malaria microscopy. The G6PD enzyme activity was measured by careSTART™ G6PD biosensor analyzer. Data was entered and analysed by SPSS.A total of 498 study participants were included in the study, of which 62% (309) were males. The overall prevalence of G6PD deficiency based on the biosensor screening was 3.6% (18/498), of which 2.9% and 4.8% were males and females, respectively. Eleven of the G6PD deficient samples had mutations confirmed by G6PD gene sequencing analysis. Mutations were detected in G267 + 119C/T, A376T, and ChrX:154535443. A significant association was found in sex and history of previous malaria infection with G6PD deficiency.The study showed that the G6PD deficient phenotype exists in Metehara even if the prevalence is not very high. G267 + 119C/T mutation is the predominant G6PD variant in this area. Therefore, malaria patient treatment using primaquine should be monitored closely for any adverse effects.The online version contains supplementary material available at 10.1186/s12936-022-04269-5. G6PD gene on X-chromosome (Xq28) . The (Xq28) , 6. The orldwide –10. Accoorldwide .Plasmodium species in the liver ..G6PD genp-value of less than 0.05 was considered as significant association between G6PDd and each contributing factor  were positive for P. vivax, and 4.2% (21) were positive for both P. falciparum and P. vivax infections. More than half of the respondents had no history of previous malaria infection. Most of the study participants developed at least two clinical malaria symptoms, with headache and muscle/joint pain accounts 98.6% (491) and 90.4% (450), respectively  G6PD activity (6.9 U/g Hb), G6PD activities < 2.07 U/g Hb, 2.07–5.52 u/g Hb, and > 5.52 U/g Hb were indicated as G6PD deficient, intermediate, and normal, respectively. G6PD activities ranged from 0.2 to 17.9 U/g Hb in males and 0.2–22.3 U/g Hb in females . Similarly, Individuals with previous history of Plasmodium infection were more likely to be G6PD deficient than those who were not previously infected with malaria   of the samples. The G267 + 119C/T, GC was the most common mutation detected in the study population. From the 11 mutations, nine of them were single base substitution and the two  were polymorphic with G267 + 119C/T, GC mutation. Therefore, chX: 154535443 may have very limited impact on G6PD enzymatic activity, whereas the role of G267 + 119C/T requires further assessment. The predominant single mutation G267 + 119C/T was detected among 36.4% of male and 63.6% of female phenotypically G6PDd individuals. It was detected in 63.6% and 36.4% of malaria positive and malaria negative phenotypically G6PDd individuals, respectively  and intermediate (01) based on phenotypic measurement were sequenced for the G6PD gene is located on the X-chromosomes. Males are hemizygous with only one copy of G6PD gene on X-chromosome, and thus can be either normal or G6PD deficient. By contrast, females have two copies of the G6PD gene on each X-chromosome, which can result in either homozygous normal, heterozygous intermediate, or homozygous deficient enzyme level [In this study, phenotypic analysis of G6PD enzyme activity indicated that 3.6% 18/498) of the study participants had < 30% enzyme activity of the AMM, which is stated as G6PD deficiency; and 56 female patients had intermediate G6PD enzyme activity (30–80%) of AMM with heterozygous gene mutation. The G6PDd was slightly higher in females (4.8%) than males 2.8%). This result contradicts the assumption that females are anticipated to be less likely affected with G6PDd given the .8%. Thisme level  showed tme level  as G6PDdme level  with a Gme level  and nati/498 of tme level  and the me level .The X-linked nature of G6PD deficiency offers an advantage for female to be less likely affected by this enzyme disorder. The higher prevalence of G6PD deficiency observed among females compared to males in this study could be due to the less optimal performance of the G6PD biosensor, which merits further validation using spectrophotometric method. G6PD enzyme activity varies among nations, regions, and even local ethnic groups. The prevalence