Arabidopsis thaliana, CONSTANS (CO) transcription factor plays a central role in regulating photoperiodic flowering. In contrast to posttranslational regulation of CO protein, still little was known about CO transcriptional regulation. Here we show that the CINCINNATA (CIN) clade of class II TEOSINTE BRANCHED 1/ CYCLOIDEA/ PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR (TCP) proteins act as CO activators. Our yeast one-hybrid analysis revealed that class II CIN-TCPs, including TCP4, bind to the CO promoter. TCP4 induces CO expression around dusk by directly associating with the CO promoter in vivo. In addition, TCP4 binds to another flowering regulator, GIGANTEA (GI), in the nucleus, and induces CO expression in a GI-dependent manner. The physical association of TCP4 with the CO promoter was reduced in the gi mutant, suggesting that GI may enhance the DNA-binding ability of TCP4. Our tandem affinity purification coupled with mass spectrometry (TAP-MS) analysis identified all class II CIN-TCPs as the components of the in vivo TCP4 complex, and the gi mutant did not alter the composition of the TCP4 complex. Taken together, our results demonstrate a novel function of CIN-TCPs as photoperiodic flowering regulators, which may contribute to coordinating plant development with flowering regulation.Photoperiod is one of the most reliable environmental cues for plants to regulate flowering timing. In  Arabidopsis thaliana responds to photoperiodic changes and flowers under long-day conditions. Based on genetic analyses using mutants defective in the photoperiodic flowering response, we learned that the transcription factor referred to as CONSTANS (CO) plays a central role in regulating the timing of flowering by directly controlling the expression of florigen (flowering-inducing substrate) gene. Long-day afternoon expression of CO is critical for this regulation; however, we had limited knowledge of CO transcriptional regulation. Here we identified that a group of plant-specific transcription factors belonging to the TCP gene family function as novel CO transcriptional activators. We demonstrated that TCP transcription factors regulate CO transcription together with known regulators of CO. Our results imply that plants utilize multiple transcription factors to precisely coordinate the expression of the key regulator gene, CO, which will directly affect flowering time.For plant adaptation to seasonal environments, a crucial developmental event is flowering, as proper timing of flowering affects reproductive success. Although plants monitor various environmental parameters to optimize this timing, photoperiod information is important for plants to regulate seasonal flowering time, because changes in photoperiod occur in a predictable manner throughout the year. The model plant  Arabidopsis thaliana, the growth-phase transition, which is observed as flowering, is promoted in long days (LD). Flowering is induced by the expression of FLOWERING LOCUS T (FT), which is induced specifically at dusk during LD , TCP4 was no longer able to induce the expression of the Luc reporter ] especially from afternoon to evening [Zeitgeber time 10 (ZT10) to ZT19] in both LD and SD  and four FBHs  were mutated, and analyzed CO expression patterns in the mutant in LD  are transcriptional activators of rs, FBHs , we nextnt in LD . The CO e mutant . This suh mutant , which ie mutant . To furtckground . OverexpQ plants . To invefternoon . Togetheme in LD , these rpression . SimilarCP lines , the indCO transcription also work together with TCP4 and/or FBH1, we performed yeast two-hybrid analysis and found that GI interacts with both TCP4 and FBH1 mainly through its N-terminal region  fused to YFPn/YFPc , which is one of the major floral integrators downstream in the photoperiodic pathway . After staining, the samples were bleached and dehydrated with a sequence of buffers: 30% ethanol, fixing solution , 80%, and 100% ethanol for 30 min each. The staining patterns of GUS activity of more than 20 individual T1 transgenic plants were analyzed, and the data from the T2, T3, and T 4 population plants which showed the representative staining patterns of the T1 populations are shown.For the construction of the y vector . Transgeeviously . Transge2, and then homogenized in 10 ml of the nuclei extraction buffer . Cross-linking reaction was performed by treating with 1% formaldehyde for 10 min at 4°C. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.15 M and incubating at 4°C for 5 min. After filtration with miracloth, and centrifugation at 10,000 g at 4°C for 10 min, nuclei-containing pellets were washed twice in the nuclei wash buffer . Isolated nuclei were lysed in the nuclei lysis buffer  and sonicated to shear DNA to an average size of 500 to 1,000 bp. The chromatin solution was diluted to 1 ml with ChIP dilution buffer . Immunoprecipitation was performed using Dynabeads Protein G (Life technologies). The beads were pretreated with anti-FLAG antibody  and incubated with chromatin solution in an ultrasonic water-bath for 20 min, followed by 1.5-h incubation at 4°C. After washing with low salt buffer , high salt buffer  and TE buffer , immunocomplexes were eluted from beads, reverse cross-linked at 65°C overnight, and treated with proteinase K that digests all proteins. DNA was extracted and eluted in 140 μl of volume using QIAquick PCR purification kit (Qiagen), according to manufacturer protocol. 3μl aliquots were used for qPCR reaction. The forward and reverse primer pairs that were used to amplify the genome sequence designated as CO amplicon 1 to amplicon 7 are shown in UBQ) for 20 sec, and 72°C extension for 15 sec. The immunoprecipitation efficiency (%) against the total input was calculated for each amplicon using the following formula: 0.02×2(Ct input-Ct ChIP) ×100.Approximately 1.5 to 2 g (fresh weight) of 10-day-old seedlings harvested at the indicated time point were ground into fine powder in liquid NCO promoter sequence, we used different combinations of forward primers  with reverse primer (5′-AAAGCTTATATCTGGTGTGAGAGA-3′). Amplified PCR products were cloned into pENTR/D-TOPO vector. CO promoter fragments were transferred to firefly luciferase (Luc) vector pFLASH (Gateway compatible version of pPZPXomegaLUC+ . After 4 h of incubation at room temperature in MES buffer, Agrobacterium solution was infiltrated into the abaxial air spaces of leaf tissue using 1-mL syringes. Two to three days after infiltration, YFP and RFP images of the tissue were analyzed with a confocal laser scanning microscope . Approximately 50 to 100 cells that express H2B-RFP were observed in each combination. We called positive interactions when more than 70% of the H2B-RFP positive cells showed reconstituted specific YFP signals, whereas no signals were observed from the combinations with GSTNLS.To generate binary vectors for the BiFC assay, the coding sequences of Ps, FBH1 , and GI FBH1 . Samples were sonicated and filtered twice through 0.45-μm pore size PVDF membrane (Millipore), followed by centrifugation to remove cell debris. The clear supernatant was incubated with anti-FLAG antibody -bound Dynabeads (Invitrogen). Once the TCP4-3F6H proteins were captured by the Dynabeads, the beads were washed three times with SII buffer with 0.1% Triton, and without the deoxycholate, protease and phosphate inhibitor mixtures. The rest of the purification steps were same as previously described [TAP-MS analysis using functional TCP4-3F6H protein was performed based on the previously described methods with slight modification . Approxiescribed .On-bead digestion for TCP4-interacting proteins was performed using the following methods. Disulfide bonds of proteins bound to Dynabeads were reduced using 50 μl of 0.2% PPS surfactant (Expedeon) in 100 mM ammonium bicarbonate with 1 μl 500 mM Tris (2-carboxyethyl) phosphine hydrochloride (Thermo Fisher Scientific) for one hour at 60°C. The sample was cooled to room temperature and the cysteine thiols alkylated by the addition of 1.1 μl of 500 mM iodoacetamide for 20 min. The beads were then digested with the addition of 1 μg trypsin overnight at 37°C with constant agitation. The supernatant containing the tryptic peptides were removed, and combined with a methanol wash of the beads. The combined supernatants were dried using vacuum centrifugation, and resuspended in 15 μl of 0.1% trifluoroacetic acid prior to LCMS analysis.m/z 400, and for MS/MS the linear ion trap provided unit resolution. The automatic gain control targets for MS in the FT was 1×106, whereas for MS/MS it was 8000, and the maximum fill times were 20 and 80 ms, respectively. The MS/MS spectra were acquired using an isolation width of 2 m/z and a normalized collision energy (NCE) of 35. MS/MS acquisitions were prevented for precursor charge states of 1, or if the charge state could not be discerned from the MS spectrum. Dynamic exclusion  was set for 30 sec.All mass spectrometry was performed on a LTQ-FT (Thermo Fisher Scientific). Three microliters of sample digest were loaded from the autosampler onto a 150-μm Kasil fritted trap packed with 3 u Dr. Maisch ReproSil-Pur C18-AQ beads to a bed length of 2 cm at a flow rate of 2 μl/min. After loading, the trap was brought on-line with a pulled fused-silica capillary tip (75-μm i.d.) also packed with the same Dr. Maisch beads mounted in an in-house constructed microspray source and placed in line with a Waters Nanoacquity binary UPLC pump plus autosampler. Peptides were eluted off the column using a gradient of 2–35% acetonitrile in 0.1% formic acid over 60 min, followed by 35–60% acetonitrile over 5 min at a flow rate of 250 μl/min. The mass spectrometer was operated using data dependent acquisition (DDA) where a maximum of seven MS/MS spectra were acquired per MS spectrum. The resolution for MS was 100,000 at q-value cutoff of 0.01. Cysteine residue masses were considered statically modified by iodoacetamide, and methionine dynamically modified by a single oxidation. Precursor mass tolerance was 10 ppm, and product ion tolerance 0.5 Da. The principle of parsimony was used for protein inference. Spectral counts per protein were then analyzed using the Fisher’s Exact test, where alpha was set at 0.01 and multiple hypothesis correction was carried out using the Bonferroni correction. Frequently identified contaminant proteins were subtracted based on previous reports [Peptide identification was performed using Comet, searching against a Uniprot Arabidopsis protein sequence database, and using Percolator with a  reports .N. benthamiana, and ChIP analyses was determined using Welch’s t-test or Student’s t-test. The statistical significance in gene expression analyses was determined by one-way ANOVA with strains as main effects. Pairwise comparisons were determined using Dunnet’s test on each time point. Student’s t-test was used to determine the statistical difference in gene expression or protein expression between two strains on each time point. The statistical significance in flowering time measurement was determined by Tukey’s honestly significant difference test. The p values are indicated in the figure legends.Statistical analyses were done using R Statistical Computing software . The statistical significance in yeast one hybrid analyses, transient assays in S1 FigTCP4 (A and B), TCP3 (C and D), CO (E and G) and FT (F and H) in LD (A and C) and SD  in 35S:TCP4 , 35S:TCP3 , and wild type (WT) plants. Time indicates hours (h) after light onset within a day. Bars above the traces represent light conditions; open and filled bars represent day and night, respectively. All expression data were normalized against IPP2 and shown relative to the average expression of each gene in WT. FT expression values in SD are shown relative to the WT average expression value in LD shown in p<0.05, Dunnett’s test). Data represent means ± SEM (n = 3). (I and J) Results of qPCR analysis that show the expression level of TCP10 (I) and CO (J) in 35S:TCP10 lines and WT harvested at ZT16 in LD. Significant differences are indicated by asterisks . Data represent means ± SEM (n = 3).(A to D) Results of qPCR analysis that show gene expression patterns of (EPS)Click here for additional data file.S2 FigTCP4 (A) and CO (B) in LD. Significant differences from WT are indicated by asterisks . Data represent means ± SEM (n = 3). (C) The representative protein profiles of mTCP4-3F6H in LD. Ponceau S-staining of RbcL is shown as the loading control. Similar results were obtained from 3 independent experiments.