Chrysanthemum sp., shoot branching is an important quality aspect as the outgrowth of axillary buds determines the final plant shape. Bud outgrowth is mainly controlled by apical dominance and the crosstalk between the plant hormones auxin, cytokinin and strigolactone. In this work the hormonal and genetic regulation of axillary bud outgrowth was studied in two differently branching cut flower Chrysanthemum morifolium (Ramat) genotypes. C17 is a split-type which forms an inflorescence meristem after a certain vegetative period, while C18 remains vegetative under long day conditions. Plant growth of both genotypes was monitored during 5 subsequent weeks starting one week before flower initiation occurred in C17. Axillary bud outgrowth was measured weekly and samples of shoot apex, stem and axillary buds were taken during the first two weeks. We combined auxin and cytokinin measurements by UPLC-MS/MS with RT-qPCR expression analysis of genes involved in shoot branching regulation pathways in chrysanthemum. These included bud development genes , auxin pathway genes , cytokinin pathway genes  and strigolactone genes (CmMAX1 and CmMAX2). Genotype C17 showed a release from apical dominance after floral transition coinciding with decreased auxin and increased cytokinin levels in the subapical axillary buds. As opposed to C17, C18 maintained strong apical dominance with vegetative growth throughout the experiment. Here high auxin levels and decreasing cytokinin levels in axillary buds and stem were measured. A differential expression of several branching genes accompanied the different hormonal change and bud outgrowth in C17 and C18. This was clear for the strigolactone biosynthesis gene CmMAX1, the transcription factor CmBRC1 and the dormancy associated gene CmDRM1, that all showed a decreased expression in C17 at floral transition and an increased expression in C18 with continuous vegetative growth. These results offer a case study for Chrysanthemum, showing an altered cytokinin to auxin balance and differential gene expression between vegetative growth with apical dominance and transition to generative growth with loss of apical dominance and axillary bud outgrowth. This suggests a conservation of several aspects of the hormonal and genetical regulation of bud outgrowth in Chrysanthemum. Furthermore, 15 previously uncharacterised genes in chrysanthemum, were described in this study. Of those genes involved in axillary bud outgrowth we identified CmDRM1, CmBRC1 and CmMAX1 as having an altered expression preceding axillary bud outgrowth, which could be useful as markers for bud activity.In the production and breeding of  Chrysanthemum morifolium (Ramat), called the florist’s chrysanthemum, is an economically important horticultural crop. Chrysanthemums show a great variation in sizes and shapes: garden and potted plants are highly branching, while cut flowers frequently show limited branching and sometimes even removal of axillary buds is required to obtain single flowered stems. Modification of plant architecture through shoot branching is a relevant factor in breeding and production of chrysanthemum. Shoot branching or axillary bud outgrowth in herbaceous shoots is regulated by a complex interaction of external factors  and plant hormone signalling [gnalling –3. The mgnalling . Removalgnalling . Auxin rgnalling ,7 in theSince the basipetal transport does not deliver auxin into the axillary buds directly, an indirect action of auxin is suggested . In liteThe canalisation model explains the inhibition of axillary bud outgrowth by the polar auxin stream in the stem that acts as an auxin sink. The shoot apex and the axillary buds are auxin sources that compete with each other for the ability to export auxin to the sink. Evidence for this model comes from the observations in Arabidopsis that strigolactones inhibit axillary bud outgrowth by reducing PIN1 mobilisation, as such restricting polar auxin transport ,12.The second messenger model states that a signal downstream of auxin is responsible for the inhibition of bud outgrowth. Both cytokinins and strigolactones control shoot branching downstream of auxins, and thus may be considered as secondary messengers. Cytokinins have a positive effect on the outgrowth of axillary buds. This is supported by observations in pea of stimulation of bud outgrowth upon exogenous application of cytokinins  and incrLAS [REVOLUTA (REV) and PHABULOSA (PHAB) . SHOOT MERISTEMLESS (STM) is another gene involved in the formation of axillary meristems [Euphorbia esula, dormant adventitious buds were shown to have upregulated STM expression after defoliation treatment to induce bud growth [In this way the physiological regulation of shoot branching involves the activity of many genes involved in the local axillary meristem maintenance and in the pathways of auxin, cytokinin and strigolactones . The forLAS , REVOLUTeristems  and can eristems . In Euphd growth .BRC1 (BRANCHED1) that is expressed locally in the axillary bud and inhibits outgrowth [BRC1. In Arabidopsis and pea, strigolactones promote BRC1 expression while cytokinins inhibit BRC1 expression [BRC1 is also involved in the floral transition as it is under control of the florigen pathway with a proposed interaction between FT (FLOWERING LOCUS T) and BRC1 whereby BRC1 is inactivated, promoting branching at floral transition [BRC1 is DRM1 (DORMANCY ASSOCIATED PROTEIN 1). DRM1 expression was shown to decrease in pea [DRM1 and BRC1 have been reported in dormant axillary buds [A central regulator of axillary bud outgrowth is the transcription factor utgrowth . Many ofpression ,28. BRC1ansition . A dormae in pea  and Arabe in pea  after thary buds ,33.TAA (TRYPTOPHAN AMINOTRANSFERASE) and YUCCA genes [TIR3 (TRANSPORT INHIBITOR RESPONSE 3) encodes a protein that is required in the mobilization of PIN1 proteins in the cell membrane [Tir3 mutant plants showed reduced auxin transport and increased shoot branching in Arabidopsis [AXR1 was shown to be responsible for the auxin induced inhibition of axillary bud growth in Arabidopsis [Auxin (IAA) biosynthesis in Arabidopsis involves the activity of the CA genes . For auxmembrane . Tir3 mubidopsis . Auxin sbidopsis ,38 leadibidopsis . AXR1 wabidopsis  and is rbidopsis . Aux/IAAbidopsis –43. The bidopsis ,45.MORE AXILLARY GROWTH (MAX) genes in Arabidopsis and it’s homologues RAMOSUS (RMS), DECREASED APICAL DOMINANCE (DAD) and DWARF (D) in pea, petunia and rice respectively. MAX3 and MAX4 convert carotenoid precursors and MAX1 that is involved in the final step of strigolactone biosynthesis ,46,47. Strigolactone signalling involves an F-Box complex with MAX2 and DWARF14 (D14), allowing the degradation of SMAX1-like proteins to enable strigolactone response [Strigolactone regulation of shoot branching involves the response ,49.IPT (ISOPENTENYLTRANSFERASE) is the rate limiting step in the pathway [HK3 and HK2. These cytokinin receptors relay a phosphor signal to RESPONSE REGULATOR proteins (RR) that act as transcription factors for downstream cytokinin responses [RR genes have been shown to be required for axillary bud activation by cytokinin [For cytokinin biosynthesis  pathway . In Arabesponses . Type A ytokinin .CmBRC1’s activity has been described in chrysanthemum with high expression in inhibited axillary buds and downregulation when buds were activated [LATERAL SUPPRESSOR LIKE (LsL) gene was isolated in chrysanthemum [LsL gene showed reduced branching and increased IAA content in the shoot tip while transformation with a sense LsL construct showed increased branching and reduced IAA content in the shoot tip [CmMAX3 showed upregulated expression in plants with inhibited axillary buds by phosphorous (Pi) starvation [CmMAX4 was studied in the strigolactone regulation of shoot branching and its expression was reported to be upregulated by exogenous auxin treatment [CmMAX2 was found to be expressed in axillary buds and stems and is necessary for the response to strigolactone, including inhibition of shoot branching . Overexpression of the CmIPT3 gene in chrysanthemum resulted in enhanced branching [In chrysanthemum only few genes involved in shoot branching have been isolated . CmBRC1’ctivated . A LATERanthemum  and planhoot tip . The strarvation . CmMAX4 reatment . CmMAX2 ranching .Aside from looking at hormone levels directly, the expression of these genes is interesting as a potential indicator of axillary bud activity. Because of the limited number of genes available in chrysanthemum, several key genes involved in the shoot branching pathways were isolated in this study and their gene expression levels were determined during branching. Two distinctive chrysanthemum genotypes with different branching patterns were analysed. In the first genotype, C17, the transition from vegetative to generative growth released apical dominance, while the second one, C18, remained vegetative. Axillary bud outgrowth measurements were related to levels of auxin and cytokinin in apex, stem and axillary bud samples in order to investigate the hormonal regulation during axillary bud outgrowth. The expression of candidate branching genes was analysed in the same samples to show the underlying transcriptional control during bud growth.Chrysanthemum morifolium (Ramat) cut flower genotypes C17 and C18, provided by Dekker Chrysanten BV., The Netherlands) were used in this study. Both genotypes are commercial cultivars that have an unrelated genetical background in their breeding history. C17 is a split type chrysanthemum, forming an inflorescence meristem at the shoot apex once a number of vegetative leaves are produced. This results in a release from apical dominance with outgrowth of subapical axillary buds. C18 is a non-split type with vegetative growth during long day (LD) conditions and generative growth under short day (SD) conditions. A diagram of the growth habit of both genotypes under LD is shown in The start of measurements and sampling was in the first week of July 2014, 2 weeks after potting. The total plant height and length of axillary buds/shoots was measured for 10 plants of each genotype during 5 consecutive weeks, referred to as: V1, T2, G3, G4, G5 (C17) or V1, V2, V3, V4, V5 (C18) with vegetative growth indicated by V, generative growth by G and the transition to generative growth indicated by T. The timing of floral transition in C17 was known from previous bud outgrowth experiments (data not shown).In total, 90 plants were sampled per genotype at the first two time points . Three replicates were sampled per time point, each replicate consisting of 15 plants. For C17 at V1, the shoot apex (the top part of the shoot above the first fully unfolded leaf) and the 30 nodal positions under the apex were sampled. At T2, the apex and 15 nodal positions under the apex were sampled, as the lateral buds below this zone had already grown out. For C18 at V1, the shoot apex and 22 underlying nodal positions were harvested, while at V2, the apex and 27 subapical nodal positions were taken. For each nodal position, the axillary buds and stems were sampled separately. Samples were frozen in liquid nitrogen and stored at -80°C. Before analysis, the samples were pooled based on the pattern of axillary bud outgrowth until week 5. They were subdivided to represent vertical positions along the stem that are at different distances and influences from apical dominance. From top to bottom these were named Apex, Zone A, Zone B and Zone C . Pooled Following a solid phase extraction, samples were analysed with UPLC-MS/MS (Acquity TQD) for quantification of free IAA and total cytokinins  as described by Prinsen et al. ,61.CmMAX1 degenerate primers were developed based on MAX1 amino acid sequences from