of G6PDd was slightly higher among Nuers (14.3%) and Anuak (12.0%) compared to the highlanders in Gambella region with no observed deficiency . MalariaPlasmodium species [P. falciparum, P. vivax, and co-infections with both P. falciparum and P. vivax, respectively [Although G6PD deficiency was postulated to have a protective effect against malaria infection, G6PD deficient individuals can still be infected with  species , 40. In ectively . G6PDd wectively . Malariaectively , no signectively . This vaBased on molecular sequencing, mutations were detected in G267 + 119C/T, A376T and ChrX: 154535443 with G267 + 119C/T being most prevalent. Although previous studies in Ethiopia reported the presence of A376G, G202A, C563T, G1116A and 485 + 37, these mutations were not identified in this study. Contrary to this study, 13% of the study participants showed G6PDA- (G202A) genotype in Brazzaville, Republic of Congo . SeveralG6PD genetic variants across the country as well as the functional significance of the variants. The low performance of the careSTART™ G6PD biosensor may also explain samples of low enzyme activity but with no observed gene mutations. Future analysis of G6PD enzyme activity by the careSTART™ G6PD biosensor would require validation using spectrophotometric method. Molecular sequencing of the G6PD enzyme would need to be done for all phenotypically G6PD normal and intermediate samples to investigate thoroughly the phenotype and genotype association. The lack of association of G6PD enzyme activities with respective genotypes advocates the need for further verification with genome-wide association testing and large sample size.Previous studies found only few variants such as G6PDA + in different parts of Ethiopia , 20, G26G6PD genetic variants in the country. Future study should aim for national-wide epidemiologic study of G6PD deficiency in Ethiopia.The prevalence of G6PD deficiency is low in the Metehara district based on phenotypic measurement. Such prevalence was significantly associated with sex and previous malaria infection history. G267 + 119C/T was the predominant genetic variant detected among the study participants. Thus, it is recommended to treat malaria patients with primaquine under close supervision and cautious follow-up for any haemolytic complications in the Metehara district. The presence of the new mutation in Ethiopia offer insights into the presence of various Additional file 1: Information sheet.Additional file 2: Consent and assent.Additional file 3: Questionnaire.Additional file 4: Laboratory procedures.


Enteroviruses (EVs) are medically important RNA viruses that cause a broad spectrum of human illnesses for which limited therapy exists. Although EVs have been shown to usurp the cellular recycling process of autophagy for pro-viral functions, the precise manner by which this is accomplished remains to be elucidated. In the current manuscript, we sought to address the mechanism by which EVs subvert the autophagy pathway using Coxsackievirus B3 (CVB3) as a model. We showed that CVB3 infection selectively degrades the autophagy cysteine protease ATG4A but not other isoforms. Exogenous expression of an N-terminally Flag-labeled ATG4A demonstrated the emergence of a 43-kDa cleavage fragment following CVB3 infection. Furthermore, bioinformatics analysis coupled with site-directed mutagenesis and in vitro cleavage assays revealed that CVB3 protease 2A cleaves ATG4A before glycine 374. Using a combination of genetic silencing and overexpression studies, we demonstrated a novel pro-viral function for the autophagy protease ATG4A. Additionally, cleavage of ATG4A was associated with a loss of autophagy function of the truncated cleavage fragment. Collectively, our study identified ATG4A as a novel substrate of CVB3 protease, leading to disrupted host cellular function and sheds further light on viral mechanisms of autophagy dysregulation. Enteroviruses (EVs) are a genus of small, non-enveloped, single-stranded positive RNA viruses of the Picornaviridae family and include poliovirus, enterovirus A71 (EV-A71), EV-D68, and coxsackievirus B3 (CVB3) ,3,4. Despol, replicates the viral genome through a negative strand intermediate to generate positive-stranded viral RNA that is subsequently packaged into icosahedral viral capsids. Eventually, viral progeny is released to infect nearby cells.A typical EV lifecycle begins when the viral capsid structure directly binds to a cell entry receptor such as the coxsackievirus and adenovirus receptor (CAR) for CVB3, resulting in the internalization of the virus . AfterwaEVs are obligate intracellular parasites that rely heavily on the cellular machinery of their hosts to replicate . The macThe autophagy pathway is a central component of cell-autonomous innate immunity that selectively targets and degrades various invading viral pathogens following infection. Nevertheless, many studies have reported that EVs can subvert the autophagy process as a viral strategy to evade host innate immunity and facilitate enhanced viral propagation. Most major EVs, such as poliovirus, coxsackievirus, enterovirus A71 and D68, have been shown to stimulate autophagosome formation upon infection, as evidenced by LC3 lipidation and puncta formation, as well as the presence of intracellular double-membraned vesicles ,15. IntrTo better understand how EV subverts autophagy, we screened canonical autophagy factors and found out that several proteins were downregulated following EV infection, including ATG4A ,18. In tHeLa and HEK293T cells obtained from American Type Culture Collection (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.The pEGFP-LC3 plasmid was purchased from addgene (#21073). The 3′Flag-ATG4A (hereby shortened as Flag-ATG4A) and 3′Flag-ATG4B (hereby shortened as Flag-ATG4B) were generated by cloning ATG4A and ATG4B into the multiple cloning sites of p3×Flag CMV10 vector with HindIII/Xbal (ATG4A) and EcoRI/BamHI (ATG4B), respectively. Mutant G374E-ATG4A was generated by site directed mutagenesis and cloning into 3′Flag plasmid. Truncated-ATG4A  was generated by cloning into 3′Flag plasmid with HindIII and KpnI. 5) for transfection overnight. For RNA interference transfection, Hela cells were transiently transfected with ATG4A siRNA  and scrambled siRNA  using lipofectamine 2000 following the manufacturer’s instruction. To assess cell viability, siRNA-treated cells were incubated with MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent  following the manufacturer’s guidelines. Briefly, cells were incubated with 10% MTS solution in complete medium for 2 h at 37 °C. SpectraMax iD3 microplate reader was used to record absorbance at 490 nm of control and treated cells.Plasmid transfections were performed using Lipofectamine 2000  according to the manufacturer’s guidelines. Briefly, 1 mg of DNA diluted in OPTI-MEM  was combined with Lipofectamine and incubated at room temperature for 15 min. DNA/lipofectamine mixture was then added dropwise to 6-well plates along with cells (5.5 × 10Seeded cells were infected with CVB3 at different multiplicities of infection (MOI) for various time points as indicated or sham-infected with DMEM. HeLa cells were infected with an MOI = 10 whereas HEK293T cells were infected with an MOI = 100 as previously reported . Viral tw/v) SDS, 10% glycerol, 0.01% (w/v) bromophenol blue, and 1.25 M of dithiothreitol). The denatured proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Proteins were transferred onto 0.45 μm nitrocellulose membrane for 80 min. at 100 V. For total protein visualization, membranes were stained with Ponceau S (0.1% w/v in 5% acetic acid) and visualized before rinsing with TBST wash buffer. After protein transfer, the membranes were incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: anti-ATG4A antibody , anti-ATG4C antibody , anti-VP1 antibody , anti-β-actin antibody , anti-LC3B antibody , anti-Flag antibody , anti-HSP90 . The membranes were then incubated with secondary antibodies for 1 h at room temperature before visualization by chemiluminescence. All primary and secondary antibodies were diluted in 2.5% BSA + Tris-buffered saline with 0.1% Tween® 20 detergent (TBST) with 1:1000 dilutions.Cells were lysed and harvested