Cytomegaloviruses (CMVs) have a highly restricted host range as they replicate only in cells of their own or closely related species. To date, the molecular mechanisms underlying the CMV host restriction remain poorly understood. However, it has been shown that mouse cytomegalovirus (MCMV) can be adapted to human cells and that adaptation goes along with adaptive mutations in several viral genes. In this study, we identify MCMV M117 as a novel host range determinant. Mutations in this gene enable the virus to cross the species barrier and replicate in human RPE-1 cells. We show that the M117 protein is expressed with early kinetics, localizes to viral replication compartments, and contributes to the inhibition of cellular DNA synthesis. Mechanistically, M117 interacts with members of the E2F transcription factor family and induces E2F target gene expression in murine and human cells. While the N-terminal part of M117 mediates E2F interaction, the C-terminal part mediates self-interaction. Both parts are required for the activation of E2F-dependent transcription. We further show that M117 is dispensable for viral replication in cultured mouse fibroblasts and endothelial cells, but is required for colonization of mouse salivary glands in vivo. Conversely, inactivation of M117 or pharmacological inhibition of E2F facilitates MCMV replication in human RPE-1 cells, whereas replacement of M117 by adenovirus E4orf6/7, a known E2F activator, prevents it. These results indicate that E2F activation is detrimental for MCMV replication in human cells. In summary, this study identifies MCMV M117 as a novel E2F activator that functions as a host range determinant by precluding MCMV replication in human cells. Human CMV is an opportunistic pathogen causing morbidity and mortality in immunocompromised individuals. It is a highly species-specific virus that replicates only in cells from humans or chimpanzees, but not in cells from mice or other laboratory animals. Mouse cytomegalovirus (MCMV), the most commonly used model to study CMV pathogenesis in vivo, is also species-specific and does not replicate in human cells. However, the causes of the CMV host species specificity have remained largely unknown. Here we show that the viral M117 protein is a major factor contributing to the MCMV host species specificity. When M117 is inactivated, MCMV acquires the ability to replicate in human cells. We further demonstrate that M117 interacts with transcription factors of the E2F family and activates E2F-dependent gene expression. While this function is needed for MCMV dissemination in mice, it is detrimental for MCMV replication in human cells. The results of this study indicate that the host range of a virus, i.e. its ability to replicate in cells from different hosts, can depend on an appropriate regulation of transcription factors. Viruses are obligate intracellular parasites. As such, they rely on suitable host cells for their replication. While some viruses can infect and replicate in cells from various tissues and different host species, others are highly adapted to their natural host and have a narrow host range ,2.Cytomegaloviruses (CMVs), representatives of the β-herpesvirus subfamily, are highly species-specific as they can replicate only in cells of their own or closely related host species . Human CA few studies have shown that MCMV is somewhat less restricted in its host range than HCMV ,7. An imAn attractive and promising strategy to identify novel host range determinants is to characterize mutations in viruses adapted to cells of a foreign host ,11. AlthOne important difference between HCMV and MCMV is their dependence on the cell cycle stage. HCMV-infected cells need to be in the G1 phase in order to express viral IE genes ,16, wherThe E2F transcription factors were first described in 1986 as DNA binding proteins activating the adenoviral E2a promoter . Since tSome viruses, such as adeno-, papilloma-, and polyomaviruses promote E2F-dependent transcription and cell cycle progression towards S phase through viral proteins   that disHere we show that the MCMV M117 protein interacts with all canonical E2F family members and activates E2F-dependent gene expression in MCMV-infected cells. M117 inactivation did not impair MCMV replication in murine cells, but massively reduced viral dissemination to the salivary glands of infected mice. By specific mutagenesis of M117 we showed that the interaction with E2F3 is of particular importance for transcriptional activation of target genes and for viral dissemination of MCMV in vivo. Intriguingly, M117 inactivation or pharmacological inhibition of E2F-dependent transcription facilitated MCMV replication in human cells while expression of the AdV E4orf6/7 protein, a known E2F activator, inhibited MCMV replication, suggesting that some E2F target proteins in human cells restrict MCMV replication.In previous work we have reported the isolation of three human cell-adapted MCMVs  and described the mutations associated with adaptation to human RPE-1 epithelial cells . All thrAccording to the MCMV genome annotation by Rawlinson  the M117To determine whether MCMV expresses M117 or m117.1 proteins during infection, we constructed recombinant MCMV viruses carrying an HA tag at the 3ˈ end of ORFs M117 or m117.1, respectively. The MCMV-M117-HA and MCMV-m117.1-HA viruses were used to infect murine fibroblasts, and expression of HA-tagged proteins was assessed by Western blot. In cells infected by MCMV-M117-HA, a protein with a molecular weight of approximately 100 kDa could be detected as early as 6 hours post infection (hpi) . The M11The M117 protein expression time course  suggesteNext, we wanted to determine the intracellular localization of the M117 protein during MCMV infection. Initial experiments performed with MCMV-HA-M117 did not yield satisfactory results as the immunofluorescence (IF) signals were very weak due to the low expression level of M117. To increase signal intensity, a mutant virus carrying a triple Flag tag at the N-terminus of M117 (MCMV-M117-FL) was created. During infection, the 3xFlag-tagged M117 protein could be detected in dot-like structures in the nuclei of infected cells. These dots colocalized with the viral E1 proteins , which aTaken together these findings suggest that M117 uses the predicted NLS for the localization in the cell nucleus but requires additional viral proteins or viral DNA, present during infection and absent in transfected cells, for its recruitment to viral replication compartments.The M117 ORF is encoded on the negative strand of the MCMV genome between M116 and M118 . Thus, i2 ratio ≥ 3 (HA-M117 vs WT) were considered as potential interaction partners. Besides M117 itself, only 4 proteins met these criteria: the viral DNA polymerase processivity factor M44 and three members of the E2F transcription factor family: E2F3, E2F4, and DP1 . Stable isotope labelling by amino acids in cell culture (SILAC) was used to compare the anti-HA immunoprecipitation products of cells infected with WT MCMV or MCMV-HA-M117. The experiment was done in duplicate including a label switch. Proteins identified by at least 2 unique peptides and a log and DP1 .To verify the candidates, M117 was immunoprecipitated from MCMV-infected cells using a different antibody (anti-Flag instead of anti-HA) and co-precipitating proteins were analyzed by immunoblot. WT MCMV, which does not express any Flag-tagged protein, and a recombinant virus expressing 3xFlag-tagged UL117 instead of M117 (MCMV-UL117-FL) were used as controls. Immunoprecipitates were separated by SDS-PAGE, blotted, and probed with antibodies specific for all canonical E2F transcription factors (E2F1-5) and DP1. Blots were also probed with antibodies specific for M44  and E1 . As shown in To determine which part of M117 is important for the interaction with E2F proteins, N- and C-terminal truncation mutants (ΔNter and ΔCter) were used for co-immunoprecipitation experiments. While deletion of the C-terminal 281 amino acids (aa) did not impair the co-precipitation of E2F transcription factors, deletion of the N-terminal 50 aa was sufficient to abrogate these interactions in MCMV infected cells . The samAs such large deletions could affect the protein’s conformation, we tested whether an alanine substitution mutation of a highly conserved motif IPP→AAA,  Figs wouTaken together, these data show that M117 interacts with E2F transcription factors via its N-terminus, whereas the C-terminus is essential for its self-interaction (dimerization). Moreover, a specific mutation leads to the loss of interaction with specific E2Fs, suggesting a change in the affinity of M117 for specific E2Fs.The E2F transcription factors have been classified as functional activators or repressors of gene expression ,56. As MTo verify the results obtained with the luciferase reporter assay, we measured the impact of MCMV infection on the transcription of selected cellular genes known to be regulated by E2Fs . NIH-3T3We also analyzed the protein levels of Cyclin A2 and Cyclin B1, representative E2F target gene products, by immunoblot. Unfortunately, none of the antibodies against mouse Cyclin E1 that we tested worked reliably in our hands. Hence we were unable to determine Cyclin E1 protein levels. As shown in To examine the importance of E2F3 in the M117-mediated E2F dependent transcription, we generated E2F3 knockout (KO) NIH-3T3 cells using CRISPR/Cas9 genome editing. Two cell clones lacking E2F3 expression and two control clones  were useTaken together, the data in Considering the clear effects of M117 on E2F-dependent gene expression  and cellTo exclude that the salivary gland phenotype of the MCMV mutant lacking M117 was caused by an unspecific effect of the deletion on neighboring genes, we infected mice with MCMV-M117stop. We also included MCMV N- and C-terminal M117 truncation mutants as well as the MCMV-M117-M4 mutant. On day 14 post infection, salivary gland titers of MCMV-M117stop, ΔNter, and ΔCter2 were below the detection limit , thus coIn the beginning of this study, we characterized M117 as a host range determinant: mutations causing the expression of C-terminally truncated M117 proteins allowed MCMV replication in human RPE-1 cells . HoweverTo understand whether the function of M117 was similar in mouse and human cells, we performed experiments similar to the ones described above. First, we checked if M117 was able to interact with the human E2F members. M117-HA was immunoprecipitated from RPE-1 infected cells and co-precipitating proteins were analyzed by immunoblot. WT MCMV, which does not express any HA-tagged proteins, was used as a control. Immunoprecipitates were separated by SDS-PAGE, blotted, and probed with antibodies specific for two E2F transcription factors, E2F3 and E2F4. As shown in Finally, we wanted to clarify whether the M117-mediated E2F activation or another hitherto unknown function of M117 precludes MCMV replication in human cells. To address this question, we tested whether HLM006474, a pan-E2F inhibitor , could fAs E4orf6/7 from AdV 5 shares functional similarities with M117, such as the interaction with E2Fs and activation of the E2F-dependent transcription ,59, we cIn this work we characterize the properties, functions and cellular interacting partners of M117, a previously uncharacterized MCMV protein. M117 has sequence similarity to pUL117, its presumed homolog in HCMV, and the two proteins show functional