Arabidopsis, tomato, poplar, soy, Medicago, vine and RicinusCmMAX3, CmDRM1, CmSTM, CmRR1, CmHK3a, CmHK3b, CmAXR1, CmAXR2, CmIAA16, CmAXR6, CmIAA12, CmPIN1, CmTIR1 and CmTIR3. Reference genes were identified by BLAST against the NCBI database. These included CmACT2, CmATUB, CmUBQ10, CmUBC, CmEF1α, CmCACS, CmEXP5, CmEXP6, CmPGK, CmPSAA and CmHH3. Sequences of CmBTUB_AB608732 and CmMTP_AB542716.1. were available for Chrysanthemum. Primers xl Genetic analyser (Applied Biosystems). BlastX [For . BlastX  was used™ automated electrophoresis DNase I according to manufacturer’s protocol . DNase treated RNA (800ng) was converted to cDNA with the iScript cDNA Synthesis kit (Bio-Rad). The no reverse-transcriptase controls (noRTs) were made using a mixture of DNase treated RNA and nuclease free water (1:1). cDNA and noRT samples were diluted (1:5) prior to use. Expression of branching genes was quantified using the LightCycler® 480 (Roche). In white plates (Bio-Rad) a final volume of 10 μl, containing 2 μl cDNA, 300nM of both primers, and 5 μl of SensiFAST™ SYBR® No-ROX (Bioline) was added. PCR amplification and melting curve analysis was done as described in De Keyser et al. [CmBRC1 [CmLSL [CmIPT3 (Acc. Nr. JQ711176.1) and CmMAX2 [CmBRC1, CmIPT, CmLsL, CmMAX1 and CmMAX2 was quantified; the second batch was used for expression analysis of CmRR1, CmAXR1, CmAXR2, CmAXR6, CmHK3a, CmHK3b, CmDRM1, CmIAA12, CmIAA16, CmMAX3, CmPIN1, CmSTM, CmTIR1 and CmTIR3; reference genes were analysed again on these cDNA samples as well. For all gene expression data analysis, qbase+ software (Biogazelle) [RNA was extracted with a modified CTAB protocol . RNA concentrationand qualitywas verified with the NanoDrop spectrophotometer r et al. . Primer  [CmBRC1 , CmLSL [1 [CmLSL , CmIPT3 d CmMAX2 . NTCs   was usedgazelle) ,68. GeneKruskal-Wallis one-way analysis of variance and Spearman correlation tests were performed with SPSS statistical software . Also in SPSS, UPGMA hierarchical clustering of the genes was done based on a Pearson correlation matrix. Discriminant analysis was performed in order to discriminate the global gene expression between tissues and time points. For statistical analysis of gene expression the log-transformed CNRQ values were used.Two different genotypes were used for branching analysis. Genotype C17 shifted from vegetative growth with apical dominance (V1) to generative growth with loss of apical dominance at T2  and consecutive outgrowth of axillary buds (G3-G5). Genotype C18 showed vegetative growth throughout all time points V1-V5. .The total plant height increased for C17 from 39.9 ± 1.5 cm at V1 to 53.1 ± 1.5 cm at T2 and 60.9 ± 2.1 cm at G3. Afterwards total length remained stable (61.0 ± 2.2 cm at G4 and 61.5 ± 2.4 cm at G5). C17 was characterised by vegetative growth at V1 and floral transition at T2. Thereafter, the shoot apex no longer initiated axillary buds and total number of nodal positions stayed at 47 from G3 to G5 . The vegPlant heights for C18 during vegetative growth from V1 to V5 were 41.8 ± 3.1 cm, 58.3 ± 3.4 cm, 72.4 ± 3.3 cm, 83.6 ± 4.4 cm and 93.5 ± 8.1 cm during V1 to V5, respectively. The shoot apex did not undergo floral transition and continuously initiated new leaves. At V5, the number of nodal positions reached a maximum of 53. A strong apical dominance was manifested during the whole vegetative growth of C18 . This waThe measurements of axillary buds were used to show the inhibition or activation of the nodal positions. Based on these measurements the nodal positions along the stem were subdivided in Zone A, Zone B and Zone C according to bud outgrowth activity and influences from apical dominance as described in Raw data of CK and IAA measurements are provided in the supplemental information Overall, IAA levels were higher in C18 than in C17, especially in apex and stem . For C17At the transition to generative growth in C17, from V1 to T2, a decrease in IAA content was observed in the apex and axillary buds of zone A, while a slight increase was seen in zone B and C. In the stem a significant decrease in auxin level occurred in zone A and B .IAA content in the shoot apex of C18 was significantly higher than in the axillary buds but did not differ from the IAA content in the stem at V1 . At V2, In C18 there was no significant difference in IAA levels between V1 and V2 in the apex and axillary buds. In the stem a significant decrease was observed in zone B” and zone C but the absolute auxin level remained high compared to the stem zone A and B of C17 .Cytokinin (CK) content was generally higher in C17 when compared to C18, especially in apex and axillary buds . CK contAt floral transition from V1 to T2 in C17, the CK content increased significantly in the axillary buds of zone A and C and in the stem of zone A .For C18 the CK content in the apex showed no significant differences with axillary buds and stem at V1 or V2 (except with zone C at V2). Between zones of axillary buds or stem no differences were seen at V1 or V2 In C18, CK levels had a decreased trend from V1 to V2 throughout the axillary buds and stem with significant changes in bud Zone B’ and stem zone C.CmDRM1 (Acc. Nr. KU528658), CmSTM (Acc. Nr. KU528663), CmMAX1 (Acc. Nr. KT124645), CmMAX3 (Acc. Nr. KU528660), CmRR1 (Acc. Nr. KU528651), CmHK3a (Acc. Nr. KU528656), CmHK3b (Acc. Nr. KU528657), CmTIR3 (Acc. Nr. KU528665), CmPIN1 (Acc. Nr. KU528662), CmAXR1 (Acc. Nr. KU528652), CmTIR1 (Acc. Nr. KU528664), CmAXR6 (Acc. Nr. KU528654), CmIAA12 (Acc. Nr. KU528659), CmAXR2 (Acc. Nr. KU528653) and CmIAA16 (Acc. Nr. KU528655), and 11 reference genes CmUBC (Acc. Nr. KX586319), CmUBQ10 (Acc. Nr. KX586320), CmEF1α (Acc. Nr. KX586321), CmACT2 (Acc. Nr. KX586322), CmATUB (Acc. Nr. KX586323), CmCACS (Acc. Nr. KX586324), CmEXP5 (Acc. Nr. KX586325), CmEXP6 (Acc. Nr. KX586326), CmPGK (Acc. Nr. KX586327), CmPSAA (Acc. Nr. KX586328) and CmHH3 (Acc. Nr. KX586329), were isolated in chrysanthemum and their sequences were deposited in the NCBI nucleotide database. The isolated branching gene sequences were verified by BLASTX against the NCBI database; the closest hits all corresponded to the gene of interest .CmBRC1 [CmLSL [CmIPT3 (Acc. Nr. JQ711176.1) and CmMAX2 [CmMAX3 there was amplification in the no-RT for the majority of the samples and the ΔCp cut-off- value of 5 was not met. Therefore CmMAX3 expression was excluded from the analysis. Similarly, one of the three biological replicates was excluded for CmAXR1 (in C17 V1 Bud Zone C), CmHK3a (in C17 V1 Bud Zone A), CmTIR3 (in C17 V1 Bud zone A and in Bud Zone C V1 and T2), CmAXR2 (in C18 V2 Bud zone B”) and CmIPT3 (in C18 V2 Bud Zone A); for these samples only 2 biological replicates were averaged.Rt-qPCR was done using the 15 isolated genes and 4 genes previously isolated in chrysanthemum: CmBRC1 , CmLSL [1 [CmLSL , CmIPT3 d CmMAX2 . These gThe expression of these genes is presented separately for C17 and C18 with significant differences between V1 and T2/V2 indicated on the figures Figs . Raw CNRCmBRC1 and CmDRM1 showed a different pattern in C17 compared to C18. A decreased trend from V1 to T2 was seen in the expression of CmBRC1  and in C18 .m zone B . In C18 V1 to V2 . CmSTM eCmMAX1 was expressed differently in C17 and C18. A decreasing trend from V1 to T2 in C17  showed similar expression patterns in C17 and C18 but with higher expression of CmHK3a in C18 compared to CmHK3b. In C17 expression increased in apex and decreased in the stem  mutants that show increased shoot branching [TB1 expression did not decrease in ccd8 (max4) mutants with increased branching phenotype [CmBRC1 expression is correlated with axillary bud activity since a decrease in expression is still seen in the stem. To further study this relationship it would require samples at short intervals within 24 hours of the floral initiation to account for the rapid changes in CmBRC1 expression.It has to be noted here that in our results the most striking decrease in pitation . Interesranching . Also inhenotype . In our CmMAX1 in C17 suggests that local strigolactone biosynthesis in the stem and axillary buds decreases during transition to generative growth and release from apical dominance, preceding bud outgrowth in the axillary buds under the apex. This is the first description of MAX1 in chrysanthemum. Given the role of strigolactones to inhibit axillary bud outgrowth [MAX1 in Arabidopsis [CmMAX1 expression. Expression levels of MAX1 during floral transition have not been previously reported but in red clover TpMAX1-1 expression as well as TpBRC1 expression were downregulated in nodal stem segments 24 hours after bud outgrowth was induced by node excision [The observed downregulation of bidopsis  and in Pbidopsis , our resexcision .CmTIR3, showed an increased expression from V1 to V2 in C18 while expression remained largely unaltered in C17 Click here for additional data file.S2 Fighttp://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi. The protein sequences for Chrysanthemum and Arabidopsis are presented together. For CmARR1 and CmTIR3, the Chrysanthemum query sequence was too short to contain a functional domain. For these sequences an alignment to the Arabidopsis protein sequence  is provided below the conserved domains.Based on NCBI conserved domain search (PDF)Click here for additional data file.S3 FigA dendrogram was constructed for both genotypes C17 and C18.(TIF)Click here for additional data file.S4 FigDiscriminant analysis was performed on the combined gene expression data of all genes that were common to all tissues. The analysis was done for both genotypes C17 and C18 at both time points together (A and D) and at the different time points: for C17: V1 (B) and T2 (C), for C18: V1 € and V2 (F). For all plots the percentage of variance explained by Function 1 and 2 are indicated as FCN1 and FCN2.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableThe accession number corresponds to the gene that was used for the BLAST search.(PDF)Click here for additional data file.S3 TableRNA concentration (ng/μl) and A260/280, A260/230 values of RNA isolated from the repeated V1 and V1/T2 shoot apex, stem and axillary bud samples of the chrysanthemum genotypes C17 and C18.(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 TableCmBRC1, CmIPT, CmLsL, CmMAX1 and CmMAX2 was analysed. Batch 2 was used for expression analysis of CmRR1, CmAXR1, CmAXR2, CmAXR6, CmHK3a, CmHK3b, CmDRM1, CmIAA12, CmIAA16, CmMAX3, CmPIN1, CmSTM, CmTIR1 and CmTIR3.In batch 1 the expression of (PDF)Click here for additional data file.S6 TableM and CV values for the 3 reference genes that were used for normalisation in the RT-qPCR analysis of branching genes in apex and axillary bud tissue and in stem samples.(PDF)Click here for additional data file.S7 TableThe fist tab shows IAA measurement and the total CK level, which is the sum of individual isoprenoid and aromatic cytokinins presented in the second tab.(XLSX)Click here for additional data file.S8 TableData are fold changes of hormone content (A-B = Zone-B/Zone-A) between mean (n = 3) IAA or CK contents and the significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S9 TableData are fold changes (A-B = Zone-B/Zone-A) between mean (n = 3) IAA or CK contents and the significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S12 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S13 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S14 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S15 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.S16 TableData are fold changes (A-B = Zone-B/Zone-A) between mean CNRQ values (n = 3). The significant difference between means by Kruskal-Wallis test is indicated by * .(PDF)Click here for additional data file.


