In this study, we investigated the antigenic and genetic characteristics of influenza viruses circulating in Bulgaria during the 2017/2018 season. The detection and typing/subtyping of influenza viruses were performed using real-time RT-PCR. Results of antigenic characterisation, phylogenetic and amino acid sequence analyses of representative influenza strains are presented. The season was characterised by the predominance of B/Yamagata viruses, accounting for 77% of detected influenza viruses, followed by A(H1N1)pdm09 (17%), B/Victoria (3.7%) and A(H3N2) (2.4%). The sequenced B/Yamagata, B/Victoria, A(H1N1)pdm09 and A(H3N2) viruses belonged to the genetic groups 3, 1A, 6B.1 and 3C.2a1, respectively. Amino acid analysis of B/Yamagata isolates revealed the presence of three changes in haemagglutinin (HA), eight changes in neuraminidase (NA) and a number of substitutions in internal proteins compared with the B/Phucket/3073/2013 vaccine virus. Despite the amino acid changes, B/Yamagata viruses remained antigenically related to the vaccine strain. B/Victoria isolates fell into a group of viruses with double deletion (Δ162–163) in HA1. Substitutions in HA and NA sequences of B/Victoria, A(H1N1)pdm09 and A(H3N2) viruses were also identified compared with the vaccine strains, including in antigenic sites. The results of this study confirm the genetic variability of circulating influenza viruses and the need for continual antigenic and molecular surveillance. Influenza is a highly contagious viral respiratory infection with the highest impact on population health in the EU/EEA compared with other infectious diseases due to its substantial incidence and associated mortality . Annual Influenza viruses are highly variable and undergo constant evolution, which proceeds by continuous replacement of genetic groups with new ones, leading to increases in the antigenic distances from the current vaccine strains and to a reduction of vaccine effectiveness. HA and NA are subjected to the strongest pressure by the host immune system resulting in amino acid changes and altered antigenicity. This process, known as ‘antigenic drift’, necessitates almost annual updating of the influenza vaccine composition in order to match the circulating strains. Previous studies have identified five major antigenic (antibody-recognizing) sites on the globular head of HA in the A(H1N1) subtype, designated as Sa, Sb, Ca 1/2, Cb, five (A-E) antigenic epitopes in the A(H3N2) subtype and four  – in the type B . A limitaims of this study are to investigate the circulation pattern of influenza viruses in Bulgaria during the 2017/2018 season, to determine their antigenic and genetic characteristics, to perform a molecular sequence analysis of the surface glycoproteins and internal proteins with the identification of amino-acid substitutions, compared with the vaccine and other reference strains.Phylogenetic and molecular analyses of influenza viruses are necessary to track their continuous evolution, to detect emergent antigenic variants, variants with increased transmissibility, virulence or variants with reduced sensitivity to antivirals. The main http://www.grippe.gateway.bg). Sentinel physicians take nose and throat swabs from a systematic selection of patients presenting with ARI and send them to the National Reference Laboratory (NRL) for influenza virus detection by real-time RT-PCR. It performs testing of clinical samples from the sentinel network and from severely ill patients hospitalised in different regions of the country. Overall positivity rates of sentinel specimens are used to estimate the start, the duration and the end of influenza activity; a 10% threshold is used to indicate the start of the seasonal epidemic (with at least 10 specimens tested). The peak of the season occurs when the positivity rate exceeds 50% [In Bulgaria, an acute respiratory infections (ARI) surveillance system is used to monitor influenza. It comprises a national sentinel network of general practitioners and paediatricians working in 218 outpatient health care facilities in all 28 major cities, regional centres and serving 381 493 people from all age groups (5.3% of the country population). During the period from November 1 to March 31, the primary care physicians report the daily number of new cases of ARI by age group, and between April and October, the data are reported on weekly basis . All samples were first tested for the presence of influenza A and B viruses. The positive for influenza A samples were subsequently screened for A(H1N1)pdm09 