with Modified Oncogene Science lysis buffer  containing protease inhibitors. Lysates were denatured in 6× sodium dodecyl sulfate protein loading buffer  CVB3 protease 2A or 3C (0.1 µg) in a cleavage assay buffer  for 2 h at 37 °C. Reactions were terminated with 6× SDS sample buffer, followed by 95 °C denaturation and subsequent Western blot analysis.CT−ΔΔ) method with control samples and presented as relative quantitation (RQ). Gene expression of 2A and VP1 was normalized to ACTB mRNA.Total RNA was extracted using RNeasy Mini Kit . To determine viral gene expression levels, quantitative PCR (qPCR) was conducted with primer pairs targeting viral 2A , VP1 , ATG4A  and ACTB . The qPCR reaction containing 1 μg of RNA was performed using the TaqMan™ RNA-to-CT™ 1-Step Kit  on a ViiA 7 Real-Time PCR System (Applied Biosystems). Samples were run in quadruplicate and analyzed using comparative CT (2Immunoprecipitation was performed using anti-FLAG magnetic beads (M8823 Sigma-Aldrich). Briefly, cell lysates were incubated with the beads overnight at 4 °C. Bounded proteins were then eluted with 1% SDS sample buffer and the reactions were terminated by incubating at 95 °C for 10 min. Immunoprecipitated samples were subjected to Western blot analysis.n > 30 cells per condition).HeLa cells were cultured in 8-well chamber slides  for 24 h. The cells were then transfected with pEGFP-LC3 together with WT-ATG4A or T-ATG4A for an additional 24 h. GFP-LC3 puncta was visualized by Zeiss LSM 880 inverted confocal microscopy and quantified using NIH Image J version 1.53c. Number of GFP-LC3 puncta-positive cells was counted and divided by total cell number to yield the percentage of GFP-LC3 puncta-positive cells  of 100 and 10, respectively, for various time-courses. The viral doses selected have been previously shown to facilitate optimal infection in respective cell type . Cell lyWe then decided to elucidate the mechanism of degradation. Since no endogenous ATG4A fragments were observed after CVB3 infection with an anti-C-terminal targeting antibody, we generated two constructs by inducing Flag tags to the N terminus of ATG4A and ATG4B. ATG4B was chosen to be cloned as it is the most active isoform of ATG4 . Flag-ATGiven that CVB3 infection resulted in the proteolytic processing of ATG4A, we next sought to identify whether viral protease is responsible for ATG4A cleavage. CVB3 expresses two cysteine proteases, 2A and 3C, that are used primarily to process the viral polyprotein. We performed an in vitro cleavage assay, in which Flag-ATG4A was transfected into HEK293T cells and the corresponding lysates were incubated with either purified 2A or 3C. Western blot analysis demonstrated no evident changes in the levels of full-length Flag-ATG4A in 3C-treated cell lysates A. Given We next set out to identify the site of cleavage by 2A. The consensus cleavage sequence of protease 2A, shown in Given that CVB3 directly cleaves ATG4A, we wondered what precise role this host factor plays during the life cycle of CVB3 infection. To address this, we silenced endogenous ATG4A by RNA interference in HeLa cells for 48 h, followed by CVB3 infection for an additional 8 h. Cell lysates were harvested and subjected to Western blot analysis to assess the efficacy of ATG4A silencing and expression level of viral capsid protein VP1. The latter was used as a proxy to measure viral replication. We found that knockdown of ATG4A was associated with significantly reduced viral protein production A. ConsisGiven that ATG4A is cleaved by viral protease 2A before glycine 374, we generated a plasmid expressing T-ATG4A (lacking amino acids 374–398) to mimic the cleaved fragment. We then assessed the effect of overexpression of WT-ATG4A and T-ATG4A on viral RNA expression. HeLa cells were transiently transfected with vector control, WT-ATG4A, or T-ATG4A. Following 24 h, cells were subjected to CVB3 infection and RNA was harvested at 1, 4, and 8 h pi. Intriguingly, quantitative assessment of viral RNA using viral VP1 primers demonstrated no significant changes between control, WT-ATG4A, and T-ATG4A expressing cells, suggesting