We show that weakening the coupling between the SwitchI (residues 25–40) and SwitchII (residues 59–75) accelerates the opening of SwitchI; however, an open conformation of SwitchI is unstable in the absence of guanine nucleotide exchange factors (GEFs) and rises up towards the bound nucleotide to close the nucleotide pocket. Both I21 and Y32, play a crucial role in SwitchI transition. We show that an open SwitchI conformation is not necessary for GDP destabilization but is required for GDP/Mg escape from the HRAS. Further, we present the first simulation study showing displacement of GDP/Mg away from the nucleotide pocket. Both SwitchI and SwitchII, delays the escape of displaced GDP/Mg in the absence of GEF. Based on these results, a model for the mechanism of GEF in accelerating the exchange process is hypothesized.HRAS regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Understanding the transition mechanism is central for the design of small molecules to inhibit the formation of RAS-driven tumors. Using a multiscale approach involving coarse-grained (CG) simulations, all-atom classical molecular dynamics (CMD; total of 3.02  HRAS, a member of the Ras superfamily, regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states The dynamic nature of the SwitchI (residues 25–40) and SwitchII (residues 59–75) regions in HRAS structure and their participation in regulation of inactive/active state transitions has been long established Essential features of the GEF-catalyzed exchange reaction have remained mostly uncharacterized due to the difficulty in crystalizing the intermediate structures. The interconversion between the GDP-GTP bound form, in the absence of regulatory proteins, has been studied computationally using targeted molecular dynamics In this paper, we use a multiscale approach including coarse-grained (CG) dynamics HRAS simulations, both CG and all-atom, were performed in GDP- (PDB id: 4Q21) and GTP-bound (PDB id: 5P21 and 3RSO) states. A CG representation of the protein as described in All-atom simulations were performed using program NAMD α atoms was used to assess the stability of HRAS in different simulations. Cross-correlation analysis for all the Cα atom pairs was performed to identify residues with correlated motions. The cross-correlation coefficient, Cij, for atom pairs, i and j, is defined as: ij is the corresponding covariance matrix element, given as:ri (t) is the coordinate of atom i at time t and Trajectory visualization and figure preparation was done using VMD α atom coordinates. In PCA, the diagonalization of covariance matrix is performed to obtain the principal components (PC), which are the eigenvectors of the covariance matrix. The inter-conformer relationships can be studied by projecting the structures onto the sub-space defined by the largest principal components.PCA was applied to examine the relationships between different structures and to determine the conformational space sampled by various simulations. In this study, PCA analysis was performed using CContact analysis was performed to determine the common contacts involved in SwitchI transition from open to closed states. The details of contact identification are given in In the PCA analysis of 46 RAS structures by Gorfe et al. using MD .Dynamics resulting from the CG simulations of the three representative structures were compared with all-atom simulations (performed at 300K). Detailed analysis assessing the stability, conformational space sampled, and the correlations arising from the CG and all-atom simulations is given in CG simulations of HRAS at different temperatures showed that the opening of SwitchI and partial melting of  regions . In agreα group in GDP preventing further opening of SwitchI , both R68 and Y71 have different orientations and do not interact with E37 and D54. No interaction among these residues is observed in the simulations at 300K as well. In fact, hydrogen bond interactions between T35 and D57 and transient interaction between Q61 and backbone of T35 are observed. Unlike 4Q21, SwitchI opening was not observed at 360K and only partial opening was observed at 400K . At 400KIn the case of 3RSO, both the We performed simulations of 4Q21 (GDP-bound) with its SwitchI modeled in an open conformation as observed in the HRAS-GEF complex In agreement with the results from CG simulations, destabilization of GDP was observed in the all-atom simulation of 4Q21-OpenSI (more about it will be said in the next section); however, it occurred only in 1/3 simulations (4Q21-OpenSI-Run2) whereas GDP/Mg remained stable in the other two runs. As predicted by CG simulations, SwitchI did rise up towards GDP in all-atom simulations to close the nucleotide pocket  with RMSα and ribose group  caused increased fluctuations of the base group . In the absence of D119 interaction, the base group was displaced from its starting position after about first 14ns , however, did not result in GDP destabilization within the simulation time. SwitchI transitions to its closed state as observed in the 4Q21-OpenSI simulations before, although with one difference. In the 4Q21-OpenSI-S17AD57A simulation, SwitchI rises up towards GDP in the first 40ns  with F28Our simulations of 4Q21S39A and 4Q21-OpenSI showed that Y32 is among the crucial players in mediating the transition of SwitchI from open to closed state and vice versa. To further understand its role in SwitchI transitions all-atom simulations of 4Q21-OpenSI-Y32A mutant were performed. While in the 4Q21-OpenSI-Y32A-Run2 SwitchI transitioned to its closed state ; an entiWe identified only the common contacts between the SwitchI residues and the surrounding regions as seen in different 4Q21-OpenSI simulations . While destabilization of GDP was observed in only 1/3 4Q21-OpenSI all-atom simulations (4Q21-OpenSI-Run2), CG simulations resulted in frequent destabilization of GDP/Mg eventually displacing them from the nucleotide pocket. The parameters for CG GDP/Mg-protein interactions were determined by studying the GDP/Mg stability in 4Q21 (closed SwitchI state); their frequent destabilization thus points to some limitations of CG simulations. Interestingly, both CG and all-atom simulations resulted in open SwitchI folding back towards its closed state  indicatiSeveral polar and hydrophobic contacts between SwitchI and residues in ulations . Of thesulations . Y32, onulations . By contulations .β atom (and thereby GDP) by at least 5–6 Å away from its starting position in the nucleotide binding pocket. The displaced GDP/Mg group was then stabilized by interaction with the residues in the two switch regions preventing its complete release from the protein within the simulation time. This is seen in the SMD/CMD run of 4Q21-OpenSI  exchange reaction, and use that knowledge to map out the specific roles of GEF in accelerating the exchange reaction. Molecular understanding of the process is essential, since recent efforts have focused on inhibiting the Ras-GEF interaction as a way to prevent Ras-driven tumors PCA analysis of available HRAS crystal structures identified a new cluster of GTP-bound structures that have low barrier for transition towards clusters represented by 4Q21 (GDP-bound) and 5P21 (GTP-bound) structures. These structures thus provide an attractive starting conformation to study and understand the transition between the GDP-GTP bound states in wild type protein.The open conformation of SwitchI, as seen in the HRAS-GEF crystal structure, is critical for GDP release I21 forms the core of the hydrophobic interactions responsible for SwitchI closing. Weakening of this core, either by mutation of I21 or its interacting partners (such as Y32), prevented or delayed the proper closing of an open SwitchI and is likely the reason for experimentally observed accelerated intrinsic nucleotide dissociation in Y32 mutants β-Ploop interaction. Further, in the presence of GEF, dissociation of Mg may precede GDP release as indicated by the ternary complex of plant G-protein Loss of base-D119 contact resulted in the destabilization of GDP, independently of the open/closed state of SwitchI. This finding correlates well with the experimental studies demonstrating a strong decrease in the nucleotide affinity in D119 mutants Based on our results, a GEF mediated exchange reaction could be thought to proceed in the following manner . First, Figure S1PCA analysis of experimental structures. Projection of structures onto the first two PCs obtained using A) 46 structure dataset and B) 71 structure dataset. Green: HRAS-GDP bound; Red, Blue: HRAS-GTP bound conformations. Experimental structures groups in to three major clusters highlighted in B.(EPS)Click here for additional data file.Figure S2RMSD and Cα atom RMSF observed in CG and all-atom simulations of 4Q21 , 5P21 , and 3RSO . Results are shown for three CG simulations  where each run was performed for 100 million time-steps at T = 0.8. RMSF calculation was done for final 95 million time-steps. Black dotted line in B and D corresponds to RMSF calculated for first 45 million time-steps. All-atom simulation was performed at T = 300K. RMSF calculation for all-atom simulation (shown in magenta) was done for 99 ns production run.(EPS)Click here for additional data file.Figure S3Projection of CG and all-atom simulations on to the first two PCs obtained from 71 structure dataset. A. 4Q21. B. 5P21. C. 3RSO . Color codes are same as in (EPS)Click here for additional data file.Figure S4Cross-correlation plot from CG and all-atom simulations. Left panel  shows the plot from CG simulations performed at T = 0.8 while right panel  shows the plot from all-atom simulations run at T = 300K.(EPS)Click here for additional data file.Figure S5Cα-RMSF from 99 ns all-atom simulations of A. 4Q21, B. 5P21, C. 3RSO at temperatures 300K (blue), 360K (red), and 400K (green). Also shown are the snapshot from higher temperature simulations (360K for 4Q21 and 3RSO and 400K for 5P21) that highlights the open SwitchI/(EPS)Click here for additional data file.Figure S6All-atom simulation of mutant 4Q21S39A and 4Q21E37A. A. Snapshot of 4Q21S39A (green), and 4Q21E37A (red) showing open SwitchI/(EPS)Click here for additional data file.Figure S7Histogram of distances between Cα atom of selected SwitchI residues and representative atoms on GDP (N9 of base and Pβ) from four different all-atom simulations: 4Q21 run at 300K (pink), 4Q21 run at 360K (blue), 4Q21E37A run at 360K (red), and 4Q21S39A run at 360K (green). Bin width was set to 0.1 Å. All four simulations have equal number of data points.(EPS)Click here for additional data file.Figure S8Coarse-grained simulation of 4Q21-OpenSI. A. 4Q21-OpenSI simulated structure at the start of the simulation (red), at the end of the simulation (blue) superimposed on 4Q21 (pink). SwitchI rises up towards GDP. GDP/Mg destabilizes and are displaced from the nucleotide pocket. Coarse grained GDP beads are shown as spheres with base (orange), ribose (yellow), PA and PB (cyan) and Mg (green). B. Sidechain hydrogen bonds formed during the simulation between residue pairs D30-R149 (blue), H27-E153 (red), T20-T35 (green), D30-K147 (black), and sidechain-mainchain hydrogen bond between S17-Y32 (magenta). C. RMSD of SwitchI residues 25–40 (in 4Q21-OpenSI) with respect to its conformation in 4Q21. Structure alignment was performed using backbone atoms N, CA, and C of core residues as defined in (EPS)Click here for additional data file.Figure S9Combined all-atom SMD/CMD trajectory of 4Q21-OpenSI at 360K. A. RMSD of GDP (with respect to its position in starting structure) in SMD simulation (blue) and CMD simulation (red). RMSD of GDP in 4Q21OpenSI-Run3 (magenta) is shown for reference. B. Distance of Pβ in simulated structure from its position in starting structure. Color codes are same as in A. C. Distance between atoms pairs K16HZ2-GDPO1B (blue), S17HG1-GDPO3B (red), and G15HN-GDPO1B (green). D. Time series plot of SMD/CMD simulation. Residues E37, D57, E62, Y64, GDP, and Mg (green sphere) are highlighted in simulated structure. GDP and Mg in starting conformation is shown in pink.(EPS)Click here for additional data file.Figure S10All-atom CMD simulation of 4Q21-OpenSI-D119A at 360K. A. RMSD of GDP with respect to its position in starting structure. B. Distance of Pβ in simulated structure from its position in starting structure.