similarities: Both are expressed with early kinetics, localize to viral replication compartments, and contribute to the blockage of host DNA synthesis. However, there are important differences: Inactivation of UL117 in HCMV lead to a profound replication defect of the virus in cell culture , whereasSimilar to E4orf6/7, the absence of M117 does not affect viral replication in cell culture, suggesting that another viral protein can compensate for the loss of M117. In the case of E4orf6/7, the AdV E1A protein provides the redundant function by inducing pRb phosphorylation ,62. We pInduction of E2F-dependent transcription by M117  is expecWhen we analyzed the ability of M117 mutant MCMVs to replicate and disseminate in vivo, we observed a profound drop in the salivary gland titers at 14 dpi , indicatFinally, we demonstrated that M117 acts as a host range determinant. Activation of the E2F-dependent transcription by M117 or by E4orf6/7 is detrimental for the replication of MCMV in human RPE-1 cells, and inhibiting this activation, either by deleting M117 or by adding an inhibitor, allows the virus to replicate in human cells Figs . The reaAltering the viral host range by adapting a virus to cells of a different host can raise biosafety concerns if a virus with zoonotic potential is involved. This is not the case for the cytomegaloviruses: They are highly adapted to their specific host and cause persistent infections only because of numerous immune evasion mechanisms , many ofIn conclusion, this study identified MCMV M117 as a new viral E2F regulator, which is important for viral fitness in vivo. M117 also acts as a host range factor that prevents MCMV replication in human RPE-1 and MRC-5 cells. Further work needs to be done to understand the function of this protein in cross-species infection as well as the differences in E2F-mediated gene regulation between human and mouse cells.All animal experiments were performed according to the recommendations and guidelines of the FELASA  and Society of Laboratory Animals (GV-SOLAS) and approved by the institutional review board and local authorities .5 PFU MCMV per mouse as described [Six to 8 week-old BALB/c female mice (Janvier laboratories) were infected intraperitoneally with 10escribed . Organs 2. RPE-1 cells are human telomerase reverse transcriptase immortalized human retinal pigment epithelial cells (ATCC CRL-4000). MRC-5 cells are primary human embryonic lung fibroblasts (ATCC CCL-171). Murine NIH-3T3 fibroblasts (CRL-1658), SVEC4-10 endothelial cells (CRL-1658), and M2-10B4 bone marrow stromal cells (CRL-1972) were obtained from the ATCC. Murine 10.1 fibroblasts are spontaneously immortalized MEFs from BALB/c mice [g, 30 min) was used. For replication kinetics, cells were infected in six-well dishes. After 4 h, cells were washed twice with phosphate-buffered saline (PBS), and fresh medium was added. Supernatants were harvested at different times post infection for virus titration on 10.1 cells using the median tissue culture infective dose (TCID50) method.All cells were maintained in complete Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 100 IU Penicillin/100 μg Streptomycin at 37°C and 5% COB/c mice  obtainedB/c mice . The GFPB/c mice ,80. All en passant mutagenesis [Mutations within the M117 locus were introduced by agenesis  into theagenesis . To recoThe M117 coding sequence was PCR-amplified from the MCMV Smith genome using a forward primer containing a HindIII restriction site and the 3xFlag sequence and a reverse primer containing an MfeI restriction site. The PCR product was cleaved with HindIII and MfeI and inserted into expression plasmid pcDNA3 (Invitrogen) digested with HindIII and EcoRI. Deletions or substitutions of nucleotides were performed by PCR-driven overlap extension . Transfewww.e-crisp.org) was used to design three guideRNAs that were cloned individually in the lentiviral vector: g1, GATGGTCTAAAGACCCCCAA; g2, GGATCTGAACAAGGCAGCAG; and g3, GGACCTCAAACTGTTAACCG. Lentiviruses were generated using standard third-generation packaging vectors in HEK-293T cells. Then, NIH-3T3 cells were transduced with an E2F3 lentiviral CRISPR/Cas9 or an empty vector (g0) in the presence of polybrene (Sigma). The cells were selected with 2.5 μg/mL puromycin (Sigma). Polyclonal cultures were subcultured to obtain single cell clones for each gRNA, and E2F3 protein expression was evaluated for each clone by Western blot analysis.The lentiviral CRISPR/Cas9 vector pSicoR-CRISPR-PuroR was used to generate E2F3 KO clones essentially as described . Briefly50/cell. Total RNA was extracted from infected cells using the innuPREP RNA Mini Kit  and contaminating DNA was removed using the TURBO DNA-free Kit (Ambion). cDNA was synthesized from 1μg of the extracted RNA by using the RevertAid H Minus Reverse Transcriptase, oligo-dT primers, and the RNase inhibitor RiboLock (Thermo Fisher Scientific). qPCR was performed on an ABI PRISM 7900HT Fast Real-Time PCR System (Applied Biosystem) using 10 ng of cDNA, the SybrGreen real time PCR Mastermix (Life technologies). To amplify mouse transcripts, the following primers were used: Gapdh (CCCACTCTTCCACCTTCGATG and GTCCACCACCCTGTTGCTGTAG), PCNA (CACGTATATGCCGAGACCTTAGC and CTCCACTTGCAGAAAACTTCACC), Cyclin A2 (GAGGGCCATCCTTGTGGACT and CACAGCCAAATGCAGGGTCT), and Cyclin E1 (CAGAGCAGCGAGCAGGAGA and GCAGCTGCTTCCACACCACT). For the amplification of human transcripts, the following primers were used: hPCNA (GGCACTCAAGGACCTCATCAAC and GTGAGCTGCACCAAAGAGACG), hGAPDH (CCCACTCCTCCACCTTTGACG and GTCCACCACCCTGTTGCTGTAG), hCyclin A2 (TGCTGGAGCTGCCTTTCATT and TGAAGGTCCATGAGACAAGGCT), and hCyclin E1 (TACACCAGCCACCTCCAGACAC and CCTCCACAGCTTCAAGCTTTTG). Transcripts were quantified using the ΔΔCt method and normalized to a housekeeping gene (Gapdh).To quantify transcripts, synchronized NIH-3T3 or RPE-1 cells were infected at a MOI of 3 TCIDFor MCMV genome quantification, total DNA was extracted from MCMV-infected RPE-1 cells using an InnuPREP DNA Mini Kit . One hundred ng of DNA was subjected to qPCR in order to quantify MCMV genome copies (primers ACTAGATGAGCGTGCCGCAT and TCCCCAGGCAATGAACAATC) and human β-actin gene copies (primers GCTGAGGCCCAGTTCTAAAT and TTCAAGTCCCATCCCAGAAAG).NIH-3T3 cells were synchronized for 48 h by serum starvation (DMEM/ 0.5% FCS) and then infected with MCMV in the presence of 10% FCS. After 1 h, cells were washed and new media containing 50 μM Ganciclovir was added. To measure both DNA content and viral protein expression, cells were first permeabilized by incubation in 75% ethanol for at least 12 h at 4°C. Afterwards, cells were stained with antibodies against IE1  and mouse-IgG-Alexa647, before staining for DNA content with propidium iodide and analyzed by flow cytometry as previously described .5 cells/well) and transfected with 1 μg pGL2-E2F and 100 ng of pRL-Renilla (Promega) using Polyfect (Qiagen). After 6 h the medium was exchanged, and after 24 h cells were infected at a MOI of 3 TCID50/cell. At 24 hpi, cells were harvested and luciferase activity was measured on a FLUOstar Omega reader (BMG Labtech) using a Dual Luciferase Reporter Assay (Promega) and according to the manufacturer’s protocol.The luciferase reporter plasmids were kindly provided by Matthias Truss . They are based on a promoter/enhancer-less pGL2-basic (Promega) modified by insertion of a TATA box and an E2F binding site . Plasmid50/cell or transfected with pcDNA3 expression plasmids using Polyfect (Qiagen). Cells were fixed in methanol for 10 min at -20°C, washed with PBS, and blocked with PBS containing 1% gelatin. Incubations with the antibodies were carried out for 1 h at room temperature (RT) in 1% gelatin/PBS. Nuclei were stained for 10 min at RT with DRAQ5 (BioStatus) diluted 1:1000 in PBS. Coverslips were mounted on microscope slides with Aqua-Poly/Mount (Polysciences) and subjected to confocal laser scanning microscopy (cLSM).NIH-3T3 cells were seeded onto glass coverslips coated for 30 min with 0.4% gelatin/PBS. On the following day, cells were infected with MCMV-3xFlagM117 at an MOI of 1 TCIDThe following antibodies were used: monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , β-actin , HA , FLAG , DP1 , and Cyclin B1 . Antibodies against MCMV gB (M55.01), IE1 (CROMA101), and M112-113 (CROMA103) were provided by Stipan Jonjic , anti-M44 (3B9.22A) was provided by Lambert Loh, University of Saskatchewan, Canada. The monoclonal antibody RSA3 against AdV 5 E4orf6/7  was kind50/cell. At 24 hpi, cells were lysed in a NP-40 buffer . Insoluble material was removed by centrifugation. After preclearing with protein G Sepharose , Flag-tagged M117 protein was precipitated with anti-Flag and PGS. Precipitates were washed 3 times with buffer 1 , 2 times with buffer 2  and 1 time with buffer 3 (10mM Tris pH 7.6), eluted by boiling in SDS-PAGE sample buffer  and subjected to SDS-PAGE and immunoblotting.Cells grown in 6-well plates were infected at an MOI of 3 TCIDFor immunoblot analysis, cells were lysed in NP-40 buffer or in SDS-PAGE sample buffer. Equal amounts of protein (NP-40 lysis) or equal volumes (sample buffer) were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane (Amersham). Proteins of interest were detected with protein-specific primary antibodies and HRP-coupled secondary antibodies by enhanced chemiluminescence (Amersham) supplemented with 10% of Lumigen TMA-6 (Bioquote Limited). All immunoblots showed are representatives of 3 or more experiments, except for 13C6,15N2-lysine / 13C6,15N4-arginine (SILAC heavy) or unlabeled lysine / arginine (SILAC light). All SILAC media were filtered before usage and stored at 4°C for up to 6 month. To ensure complete incorporation of SILAC amino acids (≥ 97%) cells were grown for at least 5 passages in SILAC medium. For immunoprecipitation, two populations were generated, one grown in light and the other in heavy media. Either the light or the heavy labelled cells were infected with the HA-M117, whereas the other labelled population was infected with the WT MCMV. After lysis in NP-40 buffer, HA tagged-M117 proteins were precipitated with rat anti-HA Affinity Matrix antibody . Precipitates were washed 3 times with minimal washing buffer  glycerol, pH 7.5) and eluted by boiling in elution buffer (1% (w/v) SDS in 50 mM Tris, 150 mM NaCl, pH 7.5). Afterwards, the samples were mixed in a light-to-heavy ratio of 1:1 for subsequent sample processing.For stable isotope incorporation, cells were grown in DMEM (high-glucose) SILAC medium supplemented with 10% dialyzed FCS, 4 mM glutamine, 100 IU penicillin/100 μg streptomycin in the presence of either 2. Proteins were digested by adding 5 μl of a 0.1 μg/μl trypsin solution (Sigma-Aldrich T-1426) followed by incubation at 37°C for 12.5 h. The reaction was stopped by adding TFA to a final concentration of 1%. Samples were dried in a vacuum centrifuge, reconstituted in 0.1% TFA and subjected to LC-MS.Cysteines were reduced in the presence of 10 mM DTT at 56 °C for 30 min and alkylated using 20 mM IAA for 30 min at RT in the dark. Proteins were precipitated by diluting the samples 1:10 with ice-cold ethanol (-40 °C) and incubation at -40 °C for 1 h. Precipitated proteins were spun down at 20,000 x g at 4 °C for 40 min and the pellet was washed with 50 μl ice-cold acetone (-40 °C). After 15 minutes of incubation at -40 °C proteins were spun down for 15 min at 20,000 x g and 4 °C. The sediment was solubilized in 7.5 μl 2 M guanidinium hydrochloride (GuHCl) and 