and A(H3N2). The genetic lineage of detected influenza B viruses was also determined by real-time RT-PCR. Primers, probes and positive controls were provided by the International Reagent Resource (IRR), USA: CDC Influenza Virus Real-time RT-PCR A/B Typing Panel (FluRUO-01); A/H3/H1pdm09; Subtyping Panel (FluRUO-09); B lineage Genotyping Panel (FluRUO-05) and Influenza B/Victoria Lineage HA Gene Deletion Panel (FluRUO-10). Amplification was performed with a Chromo 4 thermal cycler (Bio-Rad) in accordance with the protocol of WHO  [t) value <38 were considered positive.Viral nucleic acids were extracted automatically from respiratory specimens using a commercial ExiPrep Dx Viral DNA/RNA kit  in accordance with the manufacturer's instructions. Detection and typing/subtyping of influenza viruses were carried out by a real-time RT-PCR method and the SuperScript III Platinumor 30 s) . Samplest values <28 were inoculated into Madin Darby canine kidney (MDCK) and MDCK-SIAT1  [2 atmosphere and observed on a daily basis for 7 days for evidence of cytopathology. The presence of a virus in a culture was confirmed by haemagglutination assay following standard protocols using a 1% suspension of guinea pig red blood cells. The antigenicity of Bulgarian isolates was characterised using the haemagglutination inhibition (HI) assay with panels of reference viruses and antisera at the WHO Collaborating Centres (WHO-CC) in London and Atlanta. The HI titre was determined as the reciprocal of the highest dilution of antiserum that completely inhibits virus-induced haemagglutination [All real-time RT-PCR-positive clinical specimens with Csferase)  cell cultination . Viral ihttp://www.gisaid.org) [B/Yamagata: EPI_ISL_ 296666; EPI_ISL_ 296667; EPI_ISL_ 296811; EPI_ISL_ 308677; EPI_ISL_ 308678; EPI_ISL_ 314121–314127; EPI_ISL_ 314129–314132; for B/Victoria: EPI_ISL_ 314133; EPI_ISL_ 314134; for A(H1N1)pdm09: EPI_ISL_ 299022; EPI_ISL_ 299023; EPI_ISL_ 308418; EPI_ISL_ 311872–311885; EPI_ISL_ 314190–314193; EPI_ISL_ 316679; for A(H3N2): EPI_ISL_ 31260; EPI_ISL_ 312261. For the purposes of phylogenetic analyses, the study sequences, together with sequences of reference viruses whose genetic group identities were known and of viruses circulating in different countries of Europe during the 2017/2018 season, were retrieved from the GISAID. They were all aligned using the MUSCLE program embedded in the Molecular Evolutionary Genetics Analysis  software [γ distribution, HKY+G) and NA  were determined using MEGA 6.06. Phylogenetic trees were constructed using the Maximum Likelihood method within the MEGA 6.06. The reliability of the tree topology was assessed by bootstrapping with 1000 replications.Clinical specimens, positive for influenza viruses and virus isolates, were sequenced using the Sanger method at the WHO-CC, London, and using Next Generation Sequencing (NGS) at the WHO-CC, Atlanta. Sequences were deposited in the Global Initiative on Sharing All Influenza Data (GISAID) (aid.org)  under thhttp://flusurver.bii.a-star.edu.sg). The potential N-linked glycosylation sites (NGS) in the HA and NA were predicted using the NetNGlyc 1.0 web Server (http://www.cbs.dtu.dk/services/NetNGlyc) to identify sequence motifs N–X–S/T (sequon), where X can be any amino acid except proline.The amino acid sequences were generated by translating nucleotide sequences with the standard genetic code using the MEGA software. The deduced amino acid sequences of the study strains were compared with those of vaccine strains and other reference strains to identify amino acid substitutions. The amino acid identity was calculated using FluSurver (The screening of A(H1N1)pdm09 viruses for the presence of point mutation conferring H275Y oseltamivir resistance was carried out using a real-time RT-PCR assay that allowed discrimination of a single nucleotide difference between oseltamivir-sensitive and resistant viruses. Primer/probe sequences and protocol were kindly provided by Public Health England, London.http://www.grippe.gateway.bg).In the 2017/2018 season, the influenza epidemic in Bulgaria started in week 1/2018 and continued until week 9/2018. The epidemic peak was reached in the week 4/2018 with 8806 ARI cases registered and 230.83 per 10 000 population incidence rate. The highest consultation rate for ARI was observed in the 5–14 years age group, followed by the 0–4 years age group with maximum values of 722.84 and 719.84 per 10 000 population, respectively (Influenza viruses were detected in 457 (33%) of 1384 patient samples screened. Of these, 