(A and B) Results of qPCR analysis that show the gene expression pattern of (EPS)Click here for additional data file.S3 Figtcp mutants in SD. Total number of rosette leaves and cauline leaves generated from the main stem were counted when plants bolted. ns: no significance (HSD test). Data represent means ± SEM (n≥16). (B and C) Results of qPCR analysis that shows gene expression patterns of CO (B) and FT (C) in SD in higher order tcp mutants. FT expression values in SD are shown relative to the WT average expression value in LD shown in p<0.05, Dunnett’s test). Data represent means ± SEM (n = 3).(A) Flowering phenotypes of higher order (EPS)Click here for additional data file.S4 FigFBH1 (A) and FBH4 (B) in LD. Significant differences from WT are indicated by asterisks . Data represent means ± SEM (n = 3).(A and B) Results of qPCR analysis that shows gene expression patterns of (EPS)Click here for additional data file.S5 FigFBH1 (A), CO (B) and FT (C) in the 35S:FBH1, WT, and tcp-Q plants grown in LD. The data set shown in 35S:FBH1 #9, and tcp-Q. Significant differences between the 35S:FBH1 lines and their background strains are indicated by asterisks . Data represent means ± SEM (n = 3).(A to C) Results of qPCR analysis that show gene expression patterns of (EPS)Click here for additional data file.S6 Fign) and the full-length of FBH1 fused to the C-terminal half of enhanced YFP (FBH1-YFPc) were expressed in N. benthamiana leaf epidermal cells. H2B-RFP was used for the nuclear marker. Images from YFP and RFP channels were merged with bright-field (BF) images. Nuclear-localized form of GST fragments (GSTNLS) fused to YFPn or YFPc were used as negative controls. Scale bar, 20 μm. (C and E to H) Results of qPCR analysis that show gene expression patterns of FBH1 (C), CO (E and F), FT (G), and SOC1 (H) in 35S:FBH1, WT, and gi-2 grown in LD. CO expression profiles shown in (E) were enlarged in (F) to show the difference in lower values within the lines. Significant differences between the 35S:FBH1 lines and their background strains are indicated by asterisks . Data represent means ± SEM (n = 3). (D) Flowering phenotype of the 35S:FBH1 plants in LD. Total number of rosette leaves and cauline leaves generated from the main stem were counted when plants bolted. Significant differences are indicated by asterisks . Data represent means ± SEM (n = 16).(A) Results of the yeast two-hybrid assay between FBH1 and GI. The full-length of FBH1 and full-length or truncated GI fused to either the DNA-binding domain (DBD) or the activation domain (AD) of Gal4 were tested under selective  and non-selective  conditions. For GI, N and C indicate the amino acid residues1-391 and 382–1173, respectively. (B) BiFC assays of interaction between FBH1 and GI. The full-length of GI fused to the N-terminal half of enhanced YFP (GI-YFP(EPS)Click here for additional data file.S7 FigTCP4 (A), CO (B), and FT (C) in the 35S:TCP4-3F6H, WT, and gi-2 plants grown in LD. The same data set for WT and gi-2 shown in 35S:TCP4-3F6H lines and their background strains are indicated by asterisks . Data represent means ± SEM (n = 3). (D) The representative protein profiles of TCP4-3F6H in WT and gi-2 grown in LD are shown. Histone H3 protein was used as the loading control. The numbers above the images indicate time (h) after light onset within a day. (E) The quantified results of the TCP4-3F6H protein profiles shown in (D) were obtained from 3 independent biological replicates. Significant differences are indicated by asterisks . Data represent means ± SEM (n = 3).(A to C) Results of qPCR analysis that show gene expression patterns of (EPS)Click here for additional data file.S8 Figc were co-overexpressed with GST protein fused to YFPn in N. benthamiana leaf epidermal cells. H2B-RFP was used for a nuclear marker. Images from YFP and RFP channels were merged with bright-field (BF) images. Scale bar, 20 μm.Full length of TCP2, 3, 5, 10, 13, 17, and 24 fused to YFP(EPS)Click here for additional data file.S9 FigThe peptide coverages for TCP4 protein identified by MS analysis are indicated. The TCP4 peptides identified by MS analysis are highlighted in red. The percent coverage was 73%. The number of peptides recovered is shown in (EPS)Click here for additional data file.S10 Fig35S:TCP4-3F6H in either WT and gi-2 backgrounds harvested on ZT13 are shown. Amplicons located in the CO promoter are described in t-test, p<0.05). Data represent means ± SEM (n = 5).Results of ChIP analysis using (EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Phylodynamic analyses indicated that postvaccine G1P[8] strains emerged through reassortment events between human Wa- and DS-1-like rotaviruses that circulated in Malawi from the 1990s and hence were classified as atypical DS-1-like reassortants. The time to the most recent common ancestor for G1P[8] strains was from 1981 to 1994; their evolutionary rates ranged from 9.7 × 10−4 to 4.1 × 10−3 nucleotide substitutions/site/year. Three distinct G1P[8] lineages chronologically replaced each other between 1998 and 2014. Genetic drift was the likely driver for lineage turnover in 2005, whereas replacement in 2013 was due to reassortment. Amino acid substitution within the outer glycoprotein VP7 of G1P[8] strains had no impact on the structural conformation of the antigenic regions, suggesting that it is unlikely that they would affect recognition by vaccine-induced neutralizing antibodies. While the emergence of DS-1-like G1P[8] rotavirus reassortants in Malawi was therefore likely due to natural genotype variation, vaccine effectiveness against such strains needs careful evaluation.To combat the high burden of rotavirus gastroenteritis, multiple African countries have introduced rotavirus vaccines into their childhood immunization programs. Malawi incorporated a G1P[8] rotavirus vaccine (Rotarix) into its immunization schedule in 2012. Utilizing a surveillance platform of hospitalized rotavirus gastroenteritis cases, we examined the phylodynamics of G1P[8] rotavirus strains that circulated in Malawi before (1998 to 2012) and after (2013 to 2014) vaccine introduction. Analysis of whole genomes obtained through next-generation sequencing revealed that all randomly selected prevaccine G1P[8] strains sequenced (IMPORTANCE The error-prone RNA-dependent RNA polymerase and the segmented RNA genome predispose rotaviruses to genetic mutation and genome reassortment, respectively. These evolutionary mechanisms generate novel strains and have the potential to lead to the emergence of vaccine escape mutants. While multiple African countries have introduced a rotavirus vaccine, there are few data describing the evolution of rotaviruses that circulated before and after vaccine introduction. We report the emergence of atypical DS-1-like G1P[8] strains during the postvaccine era in Malawi. Three distinct G1P[8] lineages circulated chronologically from 1998 to 2014; mutation and reassortment drove lineage turnover in 2005 and 2013, respectively. Amino acid substitutions within the outer capsid VP7 glycoprotein did not affect the structural conformation of mapped antigenic sites, suggesting a limited effect on the recognition of G1-specific vaccine-derived antibodies. The genes that constitute the remaining genetic backbone may play important roles in immune evasion, and vaccine effectiveness against such atypical strains needs careful evaluation. Diarrhea is a leading cause of mortality in children under the age of 5 years globally , 2. The Reoviridae virus family. They are enveloped icosahedric viruses that contain a triple-layered capsid encasing 11 genome segments of double-stranded RNA (dsRNA). The rotavirus genome encodes six structural proteins  and five to six nonstructural proteins (NSP1 to NSP5/NSP6) .RVAs are members of the P5/NSP6) . To dateP5/NSP6) –12.  and postvaccine  rotavirus-positive stool samples collected from children presenting with acute severe diarrhea at Queen Elizabeth Central Hospital (QECH) in Blantyre, Malawi, were genotyped as part of ongoing rotavirus surveillance –30. Mult–While the concatenated sequences of all 11 genome segments of pre- and postvaccine G1P[8] rotaviruses clustered with prototype Wa- and DS-1-like strains, respectively , phylogeThe phylogenetic relationship of Malawi G1P[8] strains was inferred by using the maximum likelihood method, for which complete nucleotide sequences of RVAs available in GenBank and sequences of Wa-like (VP4 and VP7 only) and DS-1-like (the other nine genes) strains from Malawi were used. The DS-1-like genes of the postvaccine G1P[8] reassortants clustered closely with those of G2 strains that circulated simultaneously in Malawi  and not with DS-1-like G1P[8] strains identified in Southeast Asia and Japan. Sequences within each cluster were 95 to 99.8% similar .All 11 genome segments of the G1P[8] strains were undergoing purifying selection, thus potentially resulting in stabilizing selection following the purging of deleterious variants arising during error-prone rotavirus replication due to its RNA genome . The genBayesian inferences of time-measured trees were individually constructed for each of the 11 genome segments of the G1P[8] strains to further determine their evolutionary dynamics. As illustrated by phylogenetic networks, prior to the emergence of the reassortant L3 lineage, both structural and nonstructural genes segregated into at least 2 distinct lineages with a common ancestor in the mid-1990s or before , whereas VP3 had the highest mutation rates  , which also circulated in Malawi from 1997 to 2014, were used to estimate evolutionary dynamics for non-VP4 and non-VP7 genes for postvaccine G1P[8] strains . When onper year . VP2 hadWe then utilized a Gaussian Markov random field (GMRF) tree prior to investigate whether Rotarix introduction had an impact on the relative population size of the circulating G1P[8] strain. There was no evidence to suggest that vaccine introduction affected either G1P[8] genetic diversity or population size for the VP4, VP7, NSP2, NSP3, and NSP5 genes, as the peaks and troughs of their Skygrid plots exhibited similar stable profiles just before (2005 to 2012) and after (2013 to 2014) vaccine use. In contrast, genes encoding VP1 to VP3, VP6, and NSP1 had relatively stable profiles and also small effective population sizes during the postvaccine era compared to those before vaccine introduction, which could be natural, as similar downward trends were already occurring before vaccine introduction .Mapped amino acid motifs that constitute neutralizing epitopes on the outer capsid glycoprotein were compared between Rotarix and Malawian G1P[8] strains. In total, 15 lineage-defining amino acid substitutions were identified across the entire VP7 sequence, with only 5 of these being located at mapped antigenic regions 7-1a (S123N and K291R) and 7-2  . A singlThe VP7 structures of L1 to L3 strains and that of the Rotarix strain aligned perfectly when superimposed , implyinMalawi was one of the first African countries to introduce a rotavirus vaccine into its infant immunization schedule in October 2012. By 2015, vaccine coverage had exceeded 95% . We enhaWhile the prevaccine G1P8] strains had a typical Wa-like genetic constellation  strains , 34, 35,21–−4 to 1.01 × 10−3 nucleotide substitutions per site per year, were similar to those for Malawian G1P[8] strains. These rates are relatively lower than the known rates of RNA viruses  and was also present among strains circulating pre-2009, those of L2. However, it is possible that substitutions in the non-AR could affect the stability of the viral particle or protein assortment specificities, since VP6 serves as an anchor for the outer capsid VP4 and VP7 proteins, where the 260 trimers of VP7 lie directly on top of the VP6 trimers . ContactIn conclusion, genome reassortment and mutation are the major evolutionary mechanisms that influenced the genetic diversity of G1P[8] strains that circulated in Malawi from 1998 to 2014. Atypical DS-1-like G1P[8] strains emerged in 2013 through genome reassortment events between Wa- and DS-1-like human strains that can be traced back to the 1990s in Malawi. Mutations within outer capsid VP7 of Malawian G1P[8] strains compared to RV1 had no impact on the structural conformation of antigenic regions, suggesting little or no effect on the recognition of vaccine-induced antibodies. Thus, the remaining genome segments (non-G or -P) might also play an important role in immune evasion. It is likely that the atypical DS-1-like G1P[8] strains emerged through natural strain evolutionary pressure, which is unrelated to vaccine use. However, the predominance of atypical reassortant DS-1-like G1P[8] strains, which coincided with vaccine introduction, could suggest the positive selection of atypical G1P[8] strains that were undergoing purifying selection. Vaccine effectiveness against such atypical strains needs careful investigation.Stool samples collected from children aged <5 years presenting with acute gastroenteritis at QECH in