that both WT-ATG4A and T-ATG4A do not significantly perturb viral RNA production F. We useOverall, these results may suggest a complex role of ATG4A in CVB3 propagation whereby both (1) loss of endogenous ATG4A and (2) overexpression of exogenous ATG4A are associated with anti-viral effects. We then wish to gain more mechanistic insight by exploring the functional impact of the cleavage of the ATG4A.We next asked whether T-ATG4A exhibits any impairment in protein function. Since the cleavage of ATG4A before glycine 374 results in the loss of the LIR on the C-terminus, we therefore rationalized that T-ATG4A may have impaired autophagy function due to disrupted LC3/GABARAP interaction. To assess whether there are any potential functional deficits of T-ATG4A with respect to autophagy, HeLa cells were transiently transfected with either WT-ATG4A or T-ATG4A along with GFP-labelled LC3. After 24 h, confocal imaging revealed a significantly increased accumulation of GFP-LC3-puncta in T-ATG4A expressing but not in WT-ATG4A expressing cells A. The acDespite a miniscule armada of merely eleven viral proteins, EVs are capable of disrupting complex biological systems, such as the cardiovascular and central nervous system, leading to a broad spectrum of human illnesses as minor as the common cold or as severe as viral myocarditis and heart failure ,23. AmonOur results demonstrated that CVB3 infection led to the specific downregulation of ATG4A. We discovered that degradation of ATG4A is the consequence of virus-encoded protease 2A-mediated cleavage. The cleavage takes place before G374, which separates the C-terminal kinase domains from the N-terminal LIR. In this study, we focused on the N-terminal cleavage fragment (~43 kDa) as the C-terminal fragment is too small (<3 kDa) to detect by conventional Western blot analysis. Although it is possible that ATG4A may be a collateral target of the promiscuous 2A protease, findings from the current study clearly demonstrate an impaired functional autophagy through abnormalities in LC3 lipidation upon the expression of cleaved T-ATG4A supporting a potential strategy by which virus may disrupt canonical autophagy functions through direct targeting of autophagy factors.Given that CVB3 targets ATG4A, we then wondered what role ATG4A plays in CVB3 infection. Autophagy can have either pro-viral or anti-viral effect on diverse viruses. For example, Hait et al.  reportedPrevious studies have shown that pro-viral factors can be targeted by viral proteases during late infection ,19. InteIn this study, we showed that the virally mediated cleavage of the LIR domain of ATG4A leads to impaired autophagy function, including reduced LC3 delipidation and GABARAP interaction. Interestingly, the overexpression of WT-ATG4A is associated with a significant reduction in LC3 puncta whereas approximately 70% of cells expressing T-ATG4A exhibit accumulation of LC3 puncta. Increased LC3 puncta can be interpreted as either an enhancement in the autophagosome biogenesis process or a disruption of the autophagic recycling machinery, resulting in reduced clearance of LC3. We rationalize that ATG4A overexpression is likely enhancing the delipidation process whereas overexpression of T-ATG4A results in the accumulation of LC3 puncta due to an impaired LC3-II delipidation process. The truncation of ATG4A may be a viral strategy to retain autophagosome membranes and indirectly prevent lysosomal fusion. Indeed, EVs have previously been shown to utilize viral protease to disrupt the autophagosome-lysosome fusion process ,30. In tIn addition to impaired LC3 interaction, we also showed decreased physical association between T-ATG4A and GABARAP. ATG4A has been reported to have high affinity and catalytic efficiency toward GABARAP, which was confirmed in our interaction assay . NotablyPicornaviridae viral protease. The cleavage poses one of the underlying mechanisms by which EVs evade autophagy. In summary, our study identified ATG4A as a novel pro-viral host factor that is co-opted by EV protease to impair host autophagy. To our knowledge, this is the first reported cleavage of a host protease by a