(EPS)Click here for additional data file.Figure S11All atom simulation of 4Q21-OpenSI-D119A and 4Q21-D119A at 360K. A. Structure of 4Q21-OpenSI-D119A at the end of the CMD simulation  superimposed on top of starting structure . GDP/Mg destabilizes and is displaced away from its starting position  where it coordinates with E62, K16, G12, G60. Mg is trapped between E37 and D57. B. Distance between atoms pairs E62OE2-GDPH22 (blue), G12HN-GDPN7 (red), G60HN-GDPO2A (green), and K16HZ1-GDPO2A (black). C. Snapshots from simulation of 4Q21-D119A at 10, 80, and 100ns. F28 is highlighted to show interaction with base group at 10ns. Structure alignment was performed using backbone atoms N, CA, and C of core residues as defined in (EPS)Click here for additional data file.Figure S12All atom simulation of 4Q21-OpenSI-S17AD57A at 360K. A. RMSD of SwitchI residues 25–40 with respect to its conformation in 4Q21. B. Conformation at the end of the simulation. Simulated structure (red) is superimposed on 4Q21 (pink). Starting open conformation of SwitchI (residues 25–40) is also shown in blue for reference. SwitchI rises up to close the nucleotide pocket. F28 and GDP is also shown in the simulated structure.(EPS)Click here for additional data file.Figure S13All atom simulation of 4Q21-OpenSI-Y32A at 360K. A. RMSD of SwitchI residues 25–40 (in 4Q21-OpenSI-Y32A-Run2) with respect to its conformation in 4Q21. B. Conformation at the end of the 4Q21-OpenSI-Y32A-Run2 simulation. Simulated structure (red) is superimposed on 4Q21 (pink). Starting open conformation of SwitchI (residues 25–40) is also shown in blue for reference. C. Conformation at 100ns in the 4Q21-OpenSI-Y32A-Run1 simulation. SwitchI opens up even further in this simulation. Base group of GDP loses contact with D119 after about 80ns. D. RMSD of SwitchI residues 25–40 (in 4Q21-OpenSI-Y32A-Run1) with respect to its conformation in starting structure (blue) and 4Q21 (Red).(EPS)Click here for additional data file.Figure S14All atom simulation of 4Q21-OpenSI-Y32F at 360K. A. RMSD of SwitchI residues 25–40 in 4Q21-OpenSI-Y32F-Run1 (blue) and Run2 (red) with respect to its conformation in 4Q21. B. Conformation at the end of the 4Q21-OpenSI-Y32F-Run1 (blue) and Run2 (red). The starting conformation is shown in pink.(EPS)Click here for additional data file.Figure S15All atom simulation of 4Q21-OpenSI-I21S at 360K. A. RMSD of SwitchI residues 25–40 in 4Q21-OpenSI-I21S-Run1 (blue) and 4Q21-OpenSI-I21S-Run2 (red) with respect to its conformation in 4Q21. B. Conformation at the end of the 4Q21-OpenSI-I21S-Run1 simulation. Simulated structure (red) is superimposed on 4Q21 (pink). Starting open conformation of SwitchI (residues 25–40) is also shown in blue for reference. Y32 can be seen making hydrogen bonds (red dashed line) with the phosphate group of GDP. C. Conformation at the end of the 4Q21-OpenSI-I21S-Run2 simulation. Y32 forms hydrogen bonds with D54 and hydrophobic contact with L56. D. Side view of the conformation shown in C.(EPS)Click here for additional data file.Table S1List of all-atom simulations performed.(PDF)Click here for additional data file.Table S2PDB id of 25 structures added to the 46 structure dataset used for PCA.(PDF)Click here for additional data file.Table S3List of representative sidechain atoms of hydrophobic and charged residues used for contact analysis.(PDF)Click here for additional data file.Text S1Supporting Information text.(PDF)Click here for additional data file.


E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption.Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of  E1b-deleted Ads in cancer cells.Cancer is the most common cause of death in the world . The limearly (E) and late (L) genes [E1 genes, including E1a and E1b, produce critical proteins for the initiation and regulation of several viral and cellular genes. The E2 genes encode three different proteins which function directly in viral DNA replication. The E3 genes encode proteins that can modulate infected host immune responses. Products of the E4 genes encode proteins of diverse functions, such as regulating viral gene transcription, translation, mRNA nuclear export, and apoptosis pathways. The L genes of Ads encode structural proteins for packaging the viral genome into virion particles in the final stages of virus replication.Human Ads, as a common cause of respiratory tract diseases, are non-enveloped DNA viruses that can infect cells at many different cell cycle stages without integrating into host cellular chromosomes . Ad seroL) genes ,9. The EE1b gene encodes two major polypeptides of 55,000 kDa (55K) and 19,000 kDa (19K), both required for transformation of rodent cells by viral infection and DNA transfection [Ad sfection ,11. The sfection . The Ad sfection ,14,15. Esfection . ThroughE1 gene  located on the cell surface. The fiber and penton base of Ads have the functions of attachment and internalization, respectively, that facilitate the entry of the virus into the host cells by endocytosis . The lowThe replication of Ads relies on utilizing the cellular components as building blocks. The replication cycle of Ads is divided into two phases based on the onset of viral gene expression . The earThe idea of treating cancers with Ads began in the 1950s . Wild-tyE1b region which generates a premature stop codon to prevent the expression of its E1B55K protein (Ad dl1520 (ONYX-015) is a gene-attenuated oncolytic virus with an 827-bp deletion and a point mutation in the  protein  [10,25,2E1b55k region as dl1520, was generated by the Chinese company Sunway Biotech [E1a gene is driven by the alpha-fetoprotein promoter which is highly activated in primary hepatocellular carcinoma, but not in normal cells. Thus, H102 may selectively express the viral E1a gene in liver cancer, leading to specifically oncolytic replication. H102 is currently in the preclinical stage of development. H103 is an oncolytic Ad carrying a tumor antigen known as the heat shock protein (HSP) 70 gene which can stimulate an antitumor immune response while the virus selectively replicates within tumor cells. A phase I clinical trial of intratumoral injections of H103 has been conducted in a total of 27 patients with advanced-stage solid tumors [H101 (Oncorine), another Ad vector with the same deletion of the  Biotech ,28. It i Biotech ,28. The  Biotech ,29. The  Biotech . Conside Biotech ,31. Besi Biotech . In H102E1b55K-deleted Ad directly relies on virus-mediated tumor cell lysis by selective viral replication [E1b55K-deleted oncolytic Ads have been used in many clinical trials and are marketed in China, the mechanism(s) enabling this selective oncolytic replication still remains controversial. The following sections summarize three potential mechanisms proposed until now.The antitumor efficacy of lication . During lication , E3-11.6lication , and E4Olication  can causlication ,37. Aftelication ,39. UndoE1b55K-deleted oncolytic Ads could only replicate in p53-deficient tumor cells, but not in normal cells with functional p53 [p53 transcription [ARF tumor suppressor (referred to as p19ARF in murine cells) which binds to MDM2 protein and subsequently blocks MDM2-induced p53 degradation and transactivational silencing [p53 gene deletion or mutation [The original hypothesis was that onal p53 ,26,40. Aonal p53 . In responal p53 ,17,42. Ecription  or stabiilencing ,45,46. Cilencing ,48; secoilencing ,50,51; tilencing . It was ilencing ,48. Withmutation . It was mutation ,26.E1b55K-deleted Ads selectively kill cancer cells with p53 deficiency, the original proposal has been challenged by several studies. Reports have shown that E1b55K-deleted Ads are able to replicate in and kill cancer cells with wild-type p53 as efficiently as in cancer cells with p53 deficiency [ARF deficiency might determine the cancer selectivity of E1b55K-deleted Ads [ARF would neutralize MDM2-mediated p53 degradation and was considered to prevent p53 from its normal functions. Thus, p14ARF deficiency in cancer cells was indicated as the alternative molecular mechanism to allow dl1520 replication in these cancer cells with wild-type p53. However, experimental data contradicted this mechanism by showing that the replication of dl1520 is not controlled by the p53 or p14ARF status in several cancer cell lines [E1b55K-deleted or E1b55K-mutated Ads can neither efficiently induce apoptosis nor activate the transcription of downstream p53-responsive genes in Ad-infected primary cells [E1b55K-deleted Ads, E1b19K, as a homologue of BCL-2 family members, can also function as an inhibitor to E1A protein-induced apoptosis [E1b gene, it does not prevent the replication of the E1b-deleted oncolytic Ads in the majority of cells [E1b55K-deleted Ads and blocking p53 activity may not be the major requirement for viral replication. Thus far, none of the comprehensive research has been reported to provide new data to further support the p53 hypothesis.While some reports have supported the hypothesis that ficiency ,57,58,59eted Ads ,61. In cll lines . Also, ill lines ,64. Furtry cells ,66. Morepoptosis . Even ifof cells . Thus, tE1b55K-deleted oncolytic Ads lack E1B55K-mediated late viral mRNA export from the nucleus in primary cells, the viruses fail to efficiently replicate in those normal cells [E1b55K-deleted Ads. However, the molecular mechanism(s) of either selective nucleocytoplasmic transport mediated by the E1B55K/E4orf6 complex or the assembly and activity of the E3 ubiquitin-protein ligase associated with E1B55K/E4orf6 complex has not been fully understood [It was proposed that cancer cells can provide the functions of the E1B55K protein for late viral RNA export . During al cells . Permissderstood .E1b55K-deleted Ad replication in refractory cancer cells [E1b55K-deleted Ad replication.There is a report indicating that either restoring viral L4 100K protein expression or inducing heat shock responses can partially rescue er cells . In conter cells . Ad L4 1er cells ,76,77. Aer cells . In the er cells . Thus, tE1b55K-deleted Ads on late viral mRNA export, evidence has also shown neither expressing L4 100K nor inducing heat shock responses could completely restore E1b55K-deleted Ad replication to the level of wild-type Ad in a majority of cancer cell lines [Although expression of L4 100K and inducing heat shock responses may participate in determining the cancer selectivity of ll lines .E1b55K-deleted Ads are still required for further studies. Studies conducted by Goodrum and Ornelles (1999) have shown that the E1B55K function in late viral mRNA export is not correlated with the restricted replication of E1b55K-deleted Ads in cells under G0/G1 phase [et al. (2006) indicated that it appears overly simplistic to generalize that the cancer selectivity of E1b55K-deleted Ads relies on the efficiency of viral late mRNA export [Since ectopic expression of L4 100K and inducing heat shock responses cannot recover wild-type replication capacity in the absence of E1B55K, the importance of E1B55K for selective oncolytic Ad replication cannot be ignored. The exact mechanics of the interaction of E1B55K, L4 100K, and heat shock responses to regulate mammalian and late viral mRNA export which leads to cancer selectivity of G1 phase . GonzaleA export . E1B55K E1b55K-deleted Ads is restricted in HeLa cells under the G0/G1 phase, but is less restricted under the S-phase [1 phase than that in cells infected during the S-phase with either the wild-type or E1b55K-deleted Ads. A further study suggested that the E1B55K protein has a property to enable the virus to overcome the growth restrictions to its replication imposed by the cell cycle stage [E1b products affected the expression of numerous genes involved in cell cycle regulation based on a study with large-scale gene arrays [The study of Goodrum and Ornelles has shown that unlike wild-type Ad, which is able to efficiently produce viral progeny regardless of the cell cycle stages, the replication of  S-phase . Howeverle stage . In addie arrays . The lev1/S phase transition [Cyclin E gene amplification [Cyclin E is well characterized as a critical cell cycle protein to promote Gansition ,82 in eiansition  or CDK2-ansition ,85. The ansition ,87,88,89fication , overexpfication , decreasfication , togethefication ,94,95, hfication ,97.E1b55K-deleted Ads