Blantyre, Malawi, from 1998 to 2014 were utilized –29. Diar–27–Rotavirus dsRNA was extracted and purified as previously described , 56. An After denaturation of dsRNA at 95°C for 5 min, ScriptSeq RNA-Seq Library Preparation kit V2 was used to generate Illumina sequencing libraries for samples that had <2 ng/μl dsRNA . Purified cDNA generated from samples with >2 ng/μl dsRNA was subjected to standard bar-coding and library construction for Illumina sequencing using a Nextera XT DNA library preparation kit . Rotavirus VP6-specific quantitative PCR (qPCR)  and a 21de novo and reference DNA sequence assembler algorithms implemented in Geneious software v8 (http://rotac.regatools.be/) (Illumina adapter sequences were trimmed from the raw Fastq sequence data by using Cutadapt v1.2.1  and Sicktware v8 . Rotavirols.be/) .Reference nucleotide sequences for each rotavirus genome segment were retrieved from the rotavirus resource in the GenBank database . This wa−3 nucleotide substitutions/site/year, as previously reported by Zeller et al. (Neτ) or relative genetic diversity, over time. A total of 200 million Markov chain Monte Carlo (MCMC) iterations were performed and sampled every 40,000th generation. The first 20 million iterations  from the MCMC analysis, burn-in time, were discarded since these iterations may represent states that the chain explored before reaching the equilibrium state of the target distribution. The mean values and 95% HPDs of the mutation rates and the tMRCAs for each rotavirus segment were calculated from the BEAST output by using Tracer v1.6.0 (https://tree.bio.ed.ac.uk/software/tracer/). The MCC tree for each viral segment was generated by using Tree Annotator v2.1.2 (http://beast.bio.ed.ac.uk/treeannotator) and visualized by using FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and BioPython scripts . The alignments were then used to calculate the global ratio of synonymous to nonsynonymous substitutions (dN/dS) for each ORF by using single-likelihood ancestor counting (SLAC)   and a fag (SLAC) . To ideng (SLAC) , random-g (SLAC) , and fixg (SLAC)  methods g (SLAC) . The occg (SLAC)  and SBP g (SLAC) . We usedb server .To investigate the likely impact of amino acid substitutions on anti-RV1 antibody recognition due to mutations that occurred over time within the antigenic regions of Malawian G1P[8] rotaviruses, the structural conformations of the outer capsid glycoprotein of pre- and postvaccine strains were compared to that of RV1. Representative VP7 sequences for each G1P[8] lineage were utilized for protein structure modeling using Modeler version 9.17 . TemplatMG181227 to MG181941.All complete nucleotide and deduced amino acid sequences generated in this study were deposited in GenBank  under ac


Atypical antipsychotics such as olanzapine cause metabolic side effects leading to obesity and insulin resistance. The underlying mechanisms remain elusive. In this study we investigated the effects of chronic treatment of olanzapine on the fatty acid composition of plasma in mice.Twenty 8-week female Balb/c mice were randomly assigned to two groups: the OLA group and the control group. After treatment with olanzapine (10 mg/kg/day) or vehicle intraperitoneally for 8 weeks, fasting glucose, insulin levels and oral glucose tolerance test were determined. Effects on plasma fatty acid profile and plasma indices of D5 desaturase, D6 desaturase and SCD1 activity were also investigated.Chronic administration of olanzapine significantly elevated fasting glucose and insulin levels, impaired glucose tolerance, but did not increase body weight. Total saturated fatty acids and n-6 polyunsaturated fatty acids were significantly increased and total monounsaturated fatty acids were significantly decreased, while total n-3 polyunsaturated fatty acids showed no prominent changes. Chronic olanzapine treatment significantly up-regulated D6 desaturase activity while down-regulating D5 desaturase activity. Palmitic acid (C16:0), dihomo-γ-linolenic acid (C20:3n-6) and D6 desaturase were associated with an increase probability of insulin resistance, whereas nervonic acid (C24:1) and SCD1 were significantly associated with a lower insulin resistance probability.All results indicated that such drug-induced effects on fatty acid profile in plasma were relevant for the metabolic adverse effects associated with olanzapine and possibly other antipsychotics. Further studies are needed to investigate geneticand other mechanisms to explain how plasma fatty acids regulate glucose metabolism and affect the risk of insulin resistance. Olanzapine is a widely used second generation antipsychotic (SGA) drug for the treatment of schizophrenia with a low propensity for neurological side effects. HoweverThe fatty acids (FAs) in particular are highly associated with obesity, IR, and type 2 diabetes–15. SeveThe FA profile in blood partly reflects dietary FA intake, but it is also strongly determined by the endogenous FA metabolism. The D5 The FA profile can be used as an indicator of disease risk. An alteg for 10 min at 4°C. All samples were stored at –80°C until further analysis. All efforts were made to minimize suffering.Twenty 8-week female Balb/c mice weighing 20–25g  were purchased and maintained under a 12-hour light/dark cycle (lights on at 08:00 h) at 22±1°C. Animals were housed individually and allowed free access to food and water. Prior to the experiments, animals were routinely acclimated (>1 week) tolaboratory conditions to reduce potential stress effects during experiments. The animal facilities and protocol were performed in accordance with the guidelines of the Chinese Council on Animal Care and approved by the Institutional Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology (Permit Number:XH-B20150422).The number of mice was theminimum necessary to obtain significant results and inagreement with the triple R spirit for reduction of the number of animals used. Body weight was routinely recorded. Mice were anesthetized by isoflurane  and sacrificed at 9:00 am after 8 weeks of olanzapine treatment. Blood was collected into EDTA-coated tubes and plasma was isolated by centrifugation at 1,800×The primary study endpoints were the fasting insulin level and the homeostatic model assessment of insulin resistance (HOMA-IR) index. The secondary endpoint was the oral glucose tolerance test (OGTT).Mice were randomly assigned to two groups: the OLA group and the control group. The OLA group received olanzapine intraperitoneally (10mg/kg/day) in the morning (08:00–08:40h) for 8 weeks, while the control group received drug vehicle. All injection volumes were 10ml/kg.Olanzapine  was dissolved in0.1 N HCl in distilled water, adjusting to pH 6.0 with 1 N NaOH, and adding distilled water to reach the desired concentration. Vehicle was similarly pH-adjusted, distilled water. The solutions were stored at 4°C and protected from light degradation. At the time of drug administration, body weight was measured.OGTT was performed on the last day of the chronic treatment. Mice were food restricted for 14h prior to the OGTT. Mice received olanzapine one hour prior to the start of the OGTT. During the OGTT, basal blood samples and glucose measurements were obtained and then glucose was given via oral gavage (1.5g glucose/kg). Blood samples were collected via a tail snip at 30, 60, 90, and 120 min and glucose levels were determined. Blood glucose levels at 30, 60, 90, 120 min were determined via a tail snip method. Individual glucose measurements at the 5 time points during the OGTT were integrated to generate a single area under the curve (AUC) value.Fast glucose concentrations were determined using a hand-held glucometer (One Touch Ultra). Fast insulin levels were measured using commercially available kits  according to the manufacturer’s instructions. All analyses were performed by a technician blinded to treatment.0 x G0]/22.5), where I0 and G0 are fasting insulin and glucose. A larger calculated HOMA-IR value denotes greater insulin resistance.To determine insulin resistance in mice, we calculated the HOMA-IR index on the last day of every week. This equation takes into account the product of both fasting levels of glucose (expressed as mmol/L) and insulin (μU/mL) at 60 minutes post-olanzapine treatment and divides by a constant of 22.5 .All data are expressed as the mean±SD. To calculate statistical significance, Student’s vs 25.59mmol/L, p<0.001), suggesting decreased whole-body insulin sensitivity. These results , and total n-6 PUFAs  were both respectively higher in the OLA group compared to the control group. However, total MUFAs  were significantly decreased in the OLA group compared to the control group. Total n-3 PUFAs showed no significant difference between two groups. In the OLA group, we observed increased palmitic acid (C16:0) and heptadecanoic acid (C17:0) levels when compared to the control group . Linoleic acid (C18:2n-6), γ-linolenic acid (C18:3n-6), and dihomo-γ-linolenic acid (C20:3n-6) were statistically significantly increased in the plasma of OLA group as compared to the control group , while eicosadienoic acid (C20:2n-6) was significantly decreased (0.77% and 1.01%). Moreover, in the OLA group myristoleic acid (C14:1), pentadecanoic acid (C15:1), palmitoleic acid (C16:1) and heptadecanoic acid (C17:1) were significantly decreased compared to the control group , whereas nervonic acid (C24:1) was increased (0.07% and 0.05%). Our results , (C17:0), (C24:1), (C18:2n-6), (C18:3n-6) and (C20:3n-6) were higher, and the levels of FAs (C14:1), (C15:1), (C16:1), (C17:1) and (C20:2n-6) were lower in the OLA group compared to the control group. In contrast, the levels of total n-3 PUFAs showed no significant differences between the two groups. We also found that palmitic acid (C16:0), dihomo-γ-linolenic acid (C20:3n-6) and D6 desaturase were associated with an increase probability of IR, whereas nervonic acid (C24:1) and SCD1 were significantly associated with a lower IR probability.Previous work has demonstrated that chronic treatment of SGAs frequently induces excessive weight gain and obesity in schizophrenic patients and animals. HoweverIn the present study, we showed that chronic olanzapine treatment significantly increased total SFAs and decreased total MUFAs. Additionally, palmitic acid, the main contributor of total SFAs, was significantly increased and positively correlated with IR, in agreement with recent studies showing associations with impaired insulin sensitivity–36. ManyAt an exploratory level of significance we also found higher levels of the essential fatty acid linoleic acid (C18:2n-6) and total n-6 PUFAs in the OLA group. These n-6 PUFAs are linked to inflammatory signaling, insulin resistance and type 2 diabetes risk. TherefoThe FA profile in blood and tissues partly reflectsdietary FA intake, but it is also strongly determined bythe endogenous FA metabolism. The fatty acid-modifying enzymes for which connections to insulin resistance and type 2 diabetes have been shown include D5 desaturase, D6 desaturase and SCD. ClinicaRecently, it has been postulated that potential alterations in the metabolic pathway of PUFAs synthesis could constitute a fundamental trigger in the initiation and propagation of metabolic abnormalities, such as IR, 47. NumThis study has three notable limitations. First, olanzapine has a shorter plasma half-life in rats (~2.5h) than in humans. However, previous studies showed that 15 days of i.p. olanzapine at 6 mg/kg body weight in rats produced a mean plasma concentration of 12.0±4.9 ng/ml at 3h after the last daily dose, which is comparable to the plasma concentration (above 9 ng/ml) that had a greater likelihood of clinical response. MoreoveIn conclusion, our study shows that chronic olanzapine treatment induced a significant increase in fasting glucose and insulin levels and insulin resistance without body weight gain. Chronic treatment of olanzapine significantly increased total SFAs and total n-6 PUFAs, while decreased total MUFAs. Olanzapine also significantly up-regulated D6 desaturase activity while down-regulated D5 desaturase activity. Our study also find that palmitic acid (C16:0), dihomo-γ-linolenic acid (C20:3n-6) and D6 desaturase were associated with an increase probability of IR, whereas nervonic acid (C24:1) and SCD1 were significantly associated with a lower IR probability. The present data provide further support for olanzapine-mediated perturbation of fatty acid profile in plasma as a molecular mechanism involved in antipsychotic-associated metabolic adverse effects. Further studies are needed to investigate genetic and other mechanisms to explain how plasma fatty acids regulate glucose metabolism and affect the risk of IR.S1 Data(XLSX)Click here for additional data file.