10-fold diluted using 50 mM ABC and 1 mM CaCl6 and 5x104 with maximum ion injection times of 120 and 250 ms for MS and MS/MS, respectively. The dynamic exclusion was set to 12 sec and the m/z = 371.10124 lock mass was used for internal calibration.NanoLC-MS/MS was conducted using a U3000 HPLC system online coupled to a Q Exactive HF (both Thermo Scientific). Samples were loaded in 0.1% TFA on a C18 trap column  and separated on a C18 main column  using a 60 min linear gradient-program from 2.5% to 35% ACN in the presence of 0.1% FA at 60°C and a flow rate of 270 nL/min. The column effluent was introduced to the MS by nanoESI using a PicoTip emitter (new objectives) operated at 1.5 kV. The MS was operated in positive ion-mode using a top15 HCD data-dependant acquisition method with a resolution of 60,000 for MS and a resolution of 15,000 for MS/MS. The normalized collision energy was set to 27 and only ions with an assigned charge state of 2–4 were selected for fragmentation. Automatic gain control target values were set to 10Mus musculus and all Uniprot/TreEMBL entries of MCMV strain Smith and K181 . The decoy search option was enabled and mass error tolerances were set to 10 ppm and 0.02 Da for MS and MS/MS. A maximum of 2 missed cleavages was allowed, Oxididation of Met and heavy labelled Arg/Lys was set as variable modification and carbamidomethylation of Cys was set as static modifications. Results were filtered for ≤ 1% FDR at the PSM level and feature quantification was done within a 2 ppm mass precision window using the precursor ion quantifier. PSM tables were exported and further processed with Microsoft Excel and R to calculate the number of unique peptides per protein in each condition and calculate median-normalized heavy/light ratios. Proteins identified with ≥ 2 unique peptides and a log2 ratio ≥ 3 (8-fold enrichment) in both replicates were considered as potential M117 interaction partners.A database search was done using Mascot v2.4.1 implemented in Proteome Discoverer v1.4 against a merged database comprising all Uniprot entries of All the statistical analyses were performed with the GraphPad Prism 5.0 Software. A one-way ANOVA with Tukey’s multiple comparison post test was used for the analysis of the qPCR on genomic DNA. A two-way ANOVA with Bonferroni post test was used for the analysis of the luciferase reporter assays and qPCR on transcripts.S1 FigThe amino acid sequences of the M117 and its homologs in the English and Maastricht isolates of RCMV , HCMV (UL117), and Chimpanzee CMV (UL117-C) were aligned using Multalin software . Identic(TIF)Click here for additional data file.S2 Fig(A) NIH-3T3 cells were infected with MCMVs expressing 3xFlag-tagged M117 or UL117 full-length (FL) or mutant proteins. Cell lysates were subjected to immunoprecipitation (IP) using an anti-E2F3 antibody. Co-precipitating M117 proteins were detected by Western blot analysis. (B) NIH-3T3 cells were transfected with pcDNA3 expression plasmids encoding 3xFlag-tagged M117 proteins with N-terminal 50 aa deletions. Cell lysates were subjected to immunoprecipitation (IP) using an anti-Flag antibody. Co-precipitating E2F proteins were detected by Western blot analysis. (C) Schematic of the M117 mutants used in this study. ΔCter, deletion of aa 285–565; ΔCter2: frameshift of aa 449–481; ΔNter, deletion of aa 1–50 aa; ΔNter2, deletion of aa 51–100; ΔNter3, deletion of aa 101–150; ΔNter4, deletion of aa 151–200; M4, IPP→AAA substitution at positions 59–61.(TIF)Click here for additional data file.S3 Fig50/cell. Supernatants of infected cells were harvested at the indicated times post infection and titrated. The experiments were done in triplicate. Mean ±SEM are shown. DL, detection limit.Primary MEF (A) or SVEC4-10 endothelial cells (B) were infected with WT and mutant MCMV at an MOI 0.02 TCID(TIF)Click here for additional data file.S4 Fig50/cell. Three hours post infection, cells were treated with HLM006474 (+) for 24 or 48 hours or left untreated (-). Cell lysates were subjected to immunoprecipitation using an anti-Flag antibody. Co-precipitating proteins were detected by Western blot analysis. *, antibody heavy chain.Human RPE-1 cells were infected with mutant MCMVs at an MOI of 2 TCID(TIF)Click here for additional data file.


There is paucity of data on the virulence of highly pathogenic (HP) avian influenza viruses (AIV) H7 in ducks compared to HPAIV H5. Here, the virulence of HPAIV H7N1 (designated H7N1-FPV34 and H7N1-It99) and H7N7 (designated H7N7-FPV27) was assessed in Pekin and/or Muscovy ducklings after intrachoanal (IC) or intramuscular (IM) infection.The morbidity rate ranged from 60 to 100% and mortality rate from 20 to 80% depending on the duck species, virus strain and/or challenge route. All Muscovy ducklings inoculated IC with H7N7-FPV27 or H7N1-FPV34 exhibited mild to severe clinical signs resulting in the death of 2/10 and 8/10 ducklings, respectively. Also, 2/10 and 6/9 of inoculated Muscovy ducklings died after IC or IM infection with H7N1-It99, respectively. Moreover, 5/10 Pekin ducklings inoculated IC or IM with H7N1-It99 died. The level of virus detected in the oropharyngeal swabs was higher than in the cloacal swabs.Taken together, HPAIV H7 cause mortality and morbidity in Muscovy and Pekin ducklings. The severity of disease in Muscovy ducklings depended on the virus strain and/or route of infection. Preferential replication of the virus in the respiratory tract compared to the gut merits further investigation. Orthomyxoviridae. The genome of AIV contains eight gene segments, which encode at least 11 viral proteins. They are classified according to the antigenic properties of the hemagglutinin (HA) and neuraminidase (NA) proteins into 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes [Avian influenza viruses (AIV) belong to the family subtypes . Wild ansubtypes –9. Muscosubtypes , 4, 8. HHPAIV H7 were first isolated in Europe in the early 1900s and have frequently been detected in poultry and wild birds in several European countries in the last two decades. Historical outbreaks caused by several H7Nx viruses in Europe in 1902, 1927, 1934, 1980s as well as recent outbreaks in 1999, 2003 and 2015 were described –15. TherCairina moschata) after intrachoanal (IC) inoculation. Furthermore, the impact of route of infection and species of ducks on virulence of H7N1-It99 was studied in Muscovy ducks and Pekin ducks (Anas platyrhynchos domesticus) after IC or intramuscular (IM) infection. The IC route was recommended for studying the pathogenesis of AIV to simulate natural upper respiratory exposure/transmission and to ensure that each bird receives the full dose [The objective of this study was to compare the virulence of two historic HPAIV A/FPV/Dutch/27 (H7N7) (designated hereafter as H7N7-FPV27) and A/Germany/FPV/1934 (H7N1) (designated hereafter H7N1-FPV34) and the recent HPAIV A/chicken/Italy/445/1999 (H7N1) (designated hereafter H7N1-It99) in Muscovy ducks  and H7N1-FPV34 (group 2) in Muscovy ducks was assessed. The impact of duck species and inoculation route on virulence of H7N1-It99 was tested in Muscovy (groups 3 and 4) and Pekin ducks (groups 5 and 6) after IC or IM infection. All birds were observed for 10 days and clinically scored as 0 , 1 (sick), 2 (severely sick) or 3 (dead) and the pathogenicity index (PI) was calculated as a scale from 0 (avirulent) to 3 (highly virulent) .After challenge, Muscovy ducks showed diarrhea and nervous signs including circling, rolling, incoordination, steady gait and/or opisthotonus starting from 2 dpi. The morbidity rate ranged from 60 to 100% and mortality from 20 to 80%  showed mild depression at 1 dpi, which became more prominent at 2 dpi. All ducklings in this group returned to normal starting from 3 dpi except two ducklings that died or were killed for humane reasons by day 7 (Table  showed mp < 0.01) more severe and prominent signs than ducklings in group 1 and group 3 with a PI of 1.8  exhibited significantly p < 0.0 more sevSeven out of ten Muscovy ducklings inoculated IC with H7N1-It99 (group 3) showed neurological signs, while three ducklings remained healthy to the end of the experiment (PI of 0.5). Clinical signs started at 4 dpi. One bird showed mild to severe clinical signs from 4 dpi to 10 dpi. Meanwhile, four birds showed mild transient depression and recovered by 8 dpi. Two ducklings were killed at 5 and 6 dpi (and scored dead at the next day) for humane reasons after showing severe clinical signs (MDT = 6.5) (Table Muscovy ducklings inoculated IM with H7N1-It99 (group 4) showed higher morbidity and mortality compared to IC-inoculated animals (group 3). In total, six out of nine Muscovy ducklings inoculated IM with H7N1-It99 died with a PI of 1.4 and MDT of 5.7 days: three ducklings were killed at 4 dpi and one at 7 dpi. Two birds were found dead at 5 and 6 dpi. Two ducklings remained sick until 10 dpi and one bird recovered at 9 dpi  showed clinical signs (PI 0.9) where five Pekin ducklings died at 5 dpi , day 6 dpi (n = 2) and 7 dpi (n = 1) after showing mild to moderate nervous signs .Eight out of ten Pekin ducklings injected IM with H7N1-It99 (group 6) showed clinical signs (PI = 0.9) where five Pekin ducklings died at 5 dpi  and oropharyngeal (OP) swabs collected from inoculated birds at 2, 4, 7 and 10 dpi were tested by real time RT-PCR. Results are summarized in Figs. P < 0.001) at 2 dpi  –30. InteTaken together, the three European H7 viruses used in this study exhibited variable virulence in Muscovy ducklings. H7N1-FPV34 induced 80% mortality, while H7N7-FPV27 and H7N1-It99 killed only 20% of IC-inoculated ducklings. H7N1-It99 exhibited higher virulence in IM-injected Muscovy than in IC inoculated ducklings with 66.7% and 20 mortality, respectively. Furthermore, H7N1-It99 exhibited moderate virulence in Pekin ducklings with no difference between the IC or IM inoculation routes. Moreover, regardless of the challenge route, Muscovy ducks excreted higher amounts of H7N1-It99 than Pekin ducks. Findings in this study showed the variable virulence of HPAIV H7 in different duck species.The main goal of this study was to assess the virulence of three European H7 viruses in domestic ducks. Muscovy and Pekin ducklings were inoculated via the intrachoanal and/or intramuscular and were observed for 10 days post inoculation/injection (dpi). Swab samples were collected from all ducklings at 2, 4, 7 and 10 dpi and tested by generic real time RT-PCR.Viruses in this study were kindly provided by Timm C. Harder. All viruses were propagated in the allantoic sac of specific pathogen free (SPF) embryonated chicken eggs (ECE) (VALO BioMedia GmbH) according to the standard protocol . Allanto2. Cells were overlaid with semisolid BactoTM Agar (BD) containing minimal essential medium (MEM) and 4% bovine serum albumin (BSA) . All plates were incubated for 3 days at 37 °C. Cells were fixed by 10% formaldehyde containing 0.1% crystal violet. Plaques were counted and viral titers were expressed as plaque forming units per ml (PFU/ml).Virus titration was done using plaque assay. Confluent MDCKII cells in 12-well plates were infected with ten-fold serial dilutions of specified viruses for an hour at 37 °C/5% COAnimal experiments were carried out after approval by the authorized ethics committee of the State Office of Agriculture, Food Safety, and Fishery in Mecklenburg – Western Pomerania (No. 7221.3-1-060/17) and approval by the commissioner for animal