is restricted in normal cells, which may be due to the failure of efficient induction of cyclin E expression because of the deletion. We have shown that the induction of cyclin E is required for Ad replication and is correlated with the oncolytic selectivity of E1B55K-deleted Ad [E1b55K-deleted Ads to induce cyclin E expression in the absence of the E1B55K function, leading to preferential oncolytic replication [E1b55K-deleted Ads (The replication of leted Ad ,59. We aleted Ad . The inhlication . We thuseted Ads .E1b55K preferentially replicate in cancer cells and have been used in oncolytic virotherapies. However, many clinical studies have demonstrated the limited therapeutic effects in metastatic cancer patients. By far, oncolytic Ads have only shown some therapeutic benefits in the treatments of localized tumors. At least three mechanisms have been proposed so far. The original hypothesis was that, since many cancer cells lack functional p53 protein or its pathways, the E1B55K function of inhibiting p53 is not as important as it is in normal cells, and thus, E1b55K-deleted Ads could selectively replicate in cancer cells with p53 deficiency or dysfunctional pathways. The second mechanism is that cancer cells have a propensity to support the nuclear export of viral late mRNAs in the absence of the E1B55K protein, and thus allow the oncolytic replication of E1b55K-deleted Ads. The third mechanism is that cancer cells with dysregulated cyclin E allow E1b55K-deleted Ads to induce cyclin E expression, leading to preferential oncolytic replication. Understanding the molecular mechanisms underlining the cancer-selective replication of E1b55K-deleted Ads will guide us to develop new oncolytic vectors and therapeutic strategies. As the selective replication of E1b55k-deleted Ads is dependent on cyclin E dysregulation in cancer cells, better virotherapeutic efficacy may be expected when this kind of vector is used to target aggressively growing tumor cells with cyclin E overexpression. We have reported that the activity of cyclin E promoter is augmented in cancer cells during Ad infection [in vitro and in vivo [Ads with the deletion of nfection ,99. The  in vivo ,100,101.E1b55K-deleted Ads may be determined by several factors in cancer cells; some factors may be more important than others, depending on different types of tumors or cancer cells. To improve oncolytic Ad therapies for localized tumors, it is important to increase virus release from the initially targeted cells and promote Ad to spread the infection to the entire tumor mass. By further exploring the questions left in current mechanisms, strategies can be developed to overcome the limitations and ultimately build up new, powerful oncolytic Ads for virotherapy.Ad E1B55K protein has multiple functions and interacts with various cellular components, which are still not fully understood. The oncolytic replication of


Vaccinia virus A33 is an extracellular enveloped virus (EEV)-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs) by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus. Before the eradication of smallpox (variola virus) from nature, hundreds of million of people succumbed to the infection. The discovery of vaccinia virus (VACV), the active ingredient of the smallpox vaccine, ultimately led to the eradiation of smallpox from the human population. Vaccination with VACV leads to a strong antibody response that protects against variola virus. As the protective antibodies recognize viral proteins that are highly similar in sequence between the different orthopox strains, such as A33 used in this study, several antibodies have the capacity to neutralize a larger breath of orthopx viruses. In this study we have identified an anti-A33 antibody from a larger panel that exhibits a unique binding mode to A33. This antibody, A27D7, is also resistant to single amino acid changes throughout the protein and binds to engineered A33 variants that mimic ectromelia and orthopox A33 in the antibody-binding site. As the antibody further protects against ectromelia infection of mice, this antibody appears to be a potent orthopox cross-species protective antibody with therapeutic potential. Inoculation with vaccinia virus (VACV) protected against smallpox, a deadly disease caused by the related orthopoxvirus, variola (VARV) . Its sucst century. More recently a vaccinia virus clonal isolate, ACAM2000 has been used, produced using modern cell cultures . Cl. Cl50]. escribed  [50].5 cells/well into 24-well Costar plates  and EEV VACVWR neutralization was performed the following day using Mabs or pAbs samples at 10μg/ml  or 1/100  respectively in the absence or the presence of baby rabbit complement 10%   as described previously . , and the liquid was rapidly inhaled by the mouse. Mice were weighed daily to assess disease progression. Mice were euthanized if and when 25% weight loss occurred. A dose of 1x105 PFU of VACVWR was the standard dose given to 7-week-old BALB/c females. For A33 mAb protection studies, mice were treated by i.p. injection with 100 μg of antibodies 1 day before infection. Control mice received anti-A10 BG3.1 antibody, which is known to provide no protection. An additional group received anti-B5 B126 as positive control. A third group received anti-L1 M12B9, which was expected to protect against death but not weight loss , was selected based on the highest identity score in a PDB-BLAST. MR was performed using Phaser MR  and a si.3/37.4% . Refinem.3/37.4%  and rest.3/37.4% . NCS ref.4/28.2% .1 (R = 50.71%). The SEC profile of the complex suggested one Fab molecule bound to two A33 molecules , for a total MW of ~72 kDa resulting in a Matthews coefficient of 2.30 Å3/Da and 46.5% solvent. On this basis, MR was carried out with three independent components as starting models: the variable domain of the anti-tetrahydrocannabinol Lκ/HigG1 Fab fragment [pdb code 3LS4], its associated conserved domain, and one A33 monomer extracted from the previously deposited A33 dimer structure [pdb code 3K7B]. This Fab template shares the highest light chain identity (97%) with A27D7, among reported X-ray structures. Alternating cycles of manual refinement with COOT [free = 20.1/22.6%), which ranked in the 99th percentile for clashes and in the 100th percentile for geometry, compared to structures of similar resolution (Molprobity) [Initial crystals grew in 0.1 M sodium cacodylate pH 6.5, 0.2 M NaCl, 2.0 M ammonium sulfate with a protein concentration of 5.1 mg/ml . Data from a single crystal was collected at SSRL BL11-1. MR was performed with Phaser MR  and a siith COOT  and restith COOT  led to tprobity) .To assess binding kinetics of MAbs A2C7, A20G2, and A27D7 to WT-A33, we performed BLI assays using the OctetRED instrument . In a first experiment we immobilized MAbs on Anti-mouse Fc Capture (AMC) biosensors and tested binding to WT-A33 in solution. Biosensors were loaded with 10 μg/mL MAb diluted in 1X kinetics buffer  over 5 mins. MAb-loaded tips were tested against two-fold serial dilutions of WT-A33 analytes (0.78–50 nM). Binding kinetics were calculated using Octet Data Analysis 7.1 software  with curve-fitting statistics. The association steps were performed over 10 mins and dissociation steps performed over 20 mins. A negative control antibody (EE11) that targets the VACV antigen D8 was run in parallel to all assays for subtraction of background binding signal. Curves were aligned to baseline.D, kon, and koff were determined by global fitting of association and dissociation steps for all dilutions assuming a 1:1 binding model.To test the effect of the A33 mutations on antibody binding, we immobilized biotinylated A33 (BtA33) using streptavidin (SA) biosensors. SA biosensors were loaded with 1.5 μg/mL BtA33 protein in 1X kinetics buffer  over 5 mins. A33-loaded tips were tested against three-fold serial dilutions of IgG analytes (27.4 pM-20 nM). The association steps were performed over 10 mins and dissociation steps performed over 20 mins. Buffer control against biosensors loaded with WT-A33 was subtracted from raw data and curves were aligned to baseline. K2+ and Mg2+ was slowly loaded into nares (5 μl/nare). Mice subsequently were left in situ for 2 to 3 min before being returned to their cages. Mice were monitored daily for mortality and morbidity, as measured by weight change.Four- to six-week-old female C57BL/6 mice were obtained from Harlan Laboratories , housed in filter-top microisolator cages, and fed commercial mouse chow and water ad libitum. The mice were housed in an animal biosafety level 3 containment area. The day before challenge, mice were treated i.p. with 100 μg of the specified antibody. Immediately before challenge, mice were anesthetized with 0.1 ml/10 g body weight of ketamine HCl (6 mg/ml) and xylazine (0.5 mg/ml) by i.p. injections. One thousand PFU of ECTV (Moscow strain) in PBS without CaAnimal husbandry and experimental procedures were approved by the Institutional Animal Care and Use Committee of Saint Louis University  and by the Department of Laboratory Animal Care and the Animal Care Committee of the La Jolla Institute . We follow PHS Policy on humane care and use of laboratory animals.www.rcsb.org) with codes 4LQF, 4LU5, and 4M1G. The nucleotide sequences of the anti-A33 antibodies have been deposited in GenBank, and accession numbers are listed in The coordinates and structure factors of the A33:Fab complexes have been deposited in the Protein Data Bank (S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S1 FigPurified anti-A33 MAbs , or no primary antibody (-1’) were tested for binding to overlapping A33 biotinylated 20mer peptides. All seven A33 anti-A33 MAbs bind recombinant A33 protein (A33) but failed to bind any linear peptide, indicating the conformational nature of A33 epitopes.(TIF)Click here for additional data file.S2 FigLegend is the same as for (TIF)Click here for additional data file.S3 Figon), dissociation rate (koff), affinity constant (KD), and fit quality scores are reported in Real-time binding curves of MAbs A2C7, A20G2 and A27D7 to immobilized wild-type A33 and indicated A33 mutants are shown. Association (10 min) and dissociation (20 min) steps are represented. Curves are colored according to their specific antigen concentration . Association rate (k(TIF)Click here for additional data file.S4 FigVACV orthologs . VACV: vaccinia virus; VARV: variola virus; CMLV: camelpox virus; MPXV: monkeypox virus; MYXV: myxoma virus; CPXV: cowpox virus; ECTV: ectromelia virus; RFV: rabbit fibroma virus; TATV: taterapox virus; LSDV: lumpy skin disease virus; GTPV: goatpox virus; SPPV: sheeppox virus; SWPV: swinepox virus; DPV: mule deer pox virus; YMTV: Yaba monkey tumor virus; TANV: tanapox virus; YLDV: Yaba-like disease virus; MOCV: Molluscum contagiosum; ORFV: Orf virus; BSPV: bovine papular stomatitis virus. (B) GENIO/logo representation of the alignment performed with the closest A33VACV orthologs. (C) The model of A33VACV as extracted from A20G2-Fab/A33 complex structure is represented as a blue surface for conserved residues and in red and green for residues that vary among the orthopox species, as defined in (A). As expected, all variable residues are located on the surface. There are a total of 37 potential variations over the whole A33 sequence, among which eighteen can be visualized on the A33 model (residues 98–185). Amino acid positions in C can differ with alignment in B by up to 3, due to different lengths of the sequences.(A) Phylogenic cladogram of A33 orthologs. A33 sequence is overall highly conserved. All 106 sequences of A33 orthologs were downloaded from the Poxvirus Bioinformatics Resource Center server (poxvirus.org). Sequence alignment was performed with MEGA5 software using ClustalW with the GONNET similarity matrix and manual refinement. Phylogenic analysis was performed with a global gap removal to exclude gaps from alignments. Maximum Likelihood trees were constructed using the Jones-Taylor-Thornton amino acid substitution model with rates among sites Gamma distributed with Invariant sites. The robustness of trees was evaluated by bootstrap analysis with 1,000 rounds of replication. Non-redundant sequences of the clade framed in red are representative of A33 variation profile for the closest A33(TIF)Click here for additional data file.