Cardiac RF ablation is a widely accepted procedure for treatment of ventricular tachycardia and atrial fibrillation. Late gadolinium enhanced MRI (LGE-MRI) can be used to assess RF ablation lesions. However, LGE-MRI requires contrast injection and the visibility of lesions and appearance considerably change with time after the injection. Recently, native (non-contrast) T1-weighted (T1w) MRI was proposed to visualize RF lesions immediately post-ablation. The main aim of this work was to study how the visibility and volume of RF lesions in native T1w MRI changes with time after ablation.RF ablations of right and left ventricles of 2 canines were performed according to protocol approved by the local IACUC. RF lesions were created using Cool Flex catheter  at 30-40 Watts for 60 seconds. Imaging studies were performed on a 3T MRI scanner  at 0, 2, and 7 days post-ablation. Native T1w images of whole heart were acquired using 3D respiratory navigated, saturation recovery prepared GRE pulse sequence. Typical scan parameters were TR/TE=3.1/1.4 ms, flip angle of 10 degrees, TI=400 ms, voxel size=1.25x1.25x2.5 mm. Fat saturation was applied immediately before data acquisition limited to 10% of RR interval.Ablation lesions (enhanced regions) were manually segmented on native T1w images acquired at different time point. Lesion volume was calculated for each ablation (n = 9). Lesion volumes for 2 and 7 days post-ablation studies were normalized by the corresponding lesion volumes from acute (0 day) study. The visibility of ablation lesions in T1w images was quantitatively assessed using Image Intensity Ratio (IIR) metrics. IIR was calculated as a ratio of mean signal of lesion and mean signal of normal myocardium.Representative T1w images of RF ablation lesions acquired acutely, 2, and 7 days post-ablation are shown in Figure Reduction of T1 relaxation time of RF ablated myocardium has transient nature. The visibility of RF ablation lesions and volume of the corresponding enhanced regions in native T1w MRI drastically reduce with time after ablation. Non-contrast T1w MRI should be performed earlier (< 2 days) after RF ablation to achieve high contrast between ablated and normal myocardium and get an accurate estimate of lesion dimensions.


S. aureus infection and pore-forming toxins (Streptolysin and Listeriolysin). Cells detect changes in potassium level as a Danger-associated molecular pattern associated with cell damage and induce BPI expression in a p38 dependent manner. These results are further supported by in vivo findings that the BPI expression in murine intestinal epithelium is induced upon infection with bacteria which cause intestinal damage  whereas mutants that do not cause intestinal damage (STM ΔfliC and STM ΔinvC) did not induce BPI expression. Our results suggest that epithelial damage associated with infection act as a signal to induce BPI expression.As the first line of defense against invading pathogen, intestinal epithelium produces various antimicrobial proteins (AMP) that help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI) is a 55 kDa AMP that is expressed in intestinal epithelium. Dysregulation of BPI in intestinal epithelium is associated with various inflammatory diseases like Crohn’s Disease, Ulcerative colitis, and Infectious enteritis’s. In this paper, we report a direct correlation between intestinal damage and BPI expression. In Caco-2 cells, we see a significant increase in BPI levels upon membrane damage mediated by  The differentiation of self and non-self is an important aspect of both innate and adaptive immune response. Eukaryotic cells detect the presence of a pathogen by recognizing conserved microbial structures derived from pathogens collectively known as PAMPs (pathogen-associated molecular patterns). Detection of PAMPs by innate immune cells leads to the production of AMPs  and ROS (reactive oxygen species) that will help in clearance of pathogen. Bactericidal/permeability-increasing protein (BPI) is an anti-microbial protein which actively inhibits invasion by Gram-negative bacteria. The high affinity of BPI for LPS compared to its counterpart LBP (Lipopolysaccharide binding protein) makes BPI an anti-inflammatory molecule as well . In humaSalmonella Typhimurium (STM) which did not induce BPI expression in vitro, induced BPI expression in the murine intestinal epithelium in vivo which is the outcome of inflammation associated epithelial damage. Mutants of STM that cause less epithelial damage also showed less BPI expression. Together, these results indicate that intestinal epithelial cells recognize DAMPs as a signal for epithelial damage and induce BPI expression.In this study, we screened for various inflammatory signals for inducing BPI expression in intestinal epithelial cells. We found that pore-forming toxins induce BPI expression in intestinal epithelial cells. The cell damage associated changes in potassium levels act as Damage Associated Molecular Patterns (DAMPs) and induce BPI expression in a p38 dependent manner. As expected, none of the PAMPs screened induced BPI expression in epithelial cells. Interestingly, Salmonella Typhi (STY), and Staphylococcus aureus (SA) at a multiplicity of infection (MOI) of 10. Twenty-four hours post-infection, RNA was isolated and BPI expression was quantified by real-time PCR. ATLA4 (aspirin-triggered lipoxin A4) was used as a positive control in the experiment . Interestingly, BPI expression increased up to fivefold upon SA infection compared to uninfected control. As expected, BPI expression increased up to threefold upon ATLA4 treatment. Infection with STM, STY or treatment with different Pathogen Associated Molecular Patterns (PAMPs) viz LPS (100 ng), Flagellin (500 ng) and Heat Killed STM (HK STM) did not significantly influence BPI expression in Caco-2 cells.To explore the link between infection and BPI expression in intestinal epithelial cells, we analyzed the expression of BPI in Caco-2 cells upon bacterial infection. Caco-2 cells were infected with different pathogens viz STM, S. aureus at an MOI of 10. Cells were lysed at indicated time points , total protein was isolated and BPI expression was checked by western blotting . BPI expression significantly increased in a time-dependent manner in SA infected cells compared to uninfected control. There was up to fourfold increase in BPI expression within 24 h post-SA infection compared to uninfected control. SA infection induced BPI expression in HeLa cells as well, indicating a common mode of regulation in these cells . BPI expression increased in an MOI-dependent manner in Caco-2 cells as analyzed by quantifying the MFI (Mean-fluorescent intensity) of BPI expression. Maximum expression of BPI was seen at MOI of 100. ATLA4 was used as a positive control in the experiment . BPI expression was significantly high in polarized Caco-2 cells upon S. aureus infection compared to uninfected control. BPI expression remained unchanged upon STM infection. These results confirm that BPI levels are increased in intestinal epithelial cells upon SA infection.In order to evaluate BPI expression at protein level, Caco-2 cells were infected with periment . These rStaphylococcus aureus is known to produce pore-forming toxins that can damage the epithelial cells . Pretreatment of Caco-2 cells with SB203580 significantly reduced SA induced BPI expression. To confirm this result, we checked the expression of BPI under similar conditions using confocal microscopy. Cells pretreated with p38 MAPK inhibitor were infected with SA. Twenty-four hours post-infection, cells were fixed and BPI expression was analyzed by using confocal microscopy . Pretreatment of cells with SB203580 significantly reduced SA induced BPI expression as quantified by evaluating the MFI of BPI expression. These results suggest that SA induced BPI expression is in a p38 dependent manner.al cells . Epithelal cells . We hypoFigures 2C,D). SLO treatment increased BPI expression up to 2.5-fold compared to untreated control. This increased BPI expression in epithelial cells in a concentration-dependent manner further confirmed the specificity of induction . To supplement these results, we quantified BPI expression in Caco-2 cells after treating the cells with Listeriolysin O (LLO). Twenty-four hours post-treatment, BPI expression was analyzed by confocal microscopy and western blotting  for 24 h. Cells were lysed and BPI expression was quantified by western blotting (+ ionophore (Nigericin). Twenty-four hours post-treatment, cells were fixed and BPI expression was analyzed by confocal microscopy . Nigericin treatment led to significant upregulation of BPI expression compared to control as quantified by increase in MFI of BPI in nigericin treated cells . To understand the role of p38 MAPK signaling pathways in inducing nigericin mediated BPI expression, Caco-2 cells were pretreated with p38 MAPK inhibitor, and BPI expression was analyzed following nigericin treatment . Confocal analysis showed that pretreatment of cells with p38 MAPK inhibitor significantly affected nigericin induced BPI expression. To validate these results, BPI expression in Caco-2 cells post-nigericin treatment was quantified by western blotting . Nigericin treatment led to a 2.5-fold increase in BPI expression compared to untreated control. Pretreatment of cells with p38 MAPK inhibitor decreased nigericin induced BPI levels . Analysis of BPI expression in HeLa cells by western blotting revealed that Nigericin induces BPI expression in a p38 dependent manner . Treatment of Caco-2 cells with 1% cellulose couldn’t completely inhibit BPI expression mediated by SA infection. This indicates that other stress signals along with osmotic stress contribute to BPI expression in the intestinal epithelium.Three major and immediate changes associated with pore formation which induces a survival signal in epithelial cells are (a) decrease in cellular potassium levels, (b) increase in cellular calcium levels, and (c) osmotic pressure . We hypoFigures 3D). However, treatment of Caco-2 cells with LPS under similar conditions did not induce BPI expression . Interestingly, most of the PAMPs tested can induce p38 phosphorylation in Caco-2 cells, proposing involvement of p38 independent signaling pathway in inducing BPI expression . This lead to a 1.6-fold increase in BPI expression compared to untreated control even though maximum induction of BPI expression was mediated by nigericin . Transfected cells were examined by confocal microscopy for expression of membrane FLAG-tagged human TLR4 . LPS treatment, following TLR4 expression, led to increase in IL-8 mRNA levels as quantified by real time PCR . These results prove that BPI expression in epithelial cells might be exclusively mediated through DAMP signals.Previous studies have reported that epithelial cells have decreased expression of TLR4 . To undeFigure 3G). To check the contribution of BPI in limiting bacterial survival under