welfare at the FLI representing the Institutional Animal Care and Use Committee (IACUC) following the German Regulations for Animal Welfare.5 PFU via the IC or IM route as described in Table One-day old commercial Pekin and Muscovy ducklings were purchased from Czarkowski GbR, Storkow, Germany. At the FLI, swab samples were collected from all ducks and examined to exclude infection by influenza  and Salm1 to 106 pfu/mL) were run in each RT-qPCR round. The relative amount of excreted virus was quantified by plotting the Ct-values in the standard curves and results are expressed as average ± standard deviation equivalent log10 PFU/ml.OP and CL swabs were collected before infection and at 2, 4, 7 and 10 days post inoculation/injection (dpi) on swab media and stored at − 70 °C until use. Swabs medium contained (pro liter) MEM Eagle (Sigma-Aldrich), 5.6 mL BSA  and antibiotics . The RNA was extracted from swab media using NucleoMagVet® 8/96 PCR Clean-up Core Kit  in KingFisher Flex Purification System . The quantity of virus excretion in swab samples was determined using SuperScript™ III Platinum™ One-Step qRT-PCR Kit  according to the manufacturer guidelines and generic real-time reverse-transcription polymerase chain reaction (RT-qPCR) . The RT-C. sera were tested for anti-AIV nucleoprotein (NP) using enzyme-linked immunosorbent assay (ELISA) by ID screen Influenza A Antibody Competition Multispecies kit (IDvet). Plates were read in Tecan® ELISA reader. The cut-off point according to the manufacture guideline was 55%, samples between 45 and 55% were considered questionable and samples lower than 45% were considered negative.Blood was collected before infection (5 samples pro species) via wing vein puncture and at the end of the experiments from all surviving ducks after euthanization using isoflurane® (CP-Pharma). Briefly, the ducklings were gently and carefully put inside a tightly close beaker containing four to five isoflurane-soaked gauze sponges for about 2 min to ensure deep anesthesia. Complete loss of consciousness was achieved as assessed by complete suppression of pedal and ocular reflexes. Ducklings were taken out and whole blood was collected in 50 mL Falcon tubes via cutting the jugular vein using knife. Thereafter, the head was separated from the body rapidly and completely. The serum was separated from the blood after 24 h incubation in the fridge followed by centrifugation and inactivation at 72 °p value < 0.05. All analysis was done by GraphPad Prism software. Clinical scoring for mean values for each bird in 10 day-observation period was compared.Statistical differences were analyzed using non-parametric Kruskal-Wallis and Mann-Whitney Wilcoxon tests with post hoc Tukey tests. Results were considered statistically significant by any test at


Among the novel H3R antagonists examined, H3R antagonist 4 shortened the duration of tonic hind limb extension (THLE) in a dose-dependent fashion in the maximal electroshock (MES)-induced seizure and offered full protection against pentylenetetrazole (PTZ)-induced generalized tonic-clonic seizure (GTCS), following acute systemic administration . However, only H3R antagonist 13, without appreciable protective effects in MES- and PTZ-induced seizure, fully protected animals in the strychnine (STR)-induced GTCS following acute systemic pretreatment . Moreover, the protective effect observed with H3R antagonist 4 in MES-induced seizure was completely abolished when animals were co-administered with the H3R agonist (R)-α-methylhistamine . However, RAMH failed to abolish the full protection provided by the H3R antagonist 4 in PTZ-induced seizure and H3R antagonist 13 in STR-induced seizure. Furthermore, in vitro antiproliferative effects or possible metabolic interactions could not be observed for compound 4. Additionally, the predictive in silico, as well as in vitro, metabolic stability for the most promising H3R antagonist 4 was assessed. The obtained results show prospective effects of non-imidazole H3R antagonists as innovative antiepileptic drugs (AEDs) for potential single use against epilepsy.A newly developed series of non-imidazole histamine H3 receptor (H3R) antagonists ( Epilepsy is considered as a brain disease characterized by repeated seizures due to different complex causes including idiopathic (ion channel or protein defects), symptomatic stroke, head injury , metabolic disorders , or genetic mutation of DNA sequences in genes responsible for encoding various ion channels or neurotransmitter receptors ,2. In adN-methyltransferase, the histamine metabolizing enzyme in the central nervous system (CNS), using metoprine elevated the levels of brain histamine and, therefore, reduced seizure susceptibility Nα-methylhistamine at membrane preparations from human embryonic kidney (HEK)-293 cells, stably expressing the receptor. Assays ran in triplicates with seven concentrations of the test compounds 1–16  values according to the Cheng-Prusoff equation Nα-methylhistamine displacement assays on membrane preparations of HEK-293 cells, stably expressing the hH3R , with the most promising in vivo anticonvulsant effects, were further evaluated for their affinity at human histamine H1 (hH1R) and H4 (hH4R) receptors. The results show that test compounds 1–16 had an H3R affinity of 40–140 nM compared with the standard H3R antagonist PIT with an H3R affinity of 12 nM , 7 , and 13 , showed a selectivity profile toward H3Rs with at least 10-fold lower affinity at hH1- and H4Rs.The novel ligands the hH3R . Followiof 12 nM . SelecteWith regard to the MES test, the current one used in the study produced seizures in 100% of animals without mortality. Likewise, the dose of PTZ and STR used in the present study formed seizures (score 4–5) in 100% of animals without mortality.1–16 on MES-induced seizures in rats was carried out and the observed results were compared with the protective effect of the reference antiepileptic drug PHT in MES-induced seizure in rats  demonstrated a significant protection against MES-induced seizures as confirmed by one-way analysis of variance . Among the H3R antagonists tested and following post hoc analyses, compound 4 at a dose of 10 mg/kg significantly exhibited the most promising protective effect in MES-induced seizure when compared with the saline-treated group with , and provided comparable protection to that of PHT with   = 5.882; p < 0.05], , , , , and , respectively (4 were protected to a significantly lesser extent against seizures when compared with the H3R antagonist 4 (10 mg)-treated group with  , and , respectively . The results showed that co-injection with CNS penetrant histamine H3R agonist RAMH  abrogated the H3R antagonist 4 (10 mg)-provided protection with  for the comparison of saline–saline vs. 4 + RAMH  = 0.359; p = 0.563] for saline–saline vs. saline–RAMH  were assessed and compared with the protection obtained for the reference antiepileptic drug VPA in PTZ-induced seizures in rats  = 23.925; p < 0.001]   = 653.40; p < 0.001]    = 2.40; p = 0.147] for 6-treated group versus 4 + RAMH-treated group (p = 0.232 (The protective effects of H3R antagonists  in rats . The res< 0.001] . Pairwis< 0.001) . Similar< 0.001] . Moreove< 0.001) . However < 0.05) . Furthered group . Importa = 0.232  .4 with the most promising effect in MES- and PTZ-induced seizures failed to provide any appreciable protection in STR-induced seizure when compared with the saline-treated group after 30 min of observation time with   = 63.00; p < 0.001]  = 236.308; p < 0.001]  failed to exhibit protection against STR-induced seizures when compared with the saline-treated group with   were observed when 5 or 15 mg/kg of the same compound was administered with  , respectively  on proliferation of HEK-293 and hepatoma HepG2 cell lines was assessed and compared with the reference cytostatic drug doxorubicin (DX) and hepatotoxin carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (4 significantly decreased proliferation of HepG2 cells only at the highest concentration used (100 µM), however, decline in proliferation of HEK-293 cells was achieved with two doses of the same compound, namely 10 and 100 µM (p < 0.001) (The effect of H3R antagonist e (CCCP) A,B. The < 0.001) A,B.4 metabolism  showed that ~18% of this compound was converted into four metabolites, namely M-I–M-IV .All novel H3R antagonists were evaluated in the form of hydrogen oxalate salts in radioligand displacement assays. Tested H3R antagonists t 12 nM, , while t6, 9, and 15) were examined in a binding assay at the human histamine H1R and showed weak affinities (915 ≤ Ki ≤ 1338 nM), being 10-fold lower than at the H3R. As the two histamine receptors H1R and H3R play a pivotal role in the anticonvulsant activity of the central histamine , selecte4, 7, and 13) was evaluated. The observed results showed that none of the tested H3R antagonists displayed affinity for hH4R . As H3R represents the highest degree of homology with H4R , the pot4 exhibited the most promising protection against MES-induced seizures when animals were administered with 10 mg/kg i.p., and as compared with the saline-treated group of animals  was similar to that observed in the group treated with the reference antiepileptic drug PHT , and was significantly higher than that observed for H3R ligands 3, 5, 6, 7, 8, and 14   , Table 3agonists ,30,40,544, would escalate neuronal release of brain histamine, providing the protection in the MES-induced seizure in rats. The latter proposed mechanism underlying the anticonvulsant effect of H3R antagonist 4 is also in line with previous preclinical observations in animal seizure models in which high doses of several centrally acting H1R antagonists used as anti-allergic drugs promoted the development of convulsions of tested animals, indicating the involvement of H1R antagonism, and consequently, brain histaminergic neurotransmission in the seizure promotion. Noticeably, similar protective effects of imidazole-based and non-imidazole-based H3R antagonists were earlier described to be abolished either by H3R agonists or by centrally acting H1R antagonists, suggesting an interaction of the H3R antagonism-released histamine with postsynaptically located H1Rs on neurons [H3Rs are auto-receptors presynaptically positioned on histaminergic neurons with an inhibitory effect on the biosynthesis and release of histamine . Therefo neurons ,30,53,544, 7, and 11  showed full protection  or a higher dose  showed a dose-dependent protection in the PTZ-induced seizure model  was not reversed when rats were pretreated with RAMH  before PTZ challenge, suggesting that the protection observed for H3R antagonist 4 in PTZ-induced seizure is not facilitated through modulation of central histaminergic neurotransmission  as well as H3R antagonists otection , Table 3re model , Table 3smission , Table 3le, GABA .4, 7, and 11  failed to exhibit appreciable protection during 30 min of the time observation, whereas H3R antagonist 13 with moderate protection in PTZ and without any considerable protection in MES demonstrated reasonable protection following 30 min during the time observation  and hepatoma HepG2 cell lines’ proliferation. The antiproliferative activity of H3R antagonist 4 differed between both used cell lines. HEK-293 cells were more susceptible for antiproliferative activity as revealed in the statistically significant decrease of HEK-293 cell line viability (p < 0.001), which was observed at