Drosophila melanogaster. P53 also transcriptionally induces its primary negative regulator, Mdm2, which has not been found in Drosophila. In this study we identified the Drosophila gene companion of reaper (corp) as a gene whose overexpression promotes survival of cells with DNA damage in the soma but reduces their survival in the germline. These disparate effects are shared by p53 mutants, suggesting that Corp may be a negative regulator of P53. Confirming this supposition, we found that corp negatively regulates P53 protein level. It has been previously shown that P53 transcriptionally activates corp; thus, Corp produces a negative feedback loop on P53. We further found that Drosophila Corp shares a protein motif with vertebrate Mdm2 in a region that mediates the Mdm2:P53 physical interaction. In Corp, this motif mediates physical interaction with Drosophila P53. Our findings implicate Corp as a functional analog of vertebrate Mdm2 in flies.The tumor suppressor P53 is a critical mediator of the apoptotic response to DNA double-strand breaks through the transcriptional activation of pro-apoptotic genes. This mechanism is evolutionarily conserved from mammals to lower invertebrates, including  Drosophila melanogaster we identified corp as a gene that promotes the survival of such cells. Transcription of corp is activated by the P53 tumor suppressor protein, known primarily for its induction of cell death in response to DNA damage. Our experiments show that P53 regulates both pro-death and anti-death genes, and that a competition between these opposing factors determines cell fate. We find that corp functions by down-regulating P53, establishing a negative feedback loop. In vertebrates an identical mode of regulation is known: P53 up-regulates Mdm2, which physically interacts with P53 and is its primary negative regulator. We identified a protein motif on Corp that is shared with Mdm2, and is required for physical interaction between Corp and Drosophila P53. These results reinforce and strengthen the similarities of the P53 pathways and their regulation in vertebrates and in Drosophila.Organisms have exquisitely sensitive mechanisms to detect and respond to DNA damage. If DNA damage in a cell can be repaired, then that cell may resume its normal function. In multi-cellular organisms, if a cell cannot repair DNA damage it usually undergoes programmed cell death. This prevents the proliferation of cells with damaged genomes, which might otherwise differentiate incorrectly or proliferate without limit as cancer. In  Drosophila melanogaster, most cells undergo apoptosis in response to irreparable DNA damage, but a few cells escape, continue to divide and exhibit genomic instability [When cells encounter damage to their DNA in the form of DSBs, DNA damage response (DDR) pathways are triggered. The ensuing signaling cascades result in cell cycle arrest, induction of DNA repair genes, and in some cases, apoptosis. It is generally thought that, if damage cannot be repaired, cells will undergo apoptosis rather than continue to divide and propagate a damaged genome. If cells with irreparable damage do survive and proliferate, it can result in widespread genomic instability, creating an early state in the progress towards cancer –3. In Drtability –6. In oup53 gene, which is found to be mutated in most human cancers [tefu in Drosophila) phosphorylates Chk2 (encoded by lok), which in turn phosphorylates and activates P53 [p53 are prone to develop cancers, and p53 knockout mice develop cancers at an increased rate [p53-null Drosophila fail to eliminate cells with a DSB in their genome [One of the key players that controls the fate of a cell following DNA damage is the tumor-suppressor encoded by the  cancers . In respates P53 –10. Actiates P53 –17. A ceates P53 . Humans sed rate ,19,20. Sr genome .Mdm2 gene is a prominent target of P53 in mammals. It encodes a ubiquitin ligase that negatively regulates P53 and promotes its degradation, constituting a negative feedback loop [Drosophila melanogaster [Mdm2 has been identified.The ack loop –10,24,25nogaster ,21–23,26companion of reaper (corp), whose overexpression mimics the effects of p53 mutants in the soma and the germline. Our experiments indicate that Corp negatively regulates P53 protein levels. The corp gene has been previously identified as a transcriptional target of P53 [Drosophila P53. These similarities lead us to conclude that corp encodes a functional analog of vertebrate Mdm2 in flies and strengthens the similarities between the regulation and the functions of P53 in Drosophila and mammals.Here, we report the identification of a gene, t of P53 ,15,27, tBar and Telomere Loss) assay [P-element mis-expression element [eyeless-Gal4  and UAS-FLP is used to drive FLP recombinase expression in proliferating cells of the eye throughout development [Y chromosome with inverted FRT repeats (DcY(H1), or simply H1). Recombination between FRTs in inverted orientation on sister chromatids produces dicentric chromosomes which break in the subsequent mitotic division, delivering a chromosome with a single broken end to each of the two daughter cells  that produced nearly wildtype eyes in the BARTL assay (corp+ gene (CG10965). By qRT-PCR we confirmed that when Gal4 induces the P{EPgy2}CG1632EY03495 element (hereafter referred to as EP-corp+) it drives overexpression of corp+  assay  was used element  that modelopment –31. Theser cells . This reer cells . By intrTL assay . This inf corp+ . We alsoP-corp+ .corp in the BARTL assay the opposite result was obtained: the eye was completely ablated . A corp mutant was also generated by imprecise excision of a P element located in the 5’ region of the gene. This mutant, corp95B  chromosome and found that EP-corp+ produced almost wildtype eyes, similar to its effect in males  and two (T725A and T873G) encode different amino acids (L96H and L145M). These alternate amino acids are also present as polymorphisms in wildtype isolates of D. melanogaster and other Drosophila species (http://www.dpgp.org). There were also three single nucleotide differences  in introns.We sequenced the UAS-corp+ transgene that we constructed and tested (as mentioned previously) carries the canonical version, as found in CantonS. The EP-corp+, y w and w1118 strains all carry the variant allele that differs from the reference CantonS strain. Because the overexpression of either allele produces a similar large eye phenotype in the BARTL assay . Untreated discs show similar low rates of apoptosis in both the  mutants . We then element  was usedpression . These rde novo telomere addition [DcY(H1) these healed chromosomes (denoted FrY) may be detected in testcrosses to y w females by the loss of the dominant BS marker that lies distal to the inverted FRTs . To assess the effect of corp on the transmission of broken-and-healed chromosomes we induced expression of FLP by heat shock (70FLP10) during the first 24 hours of development and used nanosGal4 to drive germ cell-specific overexpression of corp+. Overexpression of corp+ blocked transmission of FrY chromosomes  and again observed a large decrease in FrY transmission relative to males with unaltered corp expression  vs. the germline (elimination of cells with broken chromosomes), there is precedent: the p535A-1-4 loss of function mutation acts similarly. Homozygous p535A-1-4 flies have almost wildtype eyes in the BARTL assay [corp and p53.Though it seems surprising that TL assay , but strTL assay . This sicorp95B; p535A-1-4 double mutants and examined them using the BARTL assay. We found that p53 is epistatic to corp, with the double mutant producing almost wildtype eyes  against corp and measured P53 protein levels by Western blot. We found that the quantity of P53 was significantly elevated following corp knockdown  gene is a prominent target of P53 following DNA damage [corp then we expect that corp overexpression should also reduce reaper induction following IR. To test this, we measured rpr mRNA levels by qRTPCR in corp+-overexpressing and corp95B mutant larvae, with and without irradiation. We found that, following IR, rpr mRNA levels decrease with corp+ overexpression and increase in corp95B mutants  and two Corp orthologs from Drosophila species (D. melanogaster and D. virilis). It identified seven similar motifs, with motifs 4 and 5 shared by Mdm2 and Corp  and CMM-2 (amino acids 14–54) in mediating the interaction between Corp and DmP53, we generated deletion constructs that lack CMM-1 (Δ58–84), CMM-2 (Δ14–54), or both (Δ14–84) . The intDrosophila. Some of these play important roles in DNA damage repair or in triggering apoptosis [Drosophila that a P53-regulated gene (apart from DNA repair genes) is not solely devoted to apoptosis. Our results show that P53 target genes act in competing pathways, defined by the hid- and reaper-mediated pro-apoptotic pathway and the corp-mediated anti-apoptotic pathway  and with several proteasomal subunits  and Dc3(FrTr61A5)1A chromosomes have been described previously by Kurzhals et al. [p535-A-1-4 by Xie et al. [Drosophila stock center: P{UAS-FLP1.D}JD1 (BL 4539), P{Gal4-ey.H}4–8 (BL 5535), P{EPgy2}CG1632EY03495 (BL 15650), P{eyFLP.N}5 (BL5576), M{3xP3-RFP.attP}ZH-86Fb; M{vas-int.B}ZH-102D (BL 23648), P{UAS-2xeGFP}AH2 (BL 6874), nanos-Gal4 [P{GMR-p53.Ex}3/TM3, Sb, Ser (BL 8417), GMR-Gal4 [P{GMR-hid}G1/CyO (BL 5771), P{GMR-rpr.H}S/TM6B, Tb (BL 5773) and P{Act5C-Gal4}17F01/TM6B, Tb (BL 3954). Two corp-RNAi stocks: v102751 and v16130 and one CG1632-RNAi stock: v106107 were obtained from the Vienna Drosophila Resource Center, Vienna, Austria (VDRC).All flies were maintained at 25°C on standard cornmeal food. Construction of the s et al.  and p53e et al. . We obta3495 BL 150, P{eyFP.N}5 BL56, M{3xP3FLP, P{70FLP}10, P{UAS-GFP} P{Act-Gal4}/CyO and y w; Sp/CyO; nanosGal4 UAS-FLP(95)/TM3, Sb. The engrailed-Gal4 stock was kindly gifted by Mark Metzstein.The following stocks were obtained from Golic lab collections: heat-shock inducible corp from CantonS flies was amplified by PCR with 5’ NotI and 3’ XbaI overhangs (primers used: Fwd-5’CATATTCGCGGCCGCATGGCCGATATCAGGAGCAG3’ and Rev- 5’CCGCGGGTCTAGACTAGATGCGAATCGAGCGCA3’) and cloned into the pUAST-w+-attB vector [attP docking sites on chromosome 3 and vasa-ΦC31 integrase on chromosome 4 (BL 23648). w+ flies were selected for establishing stable transgenic stocks.The coding region of B vector . Vector corp95B deletion mutation was generated via imprecise excision of the EY03495 P-element insertion (Baylor College of Medicine Genome Disruption Project). The DNA break points were identified by PCR amplification , treated with DNaseI  and cDNA was synthesized using RevertAid First Strand cDNA synthesis kit  according to manufacturer’s protocol. 