these conditions, bacterial survival was quantified upon BPI knock down in Caco-2 cells. Efficacy of BPI knockdown in dsRNA transfected cells was validated by western blot analysis . Knocking down of BPI in Caco-2 cells inhibited nigericin mediated bacterial killing confirming the role of BPI in bacterial killing. This result confirms that DAMP mediated BPI induction contribute to bacterial clearance.To understand whether DAMP associated BPI induction can contribute to bacterial clearance, nigericin treated Caco-2 cells were infected with STM at an MOI of 10. Bacterial numbers in Caco-2 cells were quantified by plating the cell lysates 2 h post-infection. Interestingly, treatment with nigericin led to a 65% decrease in bacterial survival compared to untreated control  . Interestingly, maximum change in BPI expression was observed in the ileum (20-fold) which is known to be a preferential entry point for STM . Conversely, there was no increase in BPI expression during SA infection  and SA infection did not cause any damage to mice intestine   .Intestinal damage during bacterial infection is an outcome of active inflammatory response against the invading pathogen . To compinvC . As expefliC. Infection with STMΔfliC showed less intestinal damage compared to STM wild type infected mice . This result suggests that inflammation associated intestinal damage act as a queue to induce BPI expression in the intestine. Next, we checked BPI expression in TLR4 knockout mice after infection with STM (WT) bacteria. STM infection in TLR4 knockout mice lead to severe damage in the intestine as seen by a significant decrease in colon length . Interestingly, infection with STM WT in these mice showed two to threefold increase in BPI expression compared to uninfected control (placebo control) . This increase in BPI level is a direct effect of epithelial damage. We couldn’t detect any change in BPI expression upon infection with STMΔinvC or STMΔfliC in these mice compared to untreated control . These results supports the notion that inflammation and associated damage induce BPI expression in murine intestinal epithelium.Two major PAMPs which can mediate an active inflammatory response during the course of Salmonella infection are LPS and flagellin . To undeFigure 1A). SA is known to produce pore-forming toxins which can target the infected cell membrane. Treatment of Caco-2 cells with pore forming toxins also lead to BPI induction in these cells. Production of BPI during pore formation might have a huge impact in regulating the microbiota-derived LPS mediated inflammatory response during epithelial damage. BPI expression associated with cell damage might restrict entry of normal microbiota during epithelial damage. Pore formation in intestinal epithelial cells leads to the alteration in cytosolic ion levels and osmotic pressure of these cells . Additionally, changes in Ca2+ levels and osmotic stress also contributed toward BPI expression to a certain extent. TLR4 overexpression and LPS treatment couldn’t induce BPI expression in Caco-2 cells  indicating that rather than PAMPs, DAMPs signal the presence of the pathogen in the gut and induce BPI expression in intestinal epithelial cells. Interestingly, inhibition of p38 MAPK  inhibited SA and Nigericin mediated BPI expression in Caco-2 cells. But, p38 is not the sole signaling pathway that leads to BPI expression in Caco-2 cells as most of the PAMPs tested can mediate p38 phosphorylation in the epithelial cell. Even though Salmonella did not induce epithelial damage in cell culture model, Salmonella infection in the mouse led to epithelial damage . These results suggest that, under resting conditions, BPI expression is tightly regulated in the distal regions of the intestine. But, during the course of infection, maximum BPI expression is seen in the distal regions of the intestine, where there is a higher chance of infiltration by gut microbiota. BPI expression was analyzed by extracting total RNA from the intestine. Since BPI is exclusively expressed in cells derived from myeloid lineage and BPI mRNA is absent in both murine neutrophils and macrophages, we believe that most changes associated with BPI mRNA are contributed by intestinal epithelial cells (invC (invasion deficient mutant) infection in mice couldn’t induce BPI expression in the ileum. STMΔinvC mutants have a defective secretion of SPI1 effectors which are important in mediating bacterial entry inside epithelial cells . Further, STM infection in TLR4-/- mice displayed a significant increase in BPI expression compared to uninfected mice, but with respect to WT mice, STM mediated BPI expression is less in TLR4-/- mice. Even though TLR4 is a major sensor that detects LPS in Gram-negative bacteria, TLR4-/- mice shows increased intestinal damage compared to WT mice during STM infection . There was no significant inflammation associated with SA infection. Under these conditions, there was no increase in BPI levels as well. Previous reports show an increase in BPI expression in Canines upon infection with E. coli , Crohn’s Disease (CD), etc. In the present study, we tried to understand a major immune modulator in the intestine, BPI. BPI acts as both anti-bacterial and anti-inflammatory agent in the intestine. Previous studies have tried to screen various inflammatory agents to study the regulation of BPI in the intestine, but none of the inflammatory signals including PAMPs and cytokines could induce BPI expression in the intestine . We screse cells . We triel damage . This dal damage . These ll damage . Interesal cells . STMΔinval cells . Since tnfection . Mice innfection . TLR4 menfection . Increasntestine . STMΔinvogenesis . In vitrf injury . We cannf injury . Even th E. coli . InteresThe animal experiments were carried out in accordance with the approved guidelines of institutional animal ethics committee at Indian Institute of Science, Bangalore, India (Registration No: 48/1999/CPCSEA). All procedures with animals were carried out in accordance with the institutional rules for animal experimentation under strict vigilance.Salmonella Typhimurium , STM ΔinvC, STM ΔfliC (Flagellin-deficient STM 14028), STY (STY ATCC CT18), SHG , SA (S. aureus ATCC 25923), and E. coli (E. coli DH5α ATCC) were grown in Luria-Bertani medium at 37°c. STM ΔinvC and STM ΔfliC were a kind gift from Michael Hensel .2. For polarizing Caco-2 cells, cells were seeded in permeable tissue culture (0.45 μTranswellR Corning) inserts for 7 days, until they form tight monolayer. For screening of PAMPs that can induce BPI expression in Caco-2 cells, 106 cells were seeded in 6 well plate. Twenty-four hours post-seeding, cells were treated with STM LPS 100 ng/mL (Sigma–Aldrich) and STM Flagellin 500 ng/mL, HK STM (Heat killed STM) or were infected with STM, STY, or SA at an MOI of 10 as explained below. Cells were treated with 100 nM ATLA4 , and was used as a positive control. To check the importance of pore-forming toxins and ion efflux in inducing BPI expression, cells were treated with Streptolysin O  for 24 h, Listeriolysin O 1 μg  for 24 h, Nigericin 6 μM (Sigma–Aldrich) and Thapsigargin 500 nm (Sigma–Aldrich) for 30 min followed by media change. p38 MAPK inhibitor, SB203580 20 nM  was added 1 h before addition of Nigericin, SLO or SA infection. For all experiments using Nigericin and ATLA4, DMSO (Sigma–Aldrich) was used as a solvent control. To check the role of osmotic stress on BPI expression, cells were treated with 1% cellulose (Sigma–Aldrich) along with SA infection. Cells were processed for either confocal microscopy or western blot as explained below. Flagellin was isolated from STM as previously described  and human cervix adenocarcinoma cell line  were maintained in DMEM (Sigma–Aldrich) containing 10% FBS  at 37°C in 5% COescribed .8 bacteria per ml . For infection with S. flexneri, mice were infected intraperitoneally with 108 bacteria per ml of SHG as described by Salmonella Shigella agar. CFU was calculated and normalized to per gram weight of intestine. For evaluating colitis in infected mice, colon length was calculated as the length between the rectum and distal colon. Mice were maintained in SPF conditions throughout the experiment.To check BPI expression in infected mice, BALB/c and C3H/Hej (TLR4 deficient) female mice aged 6 weeks were infected intragastrically with 10Cells were infected with SA, STM or STY at a MOI of 10. Extracellular bacteria were removed 30 min post-infection and cells were maintained in 100 μg/ml Gentamicin for 1 h to kill any extracellular bacteria. Infected cells were maintained in DMEM containing 10 μg/ml of Gentamicin.Salmonella Shigella agar. Percentage survival was calculated with respect to untreated control. Percentage survival was calculated as follows.For checking the effect of BPI knockdown on STM survival, Caco-2 cells were transfected with BPI dsRNA or scrambled dsRNA control. Twenty-four hours post-treatment, cells were treated with SLO (2U) or Nigericin (6 μM) for 24 h. Cells were infected with STM as described above. For quantifying bacterial survival, Caco-2 cells were lysed using 0.1% TritonX-100 (Sigma–Aldrich) 2 h post-infection. The Bacterial number in cell lysates were quantified by plating the cell lysates in In order to knockdown BPI in Caco-2 cells, BPI dsRNA was designed against three regions within the gene a) GGAGCTGAAGAGGATCAAGATTCCTGACTACTCAGACAGCTTTAAGATCAAGCATCTTGGGAAGGGGCATTATAGCTTCTACAGCATGGACATCCGTGAATTCCAGCTTCCCAGTTCCCAGATAAGCATGGTGCCCAATGTGGGCCTTAAGTTCTCCATCAGCAACGCCAATATCAAGATC b) GTGTCCACGTGC ACATCTCAAAGAGCAAAGTCGGGTGGCTGATCCAACTCTTCCACAAAAAAATTGAGTCTGCGCTTCGAAACAAGATGAACAGCCAGGTCTGCGAGAAAGTGACCAATTCTGTATCCTCCAAGCTGCAACCTTATTTCCAGACTCTGC c) GGGTCTTGAAGATGACCCTTAGAGATGACATGATTCCAAAGGAGTCCAAATTTCGACTGACA ACCAAGTTCTTTGGAACCTTCCTACCTGAGGTGGCCAAGAAGTTTCCCAACATGAAGATACAGATCCATGTCTCAGCCTCCACC. All dsRNA were obtained from chromous biotech. Transfection was done using Oligofectamine as recommended by the manufacturer . Transfection was done for 24 h in DMEM without FBS. Post-transfection, cells were maintained in DMEM with 10% FBS.