concentrations of 10 and 100 µM, whereas in the HepG2 cell line, the antiproliferative effect was achieved only at a concentration of 100 µM   A. Moreovt 100 µM B. 4 applying RLMs revealed good metabolic stability of H3R antagonist 4, as only ~18% of the substrate was metabolized and converted into four different metabolites after 120 min of incubation at 37 °C  as compared with the reference drug QD with a calculated IC50 value of 0.01 µM hexyl)azepane pharmacophore exhibited an affinity for hH3R (Ki = 69.3 nM). The in vivo anticonvulsant results revealed that H3R antagonist 4 exhibited most promising protection following acute systemic administration in MES- and PTZ-induced seizure models in rats. Moreover, the protection observed for H3R antagonist 4 in the MES-induced seizure model was fully reversed when rats were pretreated with the CNS-penetrant H3R agonist RAMH. However, RAMH failed to abrogate the protective effects observed for H3R antagonist in PTZ- or STR-induced seizure models, indicating that histaminergic pathways appear to be involved in the provided anticonvulsant efficacy of H3R antagonist 4 in only the MES-induced seizure model, but additional pharmacological properties of the compounds or their metabolites cannot be fully excluded. Moreover, ADME-Tox parameters’ screening revealed satisfying low cytotoxicity, good metabolic stability, as well as no inhibition of CYP3A4 activity and moderate inhibition of CYP2D6 activity. Therefore, the overall experimental observations provide promising potential for the novel H3R antagonist 4 to be used as a potential template for further drug design and synthesis in the search for potent and active in vivo H3R antagonists, for example, as AED drugs with a high safety profile. Nonetheless, a battery of additional seizure models with different species is still required to further corroborate the current results observed for H3R antagonist 4, and to strengthen the translational value of its potential applicability in the therapeutic management of epilepsy.The tested series of H3R antagonists showed in vitro affinity at the hH3R in the nanomolar range. The most promising H3R antagonist


Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen. The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen. H1N1 virus entered the human population from birds in 1918. It is thought to have jumped from humans to pigs in that epoch, and it was from the pig that influenza virus was first isolated in 1931 . H1N1 viin vitro and reduce virus plaque size (10–Antibodies to the hemagglutinin (HA) and neuraminidase (NA) proteins can independently provide protection from influenza disease –6. The s–que size . Anti-NA size 10– to 15.Kilbourne et al., Schild, and Couch et al. also showed that protective anti-NA antibodies are elicited in humans following natural infection , 17 and 3–In contrast to a considerable literature on human monoclonal antibodies (MAbs) against HA, the majority of MAbs targeting NA described to date are from mice and rabbits, and they show relatively limited cross-reactivity. MAbs NC10 and NC41, among the first murine MAbs against NA and specific to the N9 NA, were analyzed for functional and structural characteristics , 20. Murin vivo  and avian N9 (group 2) NAs. These exceptionally broadly reactive anti-NA MAbs offer the hope of developing vaccines that could induce them.(This article was submitted to an online preprint archive .)Two antibodies, AG7C and AF9C, were isolated from an adult  vaccinated with 2014/15 Northern Hemisphere trivalent influenza vaccine (TIV) containing A/California/7/2009 (reassortant NYMC X-179A) (H1N1), A/Texas/50/2012 (reassortant NYMC X-223) (H3N2), and B/Massachusetts/2/2012 (reassortant NYMC BX-51B), all at 15 μg/0.5 ml  . A thirdWe focused our analysis on three MAbs, AG7C, AF9C, and Z2B3, since the other antibodies were either of limited specificity or weaker in their inhibition of NA. These three MAbs were tested for the inhibition of NA activity of H1N1 viruses isolated between 1934 and 2018, using an enzyme-linked lectin assay (ELLA)  and 2, a50) of NA activity were calculated by linear interpolation. The titers yielded by a 1-mg/ml solution were then calculated and plotted for comparison to control hyperimmune sheep antisera obtained from the National Institute for Biological Standards and Controls (NIBSC)   and 3.MAb AF9C inhibited the NA activities of all H1N1 viruses tested, which were representative of those that have circulated in humans for over 100 years  and 3. M50s of ∼250 to 25 ng/ml) on the set of viruses shown, with the exception that AG7C fails to inhibit N1 NA from A/Brisbane/59/2007. In contrast, Z2B3 gave similar titers on A/PR/8/1934, A/England/195/2009, and A/England/621/2013 but had drastically reduced titers on A/USSR/90/1977 and the representative recent clade 6B.1 H1N1pdm09 viruses A/Serbia/NS-601/2014 and A/Switzerland/3330/2017, indicating that the genetic and associated antigenic drift in these viruses had resulted in a major alteration in the epitope recognized by Z2B3. The control hyperimmune sheep antiserum to A/California/07/2009 N1 showed limited cross-reactivity on recently drifted or older  viruses, with only weak activity against N1 NA from A/PR/8/1934. The sheep anti-H7N9 (A/Anhui/1/2013) antiserum contained anti-N9 NA antibodies that did not cross-react with any NAs expressed by these H1N1 viruses.The results in To avoid handling avian influenza viruses, we titrated the MAbs for inhibition of recombinant N1 NAs from a range of H5N1 viruses isolated from infected humans, representing several HA clades from pandemic virus A/Brevig Mission/1/1918 and N9 NA from H7N9 virus A/Anhui/1/2013, produced in HEK293 cells, with N1 NA from A/California/07/2009 as a positive control  and K (P17C and F4C) were found to bind N9 NA in an indirect immunofluorescence screen (not shown). W1C7 and F4C were specific for N9 NA, and W1C7 had a weak inhibitory effect on N9 in ELLA . P17C cr7 and K (de novo responses to acute H7N9 infection.Antibodies from donor Z have higher numbers of amino acid substitutions in the variable regions of heavy and light chains than do MAbs from other donors . The num4 median tissue culture infectious dose (TCID50) of A/PR/8/1934 virus  when given at a dose of 10 mg/kg 24 h before infection (P < 0.001)   and B. T4 TCID50  of X-179A virus, a reassortant containing the H1N1pdm09 viral RNA from A/California/07/2009 (P < 0.001), whereas mice receiving a nonspecific antibody had to be culled on day 5 or 6. One of 6 mice in the AG7C group was sacrificed on day 11 after losing ≈20% weight. In these prophylactic protection experiments, anti-NA MAbs were as protective as T1-3B, the positive-control anti-HA stalk MAb  variants. These substitutions abrogate Fcγ-dependent antibody-dependent cell cytotoxicity (ADCC) and complement binding and fixation . We confcal sign  and F. HWe show in this paper that broadly reactive and protective antibodies to N1 NA can be isolated from vaccinated and infected individuals, presumably due to the conservation in surface structure between N1 NAs . The twoThe very broad reactivity of these MAbs with N1 NAs, covering the complete period of H1N1 virus circulation in humans, may have been induced by exposure to the significantly different NA derived from the H1N1pdm09 virus. Both MAbs show significant sequence divergence , suggestThe third antibody, Z2B3, was isolated from a child who experienced a mild infection with H7N9 virus in 2013. It was unusual in being cross-reactive with group 1 (N1) and group 2 (N9) NAs . Two similar antibodies were isolated from this donor, both of which inhibited N1 NA with some level of cross-reaction with N9 NA , which wKEDK; PDB identifier [ID] 1NMB).MAb Z2B3 showed good reactivity with the H1N1pdm09 virus A/England/621/2013 but poor reactivity with a later clade 6B virus, A/Serbia/NS-601/2014. These two viruses showed nonconservative amino acid substitutions of only N386K and K432E in the head of NA . The forK432 was conserved prior to 2013 but underwent a K432E substitution in 2014, which became dominant thereafter. We suggest that N1 NA has been under strong evolutionary pressure from broadly cross-reactive antibodies induced by the H1N1pdm09 NA that were selected from memory B cells raised against NA(s) of earlier virus(es). Just as the conserved stalk of HA has shown a capacity for evolution under pressure from antibody selection , the NA https://www.nextstrain.org). Substitutions for Q250 are rare; Q250L was seen in 1/1,944 viruses analyzed.With this in mind, we examined the region of the NA surface recognized by broadly reactive antibodies described by Chen et al. and Gao et al. that inhibited or bound N1 NAs of viruses isolated between 1918 and 2009 but not clade 6B H1N1pdm09 viruses , 40. SomSimilarly, the comparison of NA sequences between A/Switzerland/3330/2017 (inhibited strongly by AF9C) and A/Luxembourg/2489/2019 (inhibited weakly) implicated the substitution D199N in the loss of recognition and, therefore, D199 as part of the epitope recognized by AF9C . Seven of 2,056 NA sequences from H1N1pdm09 viruses detected between 2013 and 2019 had N1 D199N/A substitutions. Further structural work to define the epitopes recognized by Z2B3, AG7C, and AF9C is in progress.in vivo via Fc-mediated functions even in the absence of neuraminidase activity  containing human variable regions linked with murine IgG2a Fc, for which protection was abrogated by the Fc mutant DA265 . Some NAactivity . HoweverIt has become clear that exposure to viruses that differ significantly from those circulating can select responses to epitopes in both HA and NA that are shared between the incoming virus and the seasonal viruses in circulation, derived from the memory B cell population , 43. Whi2 in a humidified incubator. Viruses were diluted and grown in virus growth medium (VGM), which is DMEM with 0.1% bovine serum albumin , 10 mM HEPES, and glutamine, penicillin, and streptomycin as in D10 medium.MDCK-SIAT1 cells and adherent 293T cells (ECACC) were grown in Dulbecco modified Eagle medium (DMEM)  supplemented with 10% (vol/vol) fetal calf serum , 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin . 293-F suspension cells were grown in FreeStyle 293-F expression medium  on a shaker incubator. Cells were grown at 37°C, 5% COH1N1 viruses from the years 1977 to 2019 and H3N2 viruses were obtained from the Worldwide Influenza Centre (WIC) at The Crick Institute . Other reassortant viruses and control sheep sera were obtained from the National Institute for Biological Standards and Controls (NIBSC), UK.The study was performed in compliance with good clinical practice guidelines and the Declaration of Helsinki. The protocol was approved by the Research and Ethics Committees of Chang Gung Memorial Hospital, Beijing Ditan Hospital, and the Weatherall Institute of Molecular Medicine. All subjects provided written informed consent. A list of donors with their details and isolated antibodies is in −, CD19+, CD20low/−, CD27high, and CD38high) and sorted as single cells using flow cytometry. mRNA from single plasmablasts was reverse transcribed to DNA, and VH and Vκ/λ genes were amplified using gene-specific primers and then cloned into expression vectors containing IgG1 heavy and Vκ and Vλ constant regions. Heavy and light chain plasmids were cotransfected into 293T or ExpiCHO cells  for antibody expression.MAbs were isolated from individual humans who either received seasonal influenza vaccine or were naturally infected with H7N9 virus in China or Taiwan. Peripheral blood mononuclear cells (PBMC) were collected from individual donors either a week after vaccination against influenza or from confirmed influenza virus-infected cases 10 days after onset of clinical symptoms. Antibodies were isolated from PBMC using single-cell isolation and cloning methods as described in detail previously , 47–49. 