1μl of cDNA was used per reaction in triplicates for performing qRT-PCR experiment using Maxima SYBR green/Fluorescein qPCR Master Mix  or PerfeCTa SYBR Green FastMix  in an iQ-PCR machine . Relative quantification of mRNA levels was calculated using the standard curve method. Relative copy numbers of each gene of interest (X) was calculated by normalizing cDNA levels of X over cDNA levels of Ribosomal Protein L32. Primers that were used are: Fwd-corp: 5’ GCAGCCGAGGTATGGAAATC 3’; Rev-corp: 5’ AAGCCGAGGGTCAGAAGG 3’; Fwd-p53: 5’ GCCGCCTCCTTAATCATGCC 3’; Rev-p53: 5’ GCCGAGACTGCGACGACTC 3’; Fwd-rpr: 5’ CCAGTTGTGTAATTCCGAACGA 3’; Rev-rpr:15–18 wandering third instar larvae were collected in clean 10 mm petri plates and irradiated at 4000 rads using a TORREX120D X-ray generator  or a Mark I irradiator . These larvae were returned to fresh food and incubated at 25°C until further experimental treatments.w1118 or y w) eyes, and was represented as a fraction of wildtype eye size.For determining eye sizes, the left eye of each fly was measured along the anterio-posterior axis (A) and the dorso-ventral axis (B), using a digital filar micrometer . These two measurements were used to calculate the area of an ellipse , as the area of the eye, which was then normalized to the mean area of wildtype . Briefly, dissected imaginal discs were fixed in 4% paraformaldehyde, rinsed twice in PBTW (0.3% Tween-20 in 1X PBS) for 5 minutes/rinse, post-fixed in 2:1 EtOH/1X PBS, rinsed again as before and then incubated with equilibration buffer, TdT enzyme and anti-digoxigenin Rhodamine congujate antibody in subsequent steps, according to manufacturer’s protocol. Finally, the discs were mounted in Vectashield  and photographed in Z projection. The images were exported as 8 bit TIFF files and analyzed in Adobe Photoshop. Fluorescence intensity of TUNEL staining was measured, using ImageJ software, as the total pixel intensity (referred to as Integrated density or InD) within each disc divided by the area of that disc. For wing discs the area of the entire wing disc was considered, or divided into anterior and posterior compartments, as appropriate. For eye discs, where gene expression was driven with GMR, only the region behind the morphogenetic furrow was considered for measuring InD.TUNEL staining was performed using Apoptag Red X (0.3% Triton-X in 1X PBS) twice for 30 minutes each, followed by 1 hour blocking in 5% BSA in PBTX. Next, they were incubated overnight at 4°C in primary antibody, p53-7A4  at 1:10 concentration in 5% BSA. The discs were then rinsed twice with PBTX, 30 minutes each and once in blocking buffer for 1 hour and finally incubated with Alexa Fluor 568 goat anti-mouse secondary antibody  at 1: 1000 concentration in 5% BSA for two hours. Finally, they were washed twice with PBTX as before, mounted in Vectashield  and photographed in Z projection. All images were taken at 100 ms shutter speed and at neutral density 6. Minimum and maximum intensity range was set to 200–550 for all discs, and images were exported as 8-bit TIFF files. Fluorescence was analyzed in Adobe Photoshop. P53 staining intensity was measured, using ImageJ software, as InD divided by the eye disc area, with only the region behind the morphogenetic furrow considered , according to manufacturer’s protocol. Then, the reactions were pooled together in a microcentrifuge tube and extracted with phenol-chloroform and chloroform. Finally, dsRNA was precipitated with ispropanol, dissolved in DEPC-treated H2O and quantified in a Nanodrop 1000 spectrophotometer (Thermo Scientific).To synthesize double stranded RNA for RNA interference experiments with cultured cells, PCR products not more than 700 base pairs, were made of the cDNA of interest flanked by T7 RNA polymerase sites at both ends. After gel purification of the PCR product, it was used as template for Primers used for obtaining PCR products were: Fwd_T7corp: 5’TTAATACGACTCACTATAGGGAGAATGGCCGATATCAGGAGCAG3’; Rev_T7corp: 5’TTAATACGACTCACTATAGGGAGACTAGATGCGAATCGAGCGCA3’; Fwd_T7p53: 5’TTAATACGACTCACTATAGGGAGAAGATCCAGGCGAACACGCTG3’; Rev_T7p53: 5’TTAATACGACTCACTATAGGGAGAGGCTTCCGGCACGGACTTG3’; Fwd_T7Pav: 5’TTAATACGACTCACTATAGGGAGAACAACTGCTCTTGGCAGATACC3’; Rev_T7Pav: 5’TTAATACGACTCACTATAGGGAGAAAATCCGTAACGAAACTAACCG3’.Drosophila Medium (Invitrogen) with 10% heat inactivated fetal bovine serum (HyClone) and 1X Antibiotic-Antimycotic (Invitrogen). Cells were passaged into fresh medium every 3–4 days and were discarded after passage 25 (P25).S2 cells were cultured at 25°C in Schneider’s 6 cells/ml. On day 1 they were washed and seeded in 24-well plates at 800 μl/well. 15 μg of dsRNA added to each well. The plates were then returned to the 25°C incubator. On day 4–5, cells were re-seeded in 6 well plates at 2 ml/well and retreated with 30 μg of dsRNA. As a control of dsRNA uptake rate, cells were treated with Pavarotti dsRNA, which makes them large and multinucleate[The dsRNA treatment protocol was performed as described . Cells winucleate,62. On dS2 cells, with or without dsRNA treatment, were irradiated at 4000 rads to observe any elevation/change in P53 levels from unirradiated cells, with or without dsRNA treatment. No significant changes in P53 levels were observed following irradiation, so treated cells were grouped and categorized as (I) no dsRNA control group and (II) + dsRNA experimental group for quantification.p53-dsRNA treatment experiment and 5 independent experimental sets in the corp-dsRNA treatment experiment. Out of the 3 p53-dsRNA experimental sets, 1 was following irradiation at 4000 rads and allowing 4 hours recovery before cell lysis. Likewise, out of the 5 corp-dsRNA experimental sets, 2 were following irradiation. Relative P53 levels in each experiment were calculated by normalizing total P53 protein level to total β–tubulin level.The Western results were pulled from 3 independent experimental sets in the Drosophila P53  at 1:10,000 concentration. After incubation with fluorescent goat anti-mouse secondary antibody  at 1:10,000, the membranes were scanned on an infrared Odyssey scanner (LI-COR Biosciences).S2 cells were collected and lysed in RIPA buffer containing protease inhibitor  to a final concentration of 1X. Protein concentration was measured by BCA assay  and cell lysates were mixed with sample buffer and β–mercaptoethanol to a final concentration of 1X before loading onto a 10% SDS gel at equal concentrations. Western blotting was carried out following standard procedure. Antibodies used: mouse monoclonal anti-en et al.) at 1:10The open reading frame of DmP53 was cloned into pGEX and transformed to BL21 DE3 cells. The expression was induced by addition of IPTG to a final concentration of 0.1mM in LB/Ampicillin media. Bacterial cells were harvested 4 hours following the induction and resuspended in ice-cold STE buffer  with 1.5% Sarcosyl. Cells were then lysed with sonication and subsequently incubated with STE containing 1% Triton X-100 for 30 minutes. Insoluble proteins were removed by centrifugation at 16,000g for 5 minutes. Supernatant was then incubated overnight with 50% slurry of glutathione-agarose beads at 4°C. The beads were pelleted by centrifuge at 100g and washed 4 more times with 10 ml of ice-cold PBTP (PBS with 0.1% Triton X-100 and protease inhibitors). Washed beads were then resuspended in PBTP with 0.01% sodium azide.corp-gfp-flag plasmid DNA was transfected to 2 million HeLa cells with calcium phosphate. Cells were lysed on plate with 1ml RIPA buffer at 48 hours following transfection.15ug of pRK5-Corp-HA6His was cloned into pCDNA3. In vitro synthesis were carried out using the TnT Coupled Reticulocyte Lysate Systems  following manufacturer’s instructions.in vitro synthesized protein, 1 ug GST or GST-DmP53 bound to Glutathione-agarose beads was incubated with 5 ul of synthesized protein in 500ul Binding buffer  containing BSA (0.2ug/ul). Following 1 hour incubation at RT and 1 hour incubation at 4°C, the beads were washed 4 times with Binding buffer. Beads were then pelleted at 100g, re-suspended and boiled in 30 ul sampling buffer, and resolved on SDS-PAGE gel. Following electrophoresis, the gel was fixed in (Isopropenol:dH2O:Acitic acid = 25:65:10) for 30 minutes and incubated in the Amplify Fluorographic Reagent  for 1 hour. The gel was then vacuum dried and processed for autoradiography with an intensify screen at -80°C.For For cellular extract, 1 ug GST or GST-DmP53 bound to Glutathione-agarose beads was first incubated for 30 minutes in 500ul binding buffer with 0.2ug/ul BSA. 500 ul cell lysate was then added and incubated at 4°C for overnight. Following incubation the beads were washed 4 times with 1 ml of RIPA buffer and pelleted by centrifugation at 100g for 5 minutes. Beads were then resuspended in 30ul sampling buffer and resolved on SDS-PAGE gel. Western analysis was performed with anti-Flag M2 antibody .th and 95th percentiles, top and bottom of the boxes represent 25th and 75th percentile and the horizontal line in the box represents the median, i.e., 50th percentile. The Mann-Whitney test was used in Figs Construction of graphs and calculations of statistical significance were performed using Prism 5.0 (Graphpad). In box-and-whisker plots the ends of whiskers represent 5http://meme.nbcr.net/meme/). Images were quantitatively analyzed using IMAGE J software from National Institutes of Health (http://imagej.nih.gov/ij/index.html) and images were processed using Adobe Photoshop. All line diagrams were composed using Adobe Illustrator.The MEME tool used for searching motif similarity is publicly available software Click here for additional data file.S2 Figcorp genomic region on the X chromosome and corp transcripts . Orange shading denotes the protein coding regions. The blue arrowhead indicates the site of the EY03495 EPgy2. Imprecise excision of the EY03495 element produced the corp95B allele. The extent of the corp95B deletion was determined by genomic sequencing. The nucleotide coordinates of the deleted region are X: 8,269,857–8,271,938 (Genome Release 6). Two sets of primers, Fwd1, Rev1 and Fwd2, Rev2 were used for PCR amplification of corp genomic region.  