89 (19.5%) were positive for influenza type A virus and 368 (80.5%) for type B. Among the influenza A viruses, 78 (87.6%) were A(H1N1)pdm09 and 11 (12.4%) – A(H3N2); among the type B viruses, 352 (95.7%) belonged to the Yamagata-lineage and 16 (4.3%) to the Victoria-lineage . FurtherThe first three influenza viruses of the season (B/Yamagata) were identified in week 49/2017 and the peak of influenza virus detections was reached in week 4/2018. The last influenza virus (B/Yamagata) was detected in week 12/2018.Influenza viruses were detected in all studied age groups . The larIn early January 2018, the first 19 detected influenza viruses  were sent to the WHO-CCs in London and Atlanta for further characterisation. In the course of the 2017/2018 season, a total of 52 influenza viruses were isolated on MDCK-SIAT1 cell culture. Thirty-nine representative influenza isolates were sent to the WHO-CC, London, where they were characterised in detail.A total of 16 B/Yamagata-lineage viruses was antigenically characterised by HI assay using post-infection ferret antisera raised against reference viruses. The test viruses showed 2-4-fold reduced reactivity with antisera raised against clade 3 viruses, including the antiserum raised against B/Phuket/3073/2013 strain, the component of the 2017/2018 Northern Hemisphere (NH) quadrivalent influenza vaccine .Twenty-two A(H1N1)pdm09 isolates analysed were well inhibited by the panel of antisera including the antiserum raised against A/Michigan/45/2015 strain, the A(H1N1)pdm09 component of the 2017/2018 and 2018/2019 NH vaccines, indicating antigenic similarity to the vaccine strain. Two A(H3N2) test viruses showed 2–4-fold reduced reactivity with antiserum raised against egg-propagated A/Singapore/INFIMH-16-0019/2016, the recommended vaccine component for use in the 2018–2019 NH influenza season. In contrast, both viruses reacted poorly to the antiserum against current vaccine virus, egg-propagated A/Hong Kong/4801/2014.Phylogenetic trees based on HA and NA genes were constructed. All 16 B/Yamagata-lineage viruses analysed belonged to phylogenetic clade 3, the B/Wisconsin/1/2010 – B/Phuket/3073/2013 clade . Two B/VPhylogenetic analysis of 22 A(H1N1)pdm09 isolates showed that they belonged to subclade 6B.1 represented by globally circulating A(H1N1)pdm09 viruses and clustered with the A/Michigan/45/2015 vaccine strain . Two BulThe complete HA and NA amino acid sequences of the Bulgarian influenza viruses were compared with those of corresponding vaccine strains in order to identify substitutions that could change the antigenic properties of viruses and may have had an impact on vaccine effеctiveness.Genetic analysis of Bulgarian B/Yamagata isolates indicated that the HA amino acid sequences had a high degree of similarity (amino acid identity of 99.1–99.5%) to that of the vaccine strain, B/Phuket/3073/2013. Amino acid analysis revealed the presence of three amino acid substitutions in HA with respect to the vaccine virus: L172Q, D196N  and M251V. The NA sequences contained changes in eight positions as compared with the B/Phuket/3073/2013 vaccine strain: I49M, C54Y, R65H, I171M, P244S, D342K, K373Q and S402P. The catalytic pocket and framework sites supporting the catalytic residues in NA were conserved among all 16 viruses studied . BulgariThe amino acid sequences of the internal proteins of two Bulgarian B/Yamagata viruses were compared with those of the B/Phuket/3073/2013 vaccine strain. The study sequences possessed the following amino acid substitutions: Q354H in PB2; A591S in PB1; M326V, S547G and I594V in PA; T313A in NP; M21V in M2; K110E, P114L and K176R in NS1. No substitutions were found in the proteins M1 and NEP.The HA and NA amino acid sequences of two Bulgarian isolates contained changes in four and seven positions, respectively, compared with the B/Brisbane/60/2008 vaccine strain. Two substitutions in HA (I117V and N129G) were located in the antigenic site, 120-loop. Both Bulgarian isolates fell into a group of viruses that have a six-nucleotide deletion in the HA gene segment (encoding amino acids 162 and 163) and share the substitutions N129G, I180V in HA1, R151K in HA2 and K371Q in the NA. The study viruses possessed the same 12 conserved NGS in HA  and four in NA  identified in B/Brisbane/60/2008. Three NGS  were located in the antigenic epitopes 150-loop, 160-loop and 190-helix, respectively.The sequence data of the Bulgarian isolates showed a high homology for HA protein to that of the vaccine strain, A/Michigan/45/2015 (amino acid identity