47–The L234A, L235A, and P329G (LALA-PG) amino acid substitutions  that abrMAbs were initially screened for binding to MDCK-SIAT1 cells infected with either H1N1 or H3N2 virus and for lack of binding to HA protein expressed in stably transfected MDCK-SIAT1 cells. Binding to NA was confirmed by immunoprecipitation with infected cells or binding to 293T cells transfected with the NA gene of interest.Tetrameric neuraminidase proteins were expressed from constructs based on the design of Xu et al. . In our N9 NA protein (A/Anhui/1/13) was kindly provided by Donald Benton (The Francis Crick Institute) . The expFor antibody inhibition measurements, a dilution of the NA-containing supernatant that had just reached plateau activity in the ELLA was chosen. The sequences of all the constructs and their accession numbers are shown in 2 as for tissue culture. On the next day, the contents of the plate were discarded, and the plate washed 4 times with PBS. Horseradish peroxidase-conjugated peanut agglutinin (PNA-HRP)  at 1 μg/ml was added to the wells. PNA binds to the exposed galactose after cleavage of sialic acid by NA. After 1 h of incubation and washing with PBS, the signal was developed by adding OPD (o-phenylenediamine dihydrochloride) solution , and the reaction was stopped after 5 to 15 min using 1 M H2SO4. Absorbance was read at 492 nm in a Clariostar plate reader (BMG Labtech).The ELLA was adapted from the methods described by Schulman et al.  and Sand50) of NA activity were calculated by linear interpolation. For comparison with the results for the positive-control sheep sera, titers of antibodies, with the starting concentration transformed to 1 mg/ml, were compared with the serum titers in the same graphs.The antibodies were titrated by doubling dilution, and the concentrations required to give 50% inhibition  for X-179A virus and BALB/cOlaHsd mice (n = 6/group) for PR8 virus, were purchased from Envigo, United Kingdom, and housed in individually vented cages in a special unit for infectious diseases. Mice were anesthetized with isofluorane (Abott), and 50 μl of virus was administered intranasally 24 h after the intraperitoneal administration of 10 mg/kg antibody (500 μl). Mice were observed and weighed regularly. Mice with weight loss of ≈20% or morbid clinical scores were euthanized by raising the concentration of CO2. Nonspecific IgG antibody was used as a negative control. Known HA-specific antibodies were used as positive controls. Mice were infected intranasally with lethal doses of viruses X-179A  and PR8 .Mice used in protection studies, DBA/2OlaHsd mice  or on Graphs were generated using GraphPad Prism (version 9) and Microsoft Excel 2010. Protein structures were viewed using Pymol2 .50, the midpoint between negative and plateau positive controls) derived by linear interpolation from neighboring points in the titration curve. Kaplan-Maier tests were performed to analyze the differences in mortality between experimental- and control-group mice. P values of <0.05 were considered to show a significant statistical difference.The ELLA titers were expressed as half-maximal inhibitory concentrations (IC


COL1A1 and COL1A2 have been demonstrated to be major causative factors to OI. However, the complexity of the bone formation process indicates that there are potential new pathogenic genes associated with OI. To comprehensively explore the underlying mechanism of OI, we conducted association analysis between genotypes and phenotypes of OI diseases and found that mutations in COL1A1 and COL1A2 contributed to a large proportion of the disease phenotypes. We categorized the clinical phenotypes and the genotypes based on the variation types for those 155 OI patients collected from literature, and association study revealed that three phenotypes  were enriched in two variation types . We also identified four novel variations (c.G3290A (p.G1097D), c.G3289C (p.G1097R), c.G3289A (p.G1097S), c.G3281A (p.G1094D)) in gene COL1A1 and two novel variations (c.G2332T (p.G778C), c.G2341T (p.G781C)) in gene COL1A2, which could potentially contribute to the disease. In addition, we identified several new potential pathogenic genes  based on the integration of protein–protein interaction and pathway enrichment analysis. Our study provides new insights into the association between genotypes and phenotypes of OI and novel information for dissecting the underlying mechanism of the disease.Osteogenesis imperfecta (OI), mainly caused by structural abnormalities of type I collagen, is a hereditary rare disease characterized by increased bone fragility and reduced bone mass. Clinical manifestations of OI mostly include multiple repeated bone fractures, thin skin, blue sclera, hearing loss, cardiovascular and pulmonary system abnormalities, triangular face, dentinogenesis imperfecta (DI), and walking with assistance. Currently, 20 causative genes with 18 subtypes have been identified for OI, of them, variations in  COL1A1 gene) and one α2(I) chain (encoded by COL1A2 gene). Individuals with OI have low bone mass, which results in deformity of long bones, vertebral anomalies and fractures, shortening of extremities, and skull defect (Osteogenesis imperfecta (OI) is a phenotypically and genetically heterogeneous group of bone disorders characterized by bone fragility and skeletal deformity, owing to the abnormality of type I collagen formed by two α1(I) chains . There are two general categories of mutational defects occurred in COL1A1/COL1A2. The first is missense mutation, mainly involving glycine replacement within the Gly-Xaa-Yaa repeat (the Gly-substitution missense), which results in the synthesis of collagen with abnormal structure (Current evidences demonstrate that tructure . The sectructure . ConsideCOL1A1 and COL1A2), in the past decade, a series of studies have found that a set of new non-collagen gene defects affect normal post-translational processing, molecular folding of type I collagen, fibril formation, osteoblast differentiation, and mineralization, leading to rare autosomal recessive, dominant, and X-linked forms of OI   . We manuInWeb_InBioMap is a scored human protein–protein interaction data resource. It is generated by combining interactions from eight protein interaction databases and providing confidence score for each interacting pair and relevance score for every protein. The confidence score represents a lower bound on the probability for the interaction being a true positive, and the relevance score represents a relationship between one protein and others in a specific network. We selected those interactive pairs with both confidence score of 1 and a relevance score of 1 for subsequent study .We retrieved the expression patterns for those predicted genes and pathogenic genes in different tissues from GTEx database. GTEx is a data resource platform for exploring correlations between genetic variants and gene expression in multiple human tissues .We standardized the phenotypic description for those 155 patients based on eRAM system . The colP < 0.05).WikiPathways is an open and integrative database, providing biology pathway information . WebGestCOL1A1 and COL1A2 genes. All variations information on COL1A1 and COL1A2 genes was downloaded from ANNOVAR database , insertions, and deletions with more than 10 different tools, including SIFT, CADD, PolyPhen-2, and GERP++. The pathogenic ability of each variation is predicted by those tools with defined cutoff values. SIFT is a tool that uses sequence homology to predict whether a substitution affects protein function and whether amino acid substitutions at specific positions of the protein have phenotypic effects . CADD inWe collected the allele frequency for corresponding variants from gnomAD  and ChinP < 0.05). Subsequently, we mapped those interacting gene partners of OI pathogenic genes into those significant pathways and selected those gene partners (the predicted gene set B) involved in these pathways. We then checked the expression patterns of those predicted OI-related pathogenic genes (the union of predicted gene set A and predicted gene set B). Last, we conducted literature survey to further verify those novel candidate risk genes. The whole process was displayed in To identify potential new risk factor for OI, we first mapped those 20 experimentally confirmed pathogenic genes into InWeb_InBioMap data resource to form a disease gene centralized protein–protein interaction network. Those genes with the largest confidence score (= 1) and the highest relevance score (= 1) to the OI pathogenic genes were selected to be interacting genes for OI. We then constructed a sub-network of pathogenic proteins and their interacting protein partners and focused on those interacting proteins (the predicted gene set A) that directly interact with more than one OI pathogenic proteins. In addition, we used WebGestalt to conduct pathway enrichment analysis based on Wikipathway database and selected those significant pathways (P < 0.05) between the Gly-substitution missense and other types of variation  among OI subtypes, gender, pathogenic genes, and the presence of each clinical features in OI patients. One-sided Fisher’s exact test was used to test co-occurrence relationship (P < 0.05) between two different phenotypes. All the calculated progress was conducted by R software 3.4.3.Two-sided Fisher’s exact test was used to test the difference  mainly caused by on types . We clason types . The fir1/COL1A2 , causingTo explore the associations between genotypes of pathogenic genes and OI clinical phenotypes, we first standardized the phenotypic description based on our eRAM system for those collected 155 patients (p = 1.526e-07), most OI type I were caused by the second variation type . The second variation type seemed to occur mainly in COL1A1 (p = 1.833e-13), and no variation type difference was observed between male and female patients (p = 0.7218).Based on the two types of variation, we performed statistical significance test between the two variation types and 12 phenotypes recorded in the collected 129 cases. As a result, these two variation groups were significantly different among OI type I, OI type III, and OI type IV (p = 0.4243) (p = 1), dense metaphyseal bands (p = 0.4231), vertebral fracture (p = 1), osteopenia (p = 1), hearing loss (p = 0.6024), triangular face (p = 0.389), and popcorn calcif (p = 1)  Table 1Ap = 0.02946) and DI (p = 0.03189). In addition, we also found that patients with DI were prone to have bone deformity (p = 0.0005576) or vertebral anomalies (p = 0.01832) (p = 0.06021). In addition, patients with the Gly-substitution missense had a poorer walking ability (p = 0.0001345).Results revealed that patients with the Gly-substitution missense tended to develop bone deformity (0.01832) Table 1B0.01832) . The assCOL1A1 and 110,016 possible mutations of COL1A2 in ANNOVAR. After filtering out mutations based on the cutoff value , 19 COL1A1 mutations and 5 COL1A2 mutations were kept as pathogenic variations. Strikingly, 