This ATP-gated ion channel has been shown to induce multiple metabotropic events such as changes in plasma membrane composition and morphology, ectodomain shedding, activation of lipases, kinases, and transcription factors as well as cytokine release. The specific signaling pathways and molecular mechanisms remain largely obscure. Using an unbiased genome-scale CRISPR/Cas9 screening approach in a murine T cell line, Ryoden et al.  identified three proteins involved in P2X7 regulation and signaling: Essential for Reactive Oxygen Species (EROS) is essential for P2X7 folding and maturation, and Xk and Vsp13a are required for P2X7-mediated phosphatidyl serine exposure and cell lysis. They further provide evidence for an interaction of Xk and Vsp13a at the plasma membrane and confirm the role of Xk in ATP-induced cytolysis in primary CD25 In a recent paper, the Nagata group investigated the mechanism of ATP-mediated phosphatidylserine (PtdSer) exposure in T lymphocytes. To this aim, the group created a TMEM16F−/− P2X7−/− double knockout (DKO) T cell lymphoma (WR19L) cell line, which they transformed with the murine P2X7k variant, the predominant splice form of P2X7 in WR19L WT cells [2+-dependent phospholipid scramblase and Ca2+-activated Cl− channel [2+-mediated responses in macrophages and HEK293T cells [P2X receptors (P2XRs) represent a family of trimeric, non-selective ion channels. The homomeric P2X7 subtype differs structurally and functionally from other P2X family members. Its ATP sensitivity is 10- to 100-fold lower and is therefore considered to act as a “danger signal” detector at sites of damaged tissue . UnexpecWT cells  which isWT cells . TMEM16F3T cells , 9 but w3T cells . Cells t3T cells .+ T lymphocytes from both WT and EROS−/− mice. In these unbiased screens, it was found that EROS−/− cells showed reduced P2X7 levels and therefore smaller P2X7-mediated cellular response toward ATP stimulation. This includes impaired phospholipid scrambling and dye-uptake in (DKO)-WR19L cells, reduced IL-1ß production of LPS primed human THP-1 derived macrophages and murine bone marrow–derived macrophages (BMDM), reduced caspase-1 activation in macrophages, and impaired CD62L shedding in CD4+ T cells [EROS was first described in 2017 by Thomas et al. and was shown to be essential for host defense via the phagocyte nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) by affecting the abundance of the membrane bound heterodimer of the multi-subunit complex NADPH oxidase . It rema T cells . On the  T cells  and Ryod+CD4+ T cell isolated from Xk−/− mouse spleens by 40% compared to WT cells [The second and third most abundant sgRNAs in the screen from Ryoden et al. are targeted against Xk and Vps13a. Mutations of these proteins are linked to two neurodegenerative syndromes with similar phenotypes, the McLeod syndrome (MLS) and chorea acanthocytosis (ChAc), respectively , 16. SimWT cells .2+ increase or indirectly by caspase activation and cleavage of intracellular domains [Xenopus oocytes where an influence on macropore formation and current facilitation, both hallmarks of P2X7 activation, was found [The asymmetric distribution of PtdSer and other phospholipids between inner and outer leaflet of the plasma membrane bilayer is maintained by P4-type ATPases actively positioning them to the inner leaflet –23. Duri domains . These s domains , 26. TME domains . A functas found . An intras found  and Ousias found  can be e2+ mediated by P2X7 are not essential for PtdSer exposure [In summary, using an adapted genome-scale CRISPR/Cas9 Screening protocol, Ryoden et al. identified three proteins, EROS, Xk, and Vps13a, that are important for P2X7-mediated PtdSer exposure. They show that EROS acts as a chaperone for P2X7, regulating the abundance of P2X7 in the plasma membrane, while Xk and Vps13a seem to be involved in downstream signaling of P2X7. Although they were able to show that Xk and Vps13a form a complex, the question how they are activated in the P2X7-mediated signaling cascades remains elusive as the increasing intracellular levels of Caexposure . Whether