® ViiA7TM Real-time PCR instrument. The following primers were used for detecting BPI level by real-time PCR. hBPI forward primer, 5′ATGAACAGCCAGGTCT3′, hBPI reverse primer, 5′GGTCATTACTGGCAG3′. Human actin forward primer, 5′GGTGGCTTTTAGGATGGCAAG3′, Human actin reverse primer 5′ACTGGAACGGTGAAGGTGACAG3′. Mouse BPI forward primer 5′GGTAAGAAGGAAAACAAATGCC3′, Mouse BPI reverse primer 5′AACCACCTGCTGCCAA3′. Mouse actin forward primer 5′CAGCAAGCAGCAGTACGATG3′, Mouse actin reverse primer 5′GCAGCTCAGTAACAGTCCG3′. The following primers were used to detect IL-8 expression by real time PCR. Human IL-8 forward primer 5′ATGACTTCCAAGCTGGCCGTG3′, Human IL-8 reverse primer 5′TTATGAATTCTCAGCCCTCTTCAAAAACTTCTC3′. Expression levels were quantified with respect to actin  using the 2-ΔΔct method.RNA was isolated from Caco-2 cells using TRIzol reagent (Invitrogen) as per manufacturer’s protocol. For RNA isolation from the intestine, the intestine was homogenized in bead beater in 1 ml TRIzol reagent. After DNase treatment 2 μg of RNA was used for cDNA synthesis using Tetra reverse transcriptase (Bioline). QRT PCR was performed using the Kapa SYBR Green RT-PCR kit (Kapa Biosystems) as per manufactures protocol in an Applied Biosystems6 Caco-2 cells were treated with indicated reagents or were infected with bacteria. Cells were lysed at indicated time points using RIPA buffer  containing 10% Protease inhibitor cocktail (Sigma–Aldrich). Protein obtained from cell lysates were quantified using Bradford assay and 80 μg of total protein was loaded on 10% SDS–PAGE. Proteins were resolved and transferred to PVDF membrane (Millipore). Blocking was done using 4% Bovine serum albumin  in TBST (20 mM Tris–HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20) for 1 h to prevent non-specific binding. The blots were incubated in anti-BPI antibody (Sigma–Aldrich) overnight at 4°C followed by anti-rabbit HRP  for 2 h. Antibodies were reconstituted in TBST solution containing 5% BSA. For quantifying β-Actin, blots were incubated with anti-β-Actin antibody conjugated with HRP (Sigma–Aldrich). Immunoblots were visualized by enhanced chemiluminescence reagent (Biovision) as per manufacturer’s instructions. For probing proteins in the same region of PVDF membrane, blots were incubated in stripping buffer  at 60°C on a shaker. Blots were scanned, BPI bands were quantified with respect to corresponding β-actin bands using the Multi Gauge software (FUJIFILM). Representative images were shown in illustrations.10S. aureus infection, coverslips were blocked with anti-human IgG (Sigma–Aldrich) for 2 h prior to addition of primary antibody to inhibit non-specific interaction of protein-A and anti-BPI antibody. Preliminary results confirm that the signal is specific to BPI and not a cross reacting signal from Protein-A present in S. aureus. Image acquisition was done with a Zeiss confocal microscope (LSM Meta 710). Mean fluorescent intensity of each image were quantified using Zen Blue Software.To visualize BPI expression in Caco-2 cells, cells were seeded on glass coverslips or permeable tissue culture inserts before treatment or infection. Post-treatment cells were washed with PBS and fixed with 3.5% paraformaldehyde for 15 min. Cells were permeabilized using 1% Saponin dissolved in PBS with 3% BSA. Immunostaining was done using anti-BPI antibody (Sigma–Aldrich) followed by anti-rabbit Alexa 647 antibody . To visualize TLR4 expression in Caco-2 cells, cells were stained with the anti-FLAG antibody (Sigma–Aldrich) followed by anti-rabbit Alexa 488 antibody . For experiments using Caco-2 cells were transfected with pCMV: hTLR4-FLAG  using PEI transfection reagent (Sigma–Aldrich). Twenty-four hours post-transfection, cells were washed with sterile PBS and were maintained in DMEM containing 10% FBS. Transfected cells were examined by confocal microscopy for expression of membrane FLAG-tagged human TLR4.BALB/c mice were dissected 2 days post-infection in order to evaluate the pathological effects. Intestines were fixed overnight in 4% paraformaldehyde and then dehydrated and embedded in paraffin. Paraffin-embedded intestine (ileum) was sectioned using a microtome. Intestinal sections (5 μm) were processed for hematoxylin and eosin staining to identify nuclear and protein . In a double blinded scoring, randomly selected intestinal sections were scored independently by two experts (Dr. Ritu Gambher Chadha and Dr. Joan Maria Vynetta). Histological scores were given based on the criteria explained by t-test by using Graph Pad prism 4 software.The data were subjected to statistical analysis by applying Student’s AB and DC conceived the study. AB performed the Experiments. AB and DC analyzed the data and wrote the manuscript.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.


Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. Myo1 proteins are ATP-driven actin-bound motor proteins that are commonly monomeric (single headed) in nature, unlike dimeric Myosin II Myo2) molecules  molecule. Myo1 prZebrafish embryos have thick cortical actin band, in the early stages of development (1–4 cell) –11. They50 values for various Myo1 isoforms of different organisms range from 1 to5.6 μM of PClP, whereas for Myo2, Myo5, Myo6 and -Myo7 it is above 90μM  are donS1 Text(PDF)Click here for additional data file.S1 Fig(A) Example of a two cell stage embryo, where drug was added. (B-C) Difference in cell division arrest phenotype, top-view position for (B) control by (C) 2.5 μM PClP. (D-F) Difference in cell division arrest phenotype, lateral view, extended time for (D) control by (E) 100μM blebbistatin and (F) 2.5 μM PClP. Bar 100 uM.(PDF)Click here for additional data file.S2 FigBar 50 μm.(PDF)Click here for additional data file.S3 FigActin appears mainly as dense sheet like structure in control and Myo2 inhibited treated embryos, but appeared tubular in Myo1 inhibited embryos (arrows). (bar 5 μm).(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 FigLDs at the first cleavage furrow of (A-B) Control 8 cell, (C) 2 hpf Myo1 inhibited 8 cell-sideview, (D) 2 hpf Myo1 inhibited 8 cell-topview.(PDF)Click here for additional data file.S6 Fig(A) Cartoonic representation of sidewise orientated embryo with enclosed region by dashed line represent the region in which LDs were tracked for plots in S6B–S6E Fig.  LDs’ distance from first furrow,  LDs’ distance from the yolk blastomere interface, in (PDF)Click here for additional data file.S7 Figrd furrow formation (B) Average instantaneous speed of LDs in Myo1 inhibited embryo for 30 min in the same region, encompassing 3rd furrow formation. Average of 180 LDs taken from data sets of (A) Average instantaneous speed of LDs in control for 30 min in the dotted region as in (PDF)Click here for additional data file.S1 MovieThird furrow is marked as “3”. Images were captured every 24 sec and played at 30 fps. Movie data analysed in (MOV)Click here for additional data file.S2 MovieImages were captured every 10 sec and played at 30 fps. Movie data analysed in (MOV)Click here for additional data file.S3 MovieMovie data analysed in (MOV)Click here for additional data file.S4 MovieImages were captured every 2.7 sec and played at 10 fps. Movie data analysed in (MOV)Click here for additional data file.S5 MovieImages were captured every 8 sec and played at 24 fps. Insets, full view of the side faced blastomere. Bar-50μm. Movie data were analyzed in (MOV)Click here for additional data file.S6 MovieImages were captured every 8 sec and played at 24 fps. Insets, full view of the side faced blastomere. Bar-50μm. Movie data were analyzed in (MOV)Click here for additional data file.


We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice.Influenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the IMPORTANCE The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane. Orthomyxoviridae family and has a genome composed of eight single-stranded, negative-sense RNA segments. The IAV genome encodes 10 essential proteins as well as some more recently identified accessory proteins (1Influenza A virus (IAV) is an important zoonotic pathogen that causes significant morbidity and mortality in both humans and animals. It is a member of the roteins 17. Matrixroteins 1, 9. The proteins 1. The CTroteins 1, 13. M2 roteins 1.trans-Golgi network (TGN) to prevent the premature low-pH conformational change of viral hemagglutinin (HA) during its transport to the cell surface. This function is important for highly pathogenic H7 influenza viruses, whose HA can be cleaved by ubiquitous proteases and is therefore more susceptible to low-pH-induced premature maturation  complexes from M1 and the lipid bilayers, thus completing the uncoating process during virus entry , 16. Defturation , 19 and turation . In addituration .The M2 protein is also required for virus assembly and budding. M2 is positioned at the edge of the budozone when virus budding begins and induces negative membrane curvature in a cholesterol-dependent manner , 23, thu24–Another function attributed to the M2 protein is an effect on autophagy. M2 expression during virus infection inhibits autophagosome degradation by blocking the fusion of autophagosomes and lysosomes, leading to enhanced death of virus-infected cells . It was Saccharomyces cerevisiae, three forms of the TRAPP complex, TRAPP I, TRAPP II, and TRAPP III, have been identified (–The transport protein particle (TRAPP) complex was the first multisubunit tethering complex to be identified and structurally characterized . In Saccentified –34. TRAPntified –, TRAPP Intified –, and TRAntified –. TRAPP cntified –, 37. Trantified –, is idenntified –. TRAPPC6ntified –. Howeverin vitro, and positively modulates virus virulence in mice.M2 relies on the host cell machinery to fulfill its functions during the virus life cycle. However, M2 could also be a target of the host defense system. So far, only a few host cellular proteins have been reported to physically interact with M2 –43. Ther–8 yeast colonies were screened from the library, which contained >2 × 107 independent clones, representing a >40-fold screening of the library. Putative positive colonies were selected for growth by using synthetically defined medium lacking leucine and tryptophan (SD/−Leu/−Trp), also known as double dropout (DDO) medium, and plasmids were isolated from positive colonies and sequenced. The in-frame proteins were retransformed into strains carrying the M2 and M2 CT fusion proteins to confirm the interaction with the M2 protein. One specific protein binding partner for M2, TRAPPC6A, was selected for further study due to its abundance in the screen and its specific interaction with both M2 and the M2 CT of the two different virus subtypes (NM_024108.2). Its role in modulating virus infection has never been reported.The Y2H system was used to identify host cellular proteins that interact with the IAV M2 protein. As bait, we used full-length M2 and the M2 CT domain of two different virus strains: A/Sichuan/1/2009 (SC09) (H1N1) and A/Anhui/2/05 (AH05) (H5N1). A cDNA library prepared from a mixed human cell culture  was used as prey. More than 8 × 10subtypes . Full-lein vivo. The specificity of the M2-TRAPPC6A interaction was further demonstrated by the detection of Flag-SC09 M2 in a reverse co-IP experiment using an anti-Myc MAb and by the absence of Flag-SC09 M2 on the blot from cells transfected with Flag-SC09 M2 alone  pulldown assay by using purified GST-TRAPPC6A or GST alone incubated with HEK293T lysates containing exogenously expressed SC09 M2 protein. The results showed that SC09 M2 was specifically pulled down with GST-TRAPPC6A but not GST alone , followed by Western blotting with a rabbit anti-Flag antibody for the detection of M2 and a rabbit anti-Myc antibody to reveal the presence of TRAPPC6A. TRAPPC6A-Myc was readily coimmunoprecipitated