5 of the 24 mutations have been reported to be pathogenic to OI in ClinVar, dbSNP or OIVD Database  with COMBALT revealed that 12 mutations in COL1A1 and 3 mutations in COL1A2 located in highly conserved regions among those species. Finally, we mapped the 15 candidate variations into the domains of α1(I) chain/α2(I) chain (encoded by COL1A1/COL1A2) in Ensembl, and found only four mutations (c.G3290A (p.G1097D), c.G3289C (p.G1097R), c.G3289A (p.G1097S), c.G3281A (p.G1094D)) in COL1A1 located in collagen triple helix repeat domain, which influence type I collagen formation. In contrast, two mutations (c.G2332T (p.G778C), c.G2341T (p.G781C)) in COL1A2 were not in any domain region, but located in two reported lethal spaces in COL1A2 protein sequence. We supposed that these two candidate variations also associate with OI disease.Next, we checked the frequency of those candidate variations in gnomAD and CMBD databases, and no frequency has been reported in any ethnic groups. The result indicated that all the variations are an extremely rare site with high probability of lethality for the disease. The multiple sequence alignment of ADAMTS2, ADAMTS3, ADAMTS14, COL4A6 COL5A2, COL8A1, COL19A1, COL20A1, COL21A1, COL22A1, COL24A1, COL27A1, COL28A1, TLL1, CNIH1, CNIH3, SEC16B, WNT8B) (≥1 interaction)  that interact with more than one pathogenic proteins from PPI network as the predicted gene set A (≥2 interactions). Next, we performed pathway enrichment analysis on 20 disease-causing genes and obtained 18 significant pathways (p < 0.05). Among the 18 interacting gene partners, 3 of them  fell into those 18 significant pathways (the predicted gene set B)  as the potential causal genes to OI disease. To further verify those predicted genes for OI disease, we checked the expression pattern of those 15 genes in different human tissues based on GTEx data. As related to collagen hereditary disease, most of the OI causative genes are expressed in artery aorta and transformed fibroblasts . Those three candidate genes presented similarly tissue expression pattern with their seven interactive OI pathogenic genes , especially showed high expression in transformed fibroblasts  and relevance score cutoff (= 1). The resulting network contains 20 pathogenic proteins with 18 directly interactive partners (e set B) Table 2.OI is a rare disease with bone disorders characterized by bone fragility and skeletal deformity. Clinical observation indicates that currently identified pathogenic genes and variations cannot fully decipher the phenotypic and genetic heterogeneity of the disease. To better explore the underlying mechanism of OI, we performed genotype and phenotype association analysis based on manually collected 155 patients’ data. According to the variation type, we classified mutations into two groups, one group refers to the Gly-substitution missense, and another includes frameshift, nonsense, and splicing mutation. Most of the Gly-substitution missenses result in structural abnormalities of type I collagen, whereas the second mutation group caused by the early termination of codons lead to insufficient collagen synthesis. Our results showed that the two mutation types played different roles in bone deformity, DI, and walking with assistance, which was consistent with the results of other studies .p = 0.0005576) and vertebral anomalies (p = 0.01832). Similarly, Lin et al. also reported that patients with DI were more susceptible to bone deformities and scoliosis , c.G3289C (p.G1097R), c.G3289A (p.G1097S), and c.G3281A (p.G1094D); COL1A2: c.G2332T (p.G778C) and c.G2341T (p.G781C)) locate in the conserved regions, which indicates that these four positions (amino acid position: 1094 and 1097 (on α1(I) chain); 778 and 781 (on α2(I) chain)) have a significant impact on protein structure or function. Among the 6 candidate variations, four of them (in COL1A1) locate in the triple helical region and the other two variations (in COL1A2) locate in the collagen region.For the 24 pathogenic mutations from ANNOVAR annotation, we searched different variation annotation databases, including ClinVar, dbSNP, and OIVD, to explore whether they have any disease-related report. As a result, 19 of the mutations have no report linked to OI patients. However, multiple alignment with homologous genes from different species with COMBALT showed that only six variations (A collagen triple helix is formed by three chains (two α1(I) chains and one α2(I) chain) supercoiling around the common axis and glycine, framing almost 338 Gly-Xaa-Yaa repeats in the region, is the only residue small enough to be accommodated in the limited interior of the helical space . In the COL1A1, we checked the specificity of their locations (positions of the four candidate mutations: 1094 and 1097). Evidence from the protein families database (Pfam)   demonstre (Pfam) , which ae (Pfam) . In addie (Pfam) . In all,COL1A2, we found that the two mutations locate in a special region which is enriched with lethal mutations. Previous study reported that OI-related lethal mutations normally accumulate in eight regularly spaced clusters along the chain α2(I) (For the two candidate variations in in α2(I) . Truly, in α2(I) . Abnormain α2(I) . These bin α2(I) . TherefoADAMTS2 and COL5A2 seem to be more associated with OI than COL8A1. In fact, ADAMTS2 is highly expressed in the skin, bones, tendons, and aorta and has a strong correlation with type I collagen , which will result in the polymerization of the abnormal collage fibers   . Evidence of OI)  also dem with OI .COL5A2, which encodes type V collagen, plays an important role in tissue-specific matrix assembly and the regulation of fibrillogenesis , National Natural Science Foundation of China , Clinical Application Research Funds of Capital Beijing (Z171100001017051), Beihang University & Capital Medical University Advanced Innovation Center for Big Data-Based Precision Medicine Plan (BHME- 201804) and Shanghai 111 Project (B14019).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


CaWRKY41 plays dual roles in increasing resistance to Ralstonia solanacearum infection and susceptibility to Cd stress in pepper by indirectly and directly increasing H2O2 accumulation and Zn transporter expression. Ralstonia solanacearum and Cd stress responses in pepper (Capsicum annuum). CaWRKY41 was transcriptionally up-regulated in response to Cd exposure, R. solanacearum inoculation, and H2O2 treatment. Virus-induced silencing of CaWRKY41 increased Cd tolerance and R. solanacearum susceptibility, while heterologous overexpression of CaWRKY41 in Arabidopsis impaired Cd tolerance, and enhanced Cd and zinc (Zn) uptake and H2O2 accumulation. Genes encoding reactive oxygen species-scavenging enzymes were down-regulated in CaWRKY41-overexpressing Arabidopsis plants, whereas genes encoding Zn transporters and enzymes involved in H2O2 production were up-regulated. Consistent with these findings, the ocp3 (overexpressor of cationic peroxidase 3) mutant, which has elevated H2O2 levels, displayed enhanced sensitivity to Cd stress. These results suggest that a positive feedback loop between H2O2 accumulation and CaWRKY41 up-regulation coordinates the responses of pepper to R. solanacearum inoculation and Cd exposure. This mechanism might reduce Cd tolerance by increasing Cd uptake via Zn transporters, while enhancing resistance to R. solanacearum.WRKY transcription factors have been implicated in both plant immunity and plant responses to cadmium (Cd); however, the mechanism underlying the crosstalk between these processes is unclear. Here, we characterized the roles of CaWRKY41, a group III WRKY transcription factor, in immunity against the pathogenic bacterium  Plants are frequently exposed to various biotic and abiotic stresses in their natural habitats. A variety of defense response mechanisms have evolved that protect the plant against particular stresses. These mechanisms are mediated by complex signaling pathways, which must be coordinately and tightly regulated. Common signaling pathways such as MAPK cascades  and path2−), hydrogen peroxide (H2O2), hydroxyl radical (OH•), and singlet oxygen (1O2), are partially reduced forms of molecular oxygen (O2) that typically result from the transfer of one, two, or three electrons to O2. H2O2 is the most stable ROS, with a relatively long half-life (~1 ms in the cell), and often acts as an intercellular and intracellular signal that triggers downstream responses  seedlings . This element, which is released into the agricultural ecological system as a result of urbanization and industrialization, is considered to be one of the most toxic heavy metals in the environment . WRKY proteins, which are characterized by the presence of one or two highly conserved WRKY domains, constitute one of the largest TF families. WRKY TFs are important positive and negative regulators of plant growth and development, and of defense responses to environmental stimuli (Capsicum annuum) is a solanaceous vegetable crop widely grown around the world. Blight and bacterial wilt caused by the soil-borne pathogens Phytophthora capsici and Ralstonia solanacearum, respectively, frequently reduce pepper production. Heavy metal contamination is another factor that inhibits pepper growth. Heavy metal residues are present in soils as a result of sewage irrigation and the use of heavy-metal-containing products such as pesticides and fertilizers. A better understanding of how pepper responds to heavy metal contamination would lay the foundations for developing effective countermeasures.Pepper  deficiency, because Cd toxicity-induced chlorosis resembles Fe deficiency-induced chlorosis  8# (an inbred line provided by the pepper breeding group at Fujian Agriculture and Forestry University) and CM334 (Mexican landrace of C. annuum cv. CM334), and tobacco (Nicotiana benthamiana) were imbibed in sterile water at 25±2 °C overnight and sown in a steam-sterilized soil mix  in plastic pots.Seeds of pepper , Ca (NO3)2·4H2O (1 mM), MgSO4·7H2O (1.4 mM), and KH2PO4 (0.2 mM), and the micronutrients Fe-EDTA (20 µM), H3BO3 (3 µM), (NH4)6Mo7O24 (1 µM), MnCl2 (0.5 µM), ZnSO4 (0.4 µM), and CuSO4 (0.2 µM). The pH of the solution was adjusted to 5.8, and the nutrient solution was renewed every 3 days.Pepper plants were grown in a growth room maintained at 25±2 °C with a light intensity of ~100 µmol photons mArabidopsis thaliana cultivation, wild-type , ocp3  medium  supplemented with 1% (w/v) sucrose and 0.8% agar  in the absence or presence of heavy metals or other supplements in a growth chamber maintained at 22±2 °C with a light intensity of ~100 µmol photons m−2 s−1 and a relative humidity of 70%, under a 16 h light/8 h dark cycle.For C. annuum, Solanum lycopersicum, and A. thaliana genomes were downloaded from Plant TFDB V4.0 (http://planttfdb.cbi.pku.edu.cn/index.php).The WRKY TFs were described previously  suspension of R. solanacearum. A disease index (from 0 to 5) was scored daily in the R. solanacearum-inoculated pepper plants, as follows: 0 (no wilting), 1 (1 to 20% wilted), 2 (21 to 40% wilted), 3 (41 to 60% wilted), 4 (61 to 80% wilted), and 5 (81 to 100% wilted or dead). The average values reported are based on three independent replicates, each comprising six plants. Electrolyte leakage was measured in pepper leaves at 0, 24, and 48 h post infection. For suspension inoculation, pepper plants were grown in 1.2 l black plastic beakers containing one-fifth Hoagland