Visualization of PCR results. The corp genomic region was amplified either by Fwd1, Rev1 or by Fwd 2, Rev2 primer pairs in four different genotypes, run in the four lanes on the gel, marked 1 through 4: (1) y w; (2) corp1 (not used in this work); (3) corp95B; and (4) corp2 (not used in this work). M1 is a 1 kb ladder and M2 is the 100 bp ladder. The vertical red arrow indicates the lane 3, which lacks any PCR product and corresponds to the corp95B template. The horizontal red arrowhead points to the size expected in the y w control.(A) The (TIF)Click here for additional data file.S3 FigH1 dicentrics are produced in the presence of uninduced EP-corp+; (C) EP-corp+ is induced in B+ flies without dicentric induction;  EP-corp+ is present, either uninduced (D) or induced (E) in BS flies without dicentric induction; (F) corp95B is introduced into BS background without dicentric induction; (G) corp95B is introduced into B+ flies without dicentric induction; (H) H1 dicentrics are produced in XXY(H1) females; (I) EP-corp+ is overexpressed and dicentric chromosomes are induced in XXY(H1) females; (J) dicentric chromosome formation is induced on chromosome 3 (Dc3); and (K) EP-corp+ is overexpressed and chromosome 3 dicentrics are induced. N represents the number of fly eyes scored for each genotype. The P value in S3-B represents comparison with the wildtype control, P values in S3-I and S3-K represent comparison with the graphs immediately above them, i.e., S3-H and S3-J respectively.(A) The range of eye phenotypes observed in the BARTL assay, reproduced from (TIF)Click here for additional data file.S4 Figp53-dsRNA or (B) corp-dsRNA. β–tubulin is the loading control. The efficiency of dsRNA uptake by cells was approximately 50%.  Quantitation of P53 protein levels. Protein level is quantified by measuring the integrated density of the area of the desired band of the protein (P53 and β–tubulin) on the blot divided by the band area (InD/ band area). P53 level is represented in Y axis as the ratio of InD/ band area of P53 normalized to that of β–tubulin. N represents the number of experimental sets. Data are represented as mean +SEM. Western Blots of protein extracts from S2 cells. The left lanes represent untreated cells (wildtype) while the right lanes represent cells treated with (A) (TIF)Click here for additional data file.S5 Figrpr mRNA levels were measured by qRTPCR on total cDNA extracts of irradiated and non-irradiated third instar larvae. The graph represents rpr mRNA levels with no irradiation and at different time points after irradiation, in control (blue bars) corp+ overexpressing (cyan bars) and corp95B mutant (magenta bars) larvae (as indicated). Full genotypes are indicated in the inset. The larvae were irradiated at 4000 rads and allowed to recover for 2 or 3 hours before cDNA extraction. The Y-axis indicates rpr transcript quantitation relative to the rpl transcript internal control. rpr level alterations in corp overexpressing and corp mutant larvae, compared to controls, are not statistically significant. Three biological replicates were carried out for each experiment. Data are represented as mean +SEM.(TIF)Click here for additional data file.S6 FigGMR drives overexpression of p53+ in the eye to produce very small eyes that are alleviated by p535A-1-4 mutants. Y-axis represents the fraction of a wildtype eye. N, the number of eyes measured for quantification.(TIF)Click here for additional data file.S7 Fighttp://flybase.org/cgi-bin/get_interactions.html?items=FBgn0030028&mode=ppi), shows proteins that have been identified as physically interacting with Corp. Nine of these 19 interactors are proteasome subunits, and one is an E3 ubiquitin ligase (hyd).This figure, taken from FlyBase ((TIF)Click here for additional data file.S1 Tableay w/DcY, H1; 70FLP10/SM1, Cy males X y w females by w/DcY, H1; 70FLP10/SM1, Cy males X y w corp95B females P values calculated with contingency test.(DOCX)Click here for additional data file.


Ralstonia sp. strain MD27, a novel biopolymer-degrading betaproteobacterium, was isolated from compost samples. This organism has been shown to utilize the biopolymer poly(3-hydroxybutyrate) [P(3HB)] as a carbon source for growth. We report the draft genome sequence of MD27 with an estimated total sequence length of 5.9 Mb. Ralstonia species are a diverse group that includes plant and human pathogens and other soil-dwelling organisms, such as Ralstonia eutropha (now known as Cupriavidus necator)  [P(3HB)] and other polyhydroxyalkanoate (PHA) biopolymers as part of their natural metabolism (necator) , Ralstonnacearum , and Ralickettii , 4. Ralstabolism , 6. PHA tabolism , 8.Ralstonia sp. strain MD27 was isolated from compost from the greenhouse at Wellesley College  using media containing P(3HB) as the sole carbon source. The bacterium grows using P(3HB) with a doubling time of 3.3 h. Genomic DNA of Ralstonia sp. MD27 was extracted using the Wizard Genomic DNA purification kit . Libraries were then generated using the Nextera XT sequencing kit  and sequenced by an Illumina MiSeq at Tufts University Genomics Core Facility. We obtained 2,298,090 2 × 250-bp reads (185-fold coverage), which were assembled into 72 contigs  using CLC Genomics Workbench version 7.5.Ralstonia pickettii strain 12J (99% identity based on nucleotide BLAST).The draft genome sequence of MD27 comprises 5,917,320 bp (63.6% G+C content) and was annotated using the NCBI PGAP pipeline. We predicted 5,382 protein coding sequences, 47 tRNA sequences, and three copies each of 16S, 23S, and 5S rRNA genes. The 16S rDNA sequence of MD27 was found to be most closely related to that of phaC) genes were predicted (contigs 4 and 6), both of which appear to code for the conserved lipase box necessary for activity (phaA) genes (contigs 6 and 12), and two putative acetoacetyl-CoA reductase (phaB) genes (contigs 4 and 6). Indeed, the phaCAB sequences in contig 6 appear to form a gene cluster similar to a PHA biosynthesis operon found in C. necator H16 (R. pickettii strain 12D. A putative regulatory gene (phaR) has been identified in contig 6. These findings suggest that MD27 can synthesize intracellular polyhydroxyalkanoates.Two PHA synthase  genes were identified in the MD27 genome: three intracellular depolymerase genes  and an extracellular P(3HB) depolymerase gene (contig 34) that were predicted, based on a BLAST search with the Depolymerase Engineering Database   depolymerase that is capable of breaking down highly crystalline P(3HB) and is unlike the well-studied model organism of PHA homeostasis, C. necator strain H16 .Four putative PHA depolymerase (gart.de) , to be agart.de) . Experimickettii . Ralstonrain H16 , 11, impLFZM00000000. The version described in this paper is the first version, LFZM01000000.This whole-genome shotgun sequencing project has been deposited in DDBJ/EMBL/GenBank under accession number


N-acyl Neu5Ac2en mimetics as probes for human sialidases. Herein, we describe the in vitro activity of the same set of C9 N-acyl Neu5Ac2en mimetics against sialidases expressed by influenza virus A/PR/8/34 (H1N1), A/Memphis/1/72 (H3N2), and A/Duck/313/78 (H5N3) strains. Compound 8 is identified as a promising starting point for the development of viral sialidase selective inhibitors. Multiple sequence alignment and molecular docking techniques are also performed to explore the plausible interaction of compound 8 with viral sialidases.Identification of selective influenza viral sialidase inhibitors is highly desirable in order to minimize or avoid the adverse effects due to the possible inhibition of endogenous human sialidases. We recently reported the evaluation of C9  Influenza viruses belong to the Orthomyxoviridae family and are divided into three types, namely, A, B, and C. Influenza A virus, in particular, represents a significant health risk to the public due to both its ability to spread rapidly among humans and being associated with major epidemic outbreaks . Mo. MoN-acehibitors , 38. In hibitors . ResultsN-acyl Neu5Ac2en derivatives were synthesized according to the procedures previously described. The viral sialidase inhibitory activities of compounds (5–14) were evaluated against three different strains of influenza A virus  using DANA 2 as a reference compound. Results expressed as 50% inhibitory concentration (IC50) values are presented in 2, 5, and 8)  (accession no P03468), A/Memphis/1/72 virus [H3N2] (accession no P03475), and A/Duck/313/78 virus [H5N3] (accession no P03468) were obtained from Swiss-Prot database , and the8 and DANA was built using crystal structure of zanamivir present in the active site of sialidase (PDB code: 37BE). Then compound 8 and DANA were manually docked into the active site of sialidase structure separately. The obtained complexes were then energetically minimized with 500–1000 iterations of ‘in situ ligand minimization algorithm' using the SMART MINIMIZER program. A distance constraint was applied between carboxylic group of inhibitors and conserved Arg triad in the active site. The interactions between compound 8 and active site amino acid residues were analyzed and also compared with DANA's binding mode to investigate the relative differences in interactions for understanding the affinity differences.The model of compound 5–14) are given.The spectral data of investigated compounds (