of 98.4–99.1%). Changes in four positions of HA and 12 positions of NA were identified when they were compared with the sequences of the vaccine strain. The HA protein of Bulgarian strain A/Bulgaria/962/2018 fell into a group of viruses that shared the substitutions S183P, E235D and N260D in HA1 and V193A in HA2. Eleven other Bulgarian strains shared the substitution T120A in HA1 with a large number of viruses. The substitutions S74R and S164T were located in antigenic sites Cb and Sa, respectively. The isolate A/Bulgaria/962/2018 possessed additional substitution E235D located in antigenic site Ca [Comparison of the amino acid sequences of HA protein of two Bulgarian A(H3N2) isolates with the corresponding A/Hong Kong/4801/2014 vaccine strain revealed changes at 10 positions, six of which were unusual: Q75H, R142G, N171R, I192N, M320I in HA1 and A201V in HA2. The substitutions R142G, T160K and Q75H were located in antigenic sites A, B and E, respectively. The substitution T160K, possibly associated with propagation in cell culture, resulted in a loss of NGS. Twelve potential NGS in HA  were identified. The mutation G155E in HA2 altered the potential glycosylation motif from NGT to NET. The only Bulgarian NA sequence analysed, differed from the NA of the A/Hong Kong/4801/2014 vaccine strain by 11 amino acid substitutions. The substitution N86D resulted in a loss of NGS and the substitution S245N resulted in a formation of a new NGS. Eight potential NGS in NA  were identified, with two (146 and 367) being located around the enzymatic active site [All 78 detected A(H1N1)pdm09 viruses were analysed by real-time RT-PCR with respect to the H275Y mutation in the NA sequence as a marker of the resistance to oseltamivir, none of the isolates harboured this substitution. The Bulgarian isolates did not contain other known markers of resistance or reduced susceptibility to neuraminidase inhibitors in the NA gene segment .In Bulgaria, the 2017/2018 influenza epidemic was characterised by an unexpectedly strong prevalence of B/Yamagata-lineage viruses, accounting for 77% of all positive influenza cases and a low circulation of B/Victoria-lineage (3.5%) and A(H3N2) (2.4%) viruses. The similar predominance of B/Yamagata viruses was observed in the most European countries . In BulgPrevious observations exhibit that influenza B viruses are genetically more stable, evolve more slowly and undergo antigenic drift less rapidly as compared with type A viruses , 31. TheA limited number of amino acid changes were identified in HA, NA and internal proteins of the study B/Yamagata viruses with respect to the B/Phuket/3073/2013 vaccine strain that has been in use since 2015. The antigenic regions – 120-loop (positions 116–137), 150-loop (positions 141–150) and 160-loop (positions 162–167) were identical to those in vaccine strain. One amino acid substitution and one new NGS were identified in the fourth antigenic region, 190-helix (positions 194–202) which forms part of the RBS. Amino acid variations in the internal proteins have also been found by other authors, but the biological significance of thesе substitutions has yet to be precisely elucidated , 35. DesAs regards B/Victoria isolates, more amino acid variations were identified compared with the corresponding B/Brisbane/60/2008 vaccine virus which resulted in antigenic changes. Two substitutions were located in antigenic 120-loop which is one of the most frequently changing regions , 36. TheAll Bulgarian A(H1N1)pdm09 viruses were genetically and antigenically very similar to the A/Michigan/45/2015 vaccine strain used for the first time this season as well as to the vast majority of circulating A(H1N1)pdm09 viruses worldwide. Only four amino acid substitutions in HA were identified, compared with the A/Michigan/45/2015 vaccine strain, two of them were located in antigenic regions.In Europe, influenza A(H3N2) viruses belonging to clade 3C.2a emerged at the end of the 2013/2014 season and since the 2014/2015 season has been the predominant clade. Over this period they underwent considerable genetic diversification and diverged in several subclades and subgroups: subclade 3C.2a1 (subgroups 3C.2a1a and 3C.2a1b), subclades 3C.2a2, 3C.2a3 and 3C.2a4, defined by certain amino acid substitutions , 37. In The 2017/2018 season was unique with the exclusive high activity of influenza B viruses, which occurs once every 5–10 years , 39, 40.