solution. Eight of the lateral roots were removed from each plant with a pair of scissors, and the plants were then transferred to one-fifth Hoagland solution with 108 cfu/ml R. solanacearum suspension.35S::CaWRKY41, the full-length open reading frame was cloned into pDONR207 and transferred into the pGWB2 expression vector . To construct the reporter vector (pCaWRKY41::GUS) for histochemical β-glucuronidase (GUS) analysis, the promoter of CaWRKY41 of ~2 kb in length (pCaWRKY41) was amplified via PCR from pepper genomic DNA and cloned into the pMDC163 vector (Invitrogen). The constructs 35S::CaWRKY41 and pCaWRKY41::GUS were then transformed into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw method. A. tumefaciens-mediated transformation of Arabidopsis was performed using the floral dip method . The pCambia1300-CaWRKY41-GFP construct was transformed into A. tumefaciens GV3101 and infiltrated into the fully expanded leaves of 5-week-old N. benthamiana plants. At 2 days post inoculation, green fluorescent protein (GFP) fluorescence was observed by confocal laser-scanning microscopy . For the transactivation assay, the open reading frames of CaWRKY41 (1–329) and the mutant genes CaWRKY41 (61–329), CaWRKY41 (131–329), and CaWRKY41 (192–329) were generated by PCR with specific primer pairs and cloned into pGBKT7 (Clontech) to generate various CaWRKY41 constructs . Transcriptional activation activity was determined in yeast cells transformed with these constructs grown on SD medium lacking Trp for 3 days, and a colony-lift filter assay  was performed.The coding region of CaWRKY41-silenced pepper plants were generated using tobacco rattle virus-based virus-induced gene silencing (VIGS) as described previously  and Zunla-1 (http://peppersequence.genomics.cn/page/species/blast.jsp). The fragment was cloned into the entry vector pDONR207 and then into the PYL279 vector. The vectors (PYL-279 and PYL-279-wrky41) were separately transformed into A. tumefaciens GV3101 cells, which were subsequently mixed with A. tumefaciens cells harboring PYL-192 and injected into fully expanded pepper seedling cotyledons. PYL-279-wrky41 pepper plants were subjected to experimental analysis, with PYL-279 plants (transformed with empty vector) serving as a control. Levels of H2O2 and of the expression of various genes were measured in CaWRKY41-silenced PYL-279 and PYL-279-wrky41 pepper plants grown in liquid culture.4 for 8 days. To measure the expression of various genes in plants in the presence of excess Cd supply, 7-day-old Arabidopsis seedlings were transferred to ½ MS medium without or with 25 µM CdSO4, cultured for 6 or 72 h, and harvested for use. To investigate the expression of the eight CaWRKY group III genes, pepper plants at the six-leaf stage grown in liquid culture were treated with Cd stress  and Fe deficiency (0 µM Fe-EDTA). Pepper plants at the six-leaf stage were sprayed with H2O2 (1 mM) and incubated for 0, 1, 3, 6, 12, 24, 36, and 48 h, and leaf tissue was harvested for CaWRKY41 expression analysis.To test the effect of Cd on seed germination and growth in Arabidopsis, seeds were treated by exposure to 4 °C in darkness for 3 days and then grown on ½ MS medium containing 25 µM, 50 µM, or 100 µM CdSO−1 X-Gluc, 1 mM K3Fe(CN)6, 1 mM K4Fe(CN)6, 50 mM sodium phosphate buffer pH 7.0, 10 mM Na2EDTA, and 0.1% Triton X-100) at 37 °C, destained several times in 75% (v/v) ethanol, and observed under a stereomicroscope .Leaves were stained with Trypan blue and 3, 3′-diaminobenzidine (DAB) as described previously . This one-step assay uses Amplex Red reagent  to detect H2O2. Briefly, approximately 80 mg of sample was processed and measured using an H2O2 standard curve. The fluorescence emission spectrum (590 nm) was detected at an excitation wavelength of 530 nm using a Tecan Infinite 200 Pro .Seedlings were grown on ½ MS medium for 7 days, treated with 25 µM CdSO3. Each sample was adjusted to 10 ml with Milli-Q water and then filtered through filter paper. Cd in the samples was detected by inductively coupled plasma-atomic emission spectrometry . The experiment was performed in three biological replicates.To measure the Cd contents in roots and shoots, the roots were rinsed three times (for 4 min each time) with Milli-Q water to remove Cd attached to the root surface. The root and shoot samples were weighed and digested with 0.5 ml (for roots) and 1 ml (for shoots) concentrated HNOg at 4 °C for 10 min. Then, peroxidase (POD), CAT, and APX activity were analyzed using an ELISA kit  according to the manufacturer’s instructions. Microtiter plate wells were coated with purified POD, CAT, and APX antibody, to make a solid-phase antibody, and then samples were added to the wells together with an antibody labeled with horseradish peroxidase, and an antibody–antigen–enzyme complex formed. Substrate solution was added after thorough washing, and then, using a blank well as the zero control, the absorbance was measured at 450 nm in a Tecan Infinite 200 Pro Plate Reader (Tecan).For enzymatic activity analysis, approximately 80 mg of sample was ground in liquid nitrogen using a TissueLyserII, and milled samples were homogenized in phosphate buffer  and centrifuged at 3000 × ® Premix Ex Taq™ II (TaKaRa); specific primers are listed in UBIQUITIN10 (AtUBQ10) and pepper Actin1 (CaActin1) were used for normalization.Total RNA was isolated from Arabidopsis and pepper tissues using a TaKaRa Mini BEST Universal RNA Extraction Kit . RNA (1 µg) was used as a template to synthesize cDNA with a TaKaRa PrimeScript RT-PCR Kit (TaKaRa) according to the manufacturer’s instructions. Gene expression levels were measured on a CFX96 Real-Time PCR System  using SYBRCaWRKY group III genes, we compared their nucleotide sequences to those of WRKY genes from tomato and Arabidopsis. We constructed an unrooted phylogenetic tree based on an alignment of the amino acid sequences of the group III WRKY proteins and domains from the three plant species using the neighbor-joining method. Based on this analysis, WRKYs from pepper share higher sequence similarity with WRKYs from tomato than with those from Arabidopsis (see CaWRKY group III genes is provided in To identify the phylogenetic relationships among the eight psis see . DetaileHelianthus annuus L.) and maize (Z. mays) under conditions of Fe deficiency  in the leaves and 1 HPT in the roots : wrky41] to silence CaWRKY41 in pepper. CaWRKY41 was expressed at a level approximately 3.8- and 3.2-fold lower in CaWRKY41-silenced plants than in control plants (PYL-279), in the presence and absence of Cd stress, respectively , superoxide dismutase (CaSOD1), copper zinc superoxide dismutase (CaCSD2), and APX (CaAPX1 and CaAPX2), were up-regulated at 24 HPT with Cd stress in the youngest leaves of PYL-279-wrky41 plants compared with the control. However, no difference in the expression of these genes was detected between the youngest leaves of PYL-279 and PYL-279-wrky41 under normal growth conditions  for further analysis. These CaWRKY41-OE lines were more sensitive than the WT to Cd stress  than in those of the WT after 3 or 5 days of Cd treatment. By contrast, the Fe contents in roots and shoots were similar in CaWRKY41-OE and WT plants  than in those of the WT, as revealed by DAB staining and direct H2O2 measurements  mutant, which harbors a T-DNA insertion in a homeodomain TF gene involved in increased H2O2 production in healthy plants  under pathogen inoculation and non-inoculation conditions, respectively  were triggered by R. solanacearum inoculation in the youngest leaves of PYL-279 plants at 24 h post inoculation  was higher in CaWRKY41-silenced pepper plants than in PYL-279 plants  transcription factor can be induced by H2O2, and that this plays a major role in regulating gene expression , including AtRBOHC  resulted in H2O2 accumulation, and that inhibition of H2O2 degradation conferred enhanced disease resistance (Further research is required to elucidate the mechanism underlying HCaWRKY41 . These rbidopsis . Similar2O2 accumulation, and that H2O2, and therefore the expression of CaWRKY41, might confer Cd sensitivity and resistance to R. solanacearum. In support of this notion, the Arabidopsis ocp3 mutant, which produces high levels of H2O2 and exhibits increased resistance to the necrotrophic pathogens Botrytis cinerea and Plectosphaerella cucumerina (R. solanacearum in inoculated pepper plants (R. solanacearum inoculation increased Cd uptake by the roots and leaves of pepper plants exposed to excess Cd (2O2 accumulation increases plant immunity and plant sensitivity to excess Cd.We speculate that exposure to excess Cd triggers Hcumerina . In addir plants . By contxcess Cd . TogetheCaWRKY41-overexpressing Arabidopsis plants were significantly higher than those of WT plants (CaWRKY41-overexpressing Arabidopsis plants to Cd stress is due to their high Cd contents.Increased Cd uptake or reduced levels of Cd detoxification result in cellular damage in plants (T plants , indicatAtZIP3, AtZIP4, and AtZIP9, by CaWRKY41 (CaWRKY41 was activated by Fe deficiency, the Fe content of CaWRKY41-overexpressing Arabidopsis plants did not significantly differ from that of control plants (CaWRKY41 would positively regulate disease resistance in pepper plants but promote the absorption of Cd and enhance sensitivity to Cd, which might reduce the adaptability of the plant to a heavy-metal-contaminated environment. We speculate that CaWRKY41 might have evolved to coordinate plant immunity and the absorption of essential ions, including Zn, by modulating the activity of specific ion transporters. Indeed, Zn is required for the functioning of Zn binding motif-containing proteins associated with disease resistance, including WRKY TFs (Our findings suggest that the enhanced Cd contents might be due to the up-regulation of various Zn transporter genes, such as CaWRKY41 , C, E. Il plants . It is p2O2 accumulation and the expression of CaWRKY41, as well as a positive feedback loop between these processes, are induced by R. solanacearum infection or excess Cd exposure. The increase in H2O2 accumulation and CaWRKY41 expression enhance plant immunity and sensitivity to excess Cd exposure by increasing Cd uptake via Zn transporters.Based on these findings, we propose a working model  in whichJXB online.Supplementary data are available at Fig. S1. Phylogenetic analysis of eight pepper group III WRKY proteins and Arabidopsis and tomato group III WRKY proteins.2O2 accumulation.Fig. S2. Cd stress and Fe deficiency promotes HpCaWRKY41::GUS Arabidopsis plants under normal growth conditions.Fig. S3. GUS expression in transgenic Fig. S4. CaWRKY41 is a transcriptional activator localized to the nucleus.Fig. S5. Analysis of the effects of Cd stress on plant growth using chlorophyll fluorescence imaging before the appearance of visible effects on plant growth.Fig. S6. Effect of Cd treatment on Zn concentrations in Arabidopsis.ocp3 mutant shows reduced tolerance to Cd stress.Fig. S7. The Arabidopsis ZIP members involved in Zn uptake.Fig. S8. RT–qPCR analysis of the R. solanacearum growth and R. solanacearum infection increases Cd uptake.Fig. S9. Cd inhibits Table S1. Sequences of primers used in this study.CaWRKY group III genes.Table S2. C/S-elements in the 2 kb promoter fragment of CaWRKY group III genes.Table S3. Analysis of the Supplementary Figures S1-S9Click here for additional data file.Supplementary Tables S1-S3Click here for additional data file.