with Flag-SC09 M2 , demonstM2 alone . The phyM2 alone  and C, sST alone , indicatThe localization of M2 and TRAPPC6A was examined by using immunofluorescence and confocal microscopy in A549 cells transfected with Flag-SC09 M2 and TRAPPC6A-Myc individually or in combination . M2 was To determine whether the interaction between TRAPPC6A and M2 is specific to influenza A virus or can be extended to influenza B virus, we performed an additional co-IP experiment with TRAPPC6A and BM2 of the B/Jilin/20/2003 virus. We found that TRAPPC6A was not coimmunoprecipitated with BM2 fused to green fluorescent protein (GFP) , thus deTRAPPC6AΔ is identical to full-length TRAPPC6A except for an N-terminal internal deletion of 14 amino acids. We therefore examined whether the specificity of the interaction between full-length TRAPPC6A and M2 extended to TRAPPC6AΔ. We performed a co-IP assay with HEK293T cells transfected with Flag-tagged SC09 M2 and Myc-tagged TRAPPC6A or TRAPPC6AΔ. The cell lysates were incubated with an anti-Myc MAb, followed by Western blotting to detect the coimmunoprecipitated proteins. Flag-tagged M2 was coimmunoprecipitated with both Myc-tagged TRAPPC6A and TRAPPC6AΔ . These rTo define the specific binding domain and residue for TRAPPC6A/TRAPPC6AΔ in M2, we first validated the finding of the Y2H experiment that the M2 CT mediates the interaction with TRAPPC6A in human cells. SC09 M2 was divided into two fragments according to its function, the CT fragment and the EDTM fragment, which includes the ectodomain and the transmembrane domain. Full-length M2 and the two fragments were fused to GFP in the C terminus, resulting in the construction of pEGFP-C1-SC09 M2, pEGFP-C1-SC09 M2EDTM, and pEGFP-C1-SC09 M2CT. We then coexpressed these fusion constructs with Myc-tagged TRAPPC6A in HEK293T cells and performed a co-IP experiment with a mouse anti-GFP MAb, followed by Western blotting with a rabbit anti-GFP polyclonal antibody for the detection of GFP-fused SC09 M2 and a rabbit anti-Myc antibody for the detection of TRAPPC6A. We found that both full-length SC09 M2 and the SC09 M2 CT formed a complex with TRAPPC6A but that the SC09 M2 EDTM fragment failed to associate with TRAPPC6A , indicatWe demonstrated that the residue at position 96 of M2 is critical for its interaction with TRAPPC6A/TRAPPC6AΔ. To further assess the importance of this residue for the interaction with TRAPPC6A/TRAPPC6AΔ, we genetically analyzed all of the influenza virus M2 sequences that were deposited in GenBank by 6 July 2014. Eleven different amino acids were found at position 96 among the 29,465 M2 protein sequences analyzed . StrikinWe demonstrated that both TRAPPC6A and TRAPPC6AΔ interact with M2 of IAV. To assess the role of this interaction in influenza virus replication in A549 cells, we first examined the endogenous expression of TRAPPC6A and TRAPPC6AΔ by Western blotting. We found that the rabbit anti-TRAPPC6A polyclonal antibody that we generated recognizes both TRAPPC6A and TRAPPC6AΔ. Full-length TRAPPC6A was not detected in the lysates of A549 cells; however, TRAPPC6AΔ was abundantly expressed . We therWe then assessed the effect of the overexpression of TRAPPC6AΔ on influenza virus replication. We established an A549 cell line overexpressing TRAPPC6AΔ and an A549 control cell line transduced with an empty retrovirus  and infeCollectively, these results demonstrate that endogenous TRAPPC6AΔ is essential for efficient influenza virus replication, although its overexpression provides no added benefit for virus growth.5 PFU/ml for WSN M2Del1 and 2.3 × 105 PFU/ml for the wt virus. In contrast, the virus titer for WSN M2Del2 was 6.0 × 104 PFU/ml, which was considerably lower than those for both WSN M2Del1 and the wt virus. Next, we analyzed the replication kinetics of these three viruses. A549 cells were infected with wt WSN or one of the two mutants at an MOI of 0.01, the supernatants were collected at various times, and their virus titers were assessed  (the plasmids for the M vRNA segment encode wt M2 or M2 mutants lacking 1 or 2 amino acids from the C terminus), together with the four supporting protein expression plasmids. The transfection supernatants were harvested at 48 h posttransfection and titrated by a plaque assay in MDCK cells. As shown in assessed . The WSNassessed . These din vivo, female BALB/c mice were intranasally infected with the WSN, WSN M2Del1, or WSN M2Del2 virus. The mice were observed for 14 days after inoculation, and their body weight and survival status were checked daily. When mice were infected with a low dose  of these viruses, all of the mice survived  of the viruses, all of the mice died. However, half of the mice survived when they were infected with 104 PFU of WSN M2Del2. In contrast, all of the mice died when they were infected with 104 PFU of either wt WSN or WSN M2Del1. These results indicate that the loss of the M2-TRAPPC6A/TRAPPC6AΔ interaction leads to a modest attenuation of influenza virus virulence in vivo.To determine whether the M2-TRAPPC6A/TRAPPC6AΔ interaction has any effect on the virulence of influenza virus survived . When miUpon expression, the influenza virus M2 protein is transported to the cellular plasma membrane through the protein secretion pathway to participate in virus budding and release. TRAPPC6AΔ, occasionally termed TRAPPC6A in some studies , 44, is We next determined whether TRAPPC6AΔ affects the transport of the viral HA protein, which also traffics to the plasma membrane through the Golgi apparatus. siRNA-treated A549 cells were infected with the WSN virus, and the cell surface expression level of the HA protein was determined by flow cytometry using a rabbit anti-HA polyclonal antibody at 8 and 10 h p.i. We found that the cell surface expression level of HA was very slightly increased in TRAPPC6AΔ-knocked-down cells compared with nontargeting siRNA-treated cells at both time points . PreviouThe acidic pH of the TGN is equilibrated with the pH of the cytoplasm via the activation of the M2 proton-selective ion channel, which in turn affects the kinetics of the delivery of apical membrane proteins to the cell surface and can be blocked by the addition of the ion channel blocker amantadine . To ensuWe demonstrated that endogenous TRAPPC6AΔ slows the trafficking of M2 to the cell surface, which is beneficial for virus replication. We next attempted to further determine the dynamic changes in TRAPPC6AΔ during the transport of M2 to the plasma membrane. We performed a time course confocal experiment to examine the M2-TRAPPC6AΔ interaction during virus replication. A549 cells were infected with wt WSN, WSN M2Del1, or WSN M2Del2 at an MOI of 5. At 4 h, 6 h, 8 h, 10 h, and 14 h p.i., the localization of M2 and TRAPPC6AΔ was determined. In noninfected control cells, endogenous TRAPPC6AΔ was evenly distributed in the cytoplasm (data not shown). In WSN-infected cells, M2 clearly accumulated in the area of the Golgi apparatus that stained positive for Giantin and was also dispersed in the cytoplasm at 4 h p.i.  and 9A. The influenza virus life cycle is finely regulated in order to maintain optimal virus fitness, infectivity, and transmissibility. The viral M2 protein, which is synthesized during the late stage of the virus life cycle , is tranWe demonstrated that TRAPPC6AΔ is involved in the posttranslational transport of influenza virus M2 to the plasma membrane. Flow cytometry analyses showed that siRNA knockdown of TRAPPC6AΔ increased the surface expression of M2, whereas stable overexpression of TRAPPC6AΔ reduced its surface expression. We also visualized dynamic changes in TRAPPC6AΔ during the trafficking of M2 to the plasma membrane. In WSN- and WSN M2Del1-infected cells, TRAPPC6AΔ accumulated in punctate dots, possibly transport vesicles, where it is colocalized with M2. Once the transport of M2 to the plasma membrane is complete, TRAPPC6AΔ returns to its diffuse state. Strikingly, TRAPPC6AΔ maintained its diffuse state throughout the whole infection process, and no colocalization between M2 and TRAPPC6AΔ was observed in WSN M2Del2-infected cells. Based on these data, we conclude that the physical interaction between TRAPPC6AΔ and M2 slows M2 to go through the secretory pathway and arrive at the plasma membrane. However, whether TRAPPC6AΔ alone is sufficient for this activity or whether it has to rely on the function of TRAPP as a complex to modulate M2 transport remains to be investigated.Our data demonstrated that TRAPPC6AΔ is required for the efficient growth of influenza virus. Upon the knockdown of TRAPPC6AΔ with siRNA in A549 cells, the replication of WSN virus was significantly reduced. However, the stable overexpression of TRAPPC6AΔ in A549 cells did not result in increased virus replication. These results suggest that the endogenous expression of TRAPPC6AΔ might be enough to fulfill its function in regulating M2 transport, and therefore, the overexpression of TRAPPC6AΔ provided no additional benefit. Another possibility is that the overexpression of TRAPPC6AΔ may overwhelm or alter the transport machinery of the host cells. In this case, there would also be no further promotion of virus growth when TRAPPC6AΔ was overexpressed. In fact, it has been reported that the overexpression of subunits of the TRAPP complex can disrupt the architecture of microtubules in such a way that the astral appearance becomes disorganized, frequently presenting as a circular array  (H1N1) strain of influenza virus was grown in MDCK cells cultured in MEM supplemented with 0.3% bovine serum albumin  and 0.5 μg/ml l-1-tosylamide-2-phenylmethyl chloromethyl ketone (TPCK)-treated trypsin .Human embryonic kidney cells (HEK293T), human glial cells (U251), human lung carcinoma cells (A549), human monocytic cells (THP-1), and Madin-Darby canine kidney (MDCK) cells were cultured in the following media: Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum  (HEK293T and U251), F-12K medium (Life Technologies) supplemented with 10% FBS (A549), RPMI 1640 medium (Life Technologies) supplemented with 10% FBS (THP-1), and minimum essential medium  containing 5% newborn calf serum  (MDCK). All media contained 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies). All cells were maintained in a humidified incubator containing 5% COd-galactopyranoside  and aureobasidin A (AbA)  (QDO/X/A). Blue colonies were selected and cultured in medium that lacked leucine and tryptophan (SD/−Leu/−Trp) (DDO) for plasmid extraction. The plasmids were sequenced to identify the cellular interacting proteins. To confirm the interaction between M2 and the host proteins, the bait and prey plasmids were cotransformed into the Y2HGold yeast strain. Cotransformation of pGBKT7-53 encoding the Gal4 DNA-BD fused with murine p53 (BD-p53) and pGADT7-T encoding the Gal4 activation domain fused with the simian virus 40 large T antigen (AD-T) served as a positive control. Cotransformation of pGBKT7-Lam, which encodes the Gal4-BD fused with human lamin C (BD-Lam), and AD-T served as a negative control.A matchmaker Y2H system  was used to screen for host proteins that interact with M2. The bait protein, containing the full-length M2 protein or the M2 CT domain (amino acids 44 to 97) of A/Anhui/2/05 (AH05) (H5N1) or 2009 pandemic H1N1 virus A/Sichuan/1/2009 (SC09), respectively, was expressed with an N-terminal fusion to the GAL4-binding domain (BD) in a pGBKT7 vector. Yeast strain Y2HGold was transformed with the pGBKT7-M2 or pGBKT7-M2CT bait plasmid by using the lithium acetate method and was then mated with the Y187 yeast strain transformed with a pGADT7-based cDNA library prepared from a mixed human cell culture . Transformants were selected on medium that lacked adenine, histidine, leucine, and tryptophan (SD/−Ade/−His/−Leu/−Trp) (quadruple dropout medium [QDO]). Colonies were transferred to QDO plates containing 5-bromo-4-chloro-3-indolyl-α-CY033829.1) was synthesized  and inserted into the pEGFP-C1 vector to generate the construct pEGFP-C1-BM2. pRetroX-IRES-ZsGreen1-TRAPPC6AΔ was constructed by inserting the TRAPPC6AΔ ORF into the pRetroX-IRES-ZsGreen1 vector (Clontech). pHH21-M2Del1 and pHH21-M2Del2 were constructed by PCR amplification and subsequent cloning into the pHH21 vector as described previously . These genes were subsequently cloned into the mammalian expression vector pCAGGS, resulting in constructs designated pCAGGS-TRAPPC6A and pCAGGS-TRAPPC6AΔ. Plasmids pCAGGS-TRAPPC6A-myc and pCAGGS-TRAPPC6AΔ-myc were generated by inserting the TRAPPC6A or TRAPPC6AΔ open reading frame (ORF) fused with the Myc tag sequence in the C terminus into the pCAGGS vector. The TRAPPC6A ORF was fused to the C terminus of GST in vector pGEX-6P-1 , resulting in the construct pGEX-6P-1-TRAPPC6A. Plasmid pCAGGS-SC09M2 was constructed by inserting SC09M2 into pCAGGS. Plasmid pCAGGS-Flag-M2 was generated by inserting the M2 ORF fused with the Flag tag sequence in the N terminus into pCAGGS. The constructs pCAGGS-Flag-M2Del1, pCAGGS-Flag-M2Del2, pCAGGS-Flag-M2Del3, pCAGGS-Flag-M2Del4, pCAGGS-Flag-M2Del5, pCAGGS-Flag-M2Del6, pCAGGS-Flag-M2Del12, and pCAGGS-Flag-M2Del18 were created in the same way and expressed M2 with deletions of 1, 2, 3, 4, 5, 6, 12, and 18 residues from the C terminus, respectively. The pCAGGS-Flag-M2-96Mut plasmids, containing different amino acid mutations at position 96 of M2, were PCR amplified and cloned into pCAGGS. Plasmids pEGFP-C1-M2, pEGFP-C1-M2EDTM, and pEGFP-C1-M2CT were generated by inserting full-length M2 or truncated fragments into the pEGFP-C1 vector (Clontech). The BM2 sequence of B/Jilin/20/2003 virus , mouse anti-Flag monoclonal antibody , rabbit anti-Myc polyclonal antibody , mouse anti-Myc monoclonal antibody , mouse anti-actin monoclonal antibody , rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody , rabbit anti-GFP polyclonal antibody , mouse anti-GFP monoclonal antibody , mouse anti-M2 monoclonal antibody , rabbit anti-M2 polyclonal antibody , rabbit anti-HA polyclonal antibody , rabbit anti-FGF2 monoclonal antibody , mouse anti-LAMP1 monoclonal antibody , and mouse anti-Giantin monoclonal antibody . The rabbit anti-TRAPPC6A polyclonal antibody was made and stored in our laboratory. The commercially obtained secondary antibodies used in this study were DyLight 800 goat anti-mouse IgG(H+L) , DyLight 800 goat anti-rabbit IgG(H+L) , Alexa Fluor 488 donkey anti-mouse IgG(H+L) , Alexa Fluor 488 goat anti-rabbit IgG(H+L) , and Alexa Fluor 546 donkey anti-rabbit IgG(H+L) .HEK293T cells cultured in 6-well plates were transfected with the respective plasmids (3 μg/each) by using Lipofectamine LTX and Plus reagents (Invitrogen) according to the manufacturer's instructions. At 48 h posttransfection, cells were washed three times with cold phosphate-buffered saline (PBS) (pH 7.4) and lysed with IP buffer  containing a complete protease inhibitor cocktail (1 tablet/50 ml)  for 30 min on ice. The supernatants were then mixed and rocked at 4°C overnight with the indicated primary antibodies. Protein G-agarose beads (Roche) were added, and the mixture was rocked for 6 to 8 h at 4°C. The IgG-agarose beads were washed four times with 1 ml of wash buffer , and immunoprecipitated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by Western blotting.in vitro binding assay, GST-fused TRAPPC6A or GST alone was expressed in BL21(DE3) cells. The soluble fraction of the E. coli cell lysate was purified with glutathione-Sepharose 4B  and further fractionated by cation exchange chromatography. HEK293T cells transfected with 10 μg of the pCAGGS-SC09M2 or pCAGGS plasmid were washed twice with cold PBS and lysed with IP buffer. The lysate was split into two aliquots and separately incubated with 25 μg of purified GST-TRAPPC6A or GST for 2 h at 4°C. After three washes with IP buffer, the bound proteins were extracted from the Sepharose beads by boiling in 2× SDS loading buffer, separated by SDS-PAGE, and detected by Western blotting or Coomassie blue staining.For an 2HPO4, 2 mM KH2PO4, and 0.05% Tween 20 (PBST) and incubated overnight at 4°C with the appropriate primary antibodies diluted in PBST containing 2% BSA. After washing with PBST, the blots were incubated with DyLight 800 goat anti-mouse IgG(H+L)  or DyLight 800 goat anti-rabbit IgG(H+L)  and scanned by use of an Odyssey infrared imaging system .Proteins were resolved by SDS-PAGE and electrotransferred onto nitrocellulose membranes . The blots were blocked for 1 h at room temperature with 5% skim milk in a solution containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na5 A549 cells were seeded into 12-well plates (Sigma-Aldrich) and then transfected with siRNA targeting TRAPPC6AΔ (5′-CUGUGUUGUUUGAGUUUCU-3′) or nontargeting siRNA  at a concentration of 40 nM for 48 h by using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions. The knockdown efficiency was checked by Western blotting. To study the effect of TRAPPC6AΔ knockdown on the growth of influenza virus, the WSN virus was used to infect siRNA-treated A549 cells at an MOI of 0.01. Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells.A total of 2 × 10Cell viability was determined by measuring the level of intracellular ATP with the CellTiter-Glo kit  according to the manufacturer's instructions. Briefly, A549 cells seeded into opaque-walled 96-well plates were transfected with siRNA targeting TRAPPC6AΔ or with nontargeting siRNA at a concentration of 40 nM. At 48 h posttransfection, 100 μl of CellTiter-Glo reagent was added directly into each well, and the contents were mixed for 2 min on a shaker to induce cell lysis. After a 10-min incubation at room temperature, luminescence was recorded with a GloMax 96 microplate luminometer (Promega).The pRetroX-IRES-ZsGreen1-TRAPPC6AΔ retroviral plasmid and an insertless pRetroX-IRES-ZsGreen1 control retroviral vector were independently transfected into the AmphoPack-293 packaging cell line (Clontech) by using Lipofectamine LTX and Plus reagents. Viral supernatants from transfectants were used to transduce A549 cells. After 48 h, transduction was repeated to enrich for transductants. GFP-positive cells were then harvested 48 h later by using a MoFlo XDP cell sorter (Beckman Coulter), propagated, and examined for TRAPPC6AΔ overexpression by Western blotting. To study the effect of TRAPPC6AΔ overexpression on the growth of influenza virus, the WSN virus was used to infect the TRAPPC6AΔ-overexpressing A549 cell line or an A549 control cell line transduced with empty retrovirus at an MOI of 0.01. Supernatants were collected at 24 and 48 h p.i., and virus titers were determined by plaque assays on MDCK cells.The mutant WSN viruses WSN M2Del1and WSN M2Del2 were generated from plasmids as described previously . BrieflyA549 cells (either untreated or treated with siRNA targeting TRAPPC6AΔ for 48 h as described above) were infected with the wt WSN virus or one of the mutant WSN viruses WSN M2Del1 and WSN M2Del2 at an MOI of 0.01. After 1 h of adsorption, removal of the inoculum, and one wash with F-12K medium (0.3% BSA), the cells were incubated with F-12K medium (0.3% BSA) at 37°C. The virus-containing culture supernatant was collected at various time points and titrated by plaque assays in MDCK cells.A549 cells grown in glass-bottom dishes were transiently transfected with the indicated plasmids by using Lipofectamine LTX and Plus reagents or were infected with WSN, WSN M2Del1, or WSN M2Del2 virus at an MOI of 5. At 24 h posttransfection or the indicated time points p.i., cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 30 min at room temperature, and permeabilized with 0.5% Triton X-100 in PBS for 30 min. After such treatment, A549 cells were incubated with blocking buffer (5% BSA in PBS) for 1 h at room temperature. Primary antibodies  were diluted in blocking buffer and incubated with cells for 2 h. The cells were then washed three times with PBS and incubated with secondary antibodies [Alexa Fluor 488 donkey anti-mouse IgG(H+L) and Alexa Fluor 546 donkey anti-rabbit IgG(H+L)] for 1 h. The cells were then washed three times with PBS and incubated with DAPI  for 15 min. Cells were examined by using a Leica SP2 confocal system .To examine whether the regulation of TRAPPC6AΔ expression affects the surface expression of M2, HA, or the cellular protein FGF2, the WSN virus was used to infect the indicated cells at an MOI of 3. The cells were collected at 8 and 10 h p.i. In an independent experiment, infected cells were subjected to an additional step of replacing the culture medium with medium supplemented with 25 μM amantadine (Sigma-Aldrich) at 2 h p.i. and then collected at 8 and 10 h p.i. The collected cells were fixed with 4% PFA for 30 min and then stained for the surface expression of M2, HA, or FGF2 with the corresponding primary and secondary antibodies , without permeabilization treatment. After washing, the cell suspensions were subjected to flow cytometry on a FACSAria flow cytometer . The data were analyzed by using FlowJo software.2 and intranasally infected with the wt WSN virus or the M2 deletion mutants WSN M2Del1 and WSN M2Del2 in 50 μl of PBS, with doses ranging from 103 to 106 PFU per mouse. Four mice were infected with each dose and observed for 14 days p.i., and their body weight and survival status were checked daily.Six-week-old female BALB/c mice  were lightly anesthetized with COPlaque assays were performed on MDCK cells in 12-well plates according to standard procedures. Briefly, cells were infected with serial 10-fold dilutions of the virus supernatants in 1× MEM (0.3% BSA) for 1 h at 37°C. The inoculum was removed, and the cells were washed with PBS and overlaid with 1% SeaPlaque agarose  in 1× MEM . After 2 to 3 days of incubation, the cells were fixed with formalin in PBS, and plaques were counted.t test. A P value of <0.05 was considered statistically significant.Results were analyzed for statistical significance by using Student's