Hyaloperonospora arabidopsidis does not induce necrosis. We determined the crystal structure of HaNLP3 and showed that it adopts the NLP fold. However, the conformations of the loops surrounding the GIPC headgroup-binding cavity differ from those of cytotoxic Pythium aphanidermatum NLPPya. Essential dynamics extracted from μs-long molecular dynamics (MD) simulations reveals a limited conformational plasticity of the GIPC-binding cavity in HaNLP3 relative to toxic NLPs. This likely precludes HaNLP3 binding to GIPCs, which is the underlying reason for the lack of toxicity. This study reveals that mutations at key protein regions cause a switch between non-toxic and toxic phenotypes within the same protein scaffold. Altogether, these data provide evidence that protein flexibility is a distinguishing trait of toxic NLPs and highlight structural determinants for a potential functional diversification of non-toxic NLPs utilized by biotrophic plant pathogens.Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic microorganisms. They trigger necrosis in various eudicot plants upon binding to plant sphingolipid glycosylinositol phosphorylceramides (GIPC). Interestingly, HaNLP3 from the obligate biotroph oomycete  Hyaloperonospora arabidopsidis that could explain how these proteins evolved a range of new functions in variety of pathogens with different lifestyles.The rhizosphere and phyllosphere of terrestrial plants are home to a number of microorganisms, many of which are potentially pathogenic at certain stages during their associations with plants. The pathogens use diverse routes to penetrate physical barriers and colonize host plants with different lifestyles. Necrosis and ethylene inducing peptide 1 (Nep1)-like proteins (NLPs) are widely distributed among prokaryotic and eukaryotic organisms, like fungi, bacteria and oomycetes, and may infect a range of different crops, such as potato, tomato, soybean and tobacco, thus posing major threat to agriculture worldwide. It was shown that NLPs function as cytolytic toxins that induce plasma membrane leakage by binding to plant membrane sphingolipid receptor. Interestingly, non-toxic NLP proteins can also be secreted by several hemibiotrophic and obligate biotrophic pathogens. This study provides a structural and functional characterization of a non-cytotoxic HaNLP3 protein from  Necrosis- and ethylene-inducing peptide 1-like proteins (NLPs) have been found in plant-associated prokaryotic (bacteria) and eukaryotic  microorganisms and are widely known to induce necrosis and ethylene production in eudicot plants . NLPs shPythium aphanidermatum (NLPPya) revealed a central β-sandwich surrounded by α-helices [Moniliophthora perniciosa exhibited a similar structure [et al. (2017) we showed that binding of the GIPC terminal hexose moiety to NLPPya induces several conformational changes within the toxin that may precede membrane attachment and host cell lysis. Similar to actinoporins, the interaction of NLPPya with the membrane is stabilized via a tryptophan residue in the loop adjacent to the sphingolipid binding site. Importantly, monocots are unaffected by NLP proteins because their plasma membranes mostly lack series A GIPCs [The first crystal structure of a toxic type 1 NLP from the oomycete -helices . MpNEP2 tructure . Comparitructure . Actinoptructure –10. The tructure . GIPCs ctructure . The typtructure , 13. In  A GIPCs .Hyaloperonospora arabidopsidis, a downy mildew pathogen of Arabidopsis, is an obligate biotrophic oomycete [H. arabidopsidis secretome analysis revealed NLPs as one of many secreted protein families [Arabidopsis infection, HaNLP3 does not induce necrosis on plant cells [oomycete . H. arabfamilies . None offamilies . Based ont cells , 15. ThePya, both members of the oomycete type 1 NLP group. A detailed comparison of the two protein structures allowed us to assign a functional role to specific protein structural elements. These findings demonstrate how differences within the same protein scaffold fine-tune functional plasticity of NLP proteins, further illuminating the mechanistic mode of action of toxic NLPs.The aim of the current study was to provide structural and functional information on similarities and differences between non-toxic HaNLP3 and toxic NLPPya and HaNLP3 is due to inability of the latter to bind the headgroup of plant sphingolipids. As opposed to toxic NLPPya, HaNLP3 was unable to bind to multilamellar vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and tobacco leaf GIPCs (POPC:GIPCs) Fig 1A). t HaNLP3 . In addiLP3 (Pya . Importagomyelin .Pya [Pya-glucosamine complex [α atoms of HaNLP3 and apo-NLPPya (PDB ID 3GNZ), NLPPya in complex with glucosamine (PDB ID 5NNW) and MpNEP2 (PDB ID 3ST1) are 1.1 Å, 1.1 Å and 1.5 Å, respectively. The fold of the conserved loops of NLP proteins, L1-L3, is well defined in HaNLP3  with PNGase F, resulting in significantly faster SDS-PAGE migration , NLPPyaS96M, T97L, G98L, I99M (L2 loop), NLPPyaS153H, T154S, W155F, NLPPyaW155F, and NLPPyaW155S  mutants. Liposome sedimentation assays employing POPC:GIPCs multilamellar vesicles confirmed compromised membrane binding ability of NLPPyaW155F, NLPPyaS153H, T154S, W155F, NLPPyaS96M, T97L, G98L, I97M, NLPPyaW155S, but not of NLPPyaA127W   simulations in explicit solvent. These simulations revealed that the two proteins display a remarkably different conformational flexibility (measured as root mean square fluctuation (RMSF) per residue) of the L1, L2 and L3 and Lc1 loops lining the GIPC headgroup binding cavity, with the L2 and L3 being significantly more flexible in NLPPya  connecting L3 with Lc1 and Lc3  of NLPPya, pictured by vectors indicating the direction and the amplitude of the motion  of the NLPe motion , underpie motion . The opee motion , and it Pya in terms of the coupled motion of different loops. As a result, the essential dynamics of MpNEP2 displays an opening/closing of the sugar headgroup-binding cavity mostly ascribable to Lc1 and Lc2 , Lc1 and L2 from NLPPya (variant 2) and Lc1, L2 and L3 loops from NLPPya (variant 3), respectively, were introduced biotrophic pathogens, suggesting functional diversification within this microbial protein superfamily , 15, 27.H. arabidopsidis in the early stages of Arabidopsis thaliana infection [Pya, HaNLP3 represents an excellent model protein to investigate differences between toxic and non-toxic NLP variants. Recently, we proposed that the principal functional feature of toxic NLPs is their association with the GIPC headgroup, followed by disruption of plasma membrane integrity which leads to cell death in eudicot plants [Pya and non-toxic HaNLP3 protein is in GIPC headgroup recognition. In contrast to NLPPya, the HaNLP3 protein does not bind to GIPCs alone or GIPCs embedded in liposomes, consistent with its inability to cause necrosis of tobacco leaves.This study provides the first functional and structural analysis of non-toxic oomycete type 1 HaNLP3, produced by nfection . Due to t plants . Here wePya is highly conserved, there are important structural differences between the two types of NLPs. For example, HaNLP3 is glycosylated, contains an additional cysteine bond, and lacks a bivalent metal ion. There is no information available about HaNLP3 glycosylation patterns when produced in its native host H. arabidopsidis. Due to expression of recombinant HaNLP3 in P. pastoris we subsequently performed protein deglycosylation assays to exclude possible effects of glycosylation on toxicity. We showed that deglycosylation or preincubation with metal ions did not affect HaNLP3 non-toxicity; nor did the introduction of an analogous disulfide bond in NLPPya change its toxicity. Importantly, comparing the surfaces of HaNLP3 and NLPPya proteins showed a volume disparity in the Mg2+ and hexose binding cavity. In HaNLP3, the cavity volume is significantly smaller than in NLPPya. Structural differences based on variation at the level of amino acid sequences between HaNLP3 and NLPPya in the Lc1, L2 and L3 loops appear to be critical for the gain/loss of necrotic activity. Several amino acids that are presumably important for toxicity have already been identified based on toxic and non-toxic NLP amino acid sequence comparisons; yet the effect of amino acid substitutions on necrosis was tested mostly by Agrobacterium-mediated plant transformations [Pya resulted in reduced GIPC binding ability. Furthermore, the NLPPyaP41A, D44N, N48E mutant, functionally and structurally characterized in this work, pinpoints another important structural element to regulate stability and functional dynamics of the protein. This underlines how additional switch elements affecting Mg2+ and GIPC headgroup binding may be spread across distinct protein regions.We have solved the HaNLP3 crystal structure to explore the molecular mechanism of HaNLP3 non-toxicity. While the overall fold between HaNLP3 and toxic NLPrmations –33. In tPya. Indeed, the essential dynamics of NLPPya revealed a collective motion of the Lc1, L1, L2 and L3 loops, which may enable opening and closing of the lipid-binding cavity. Such a dynamic arrangement is most likely a prerequisite for the capability of NLPPya to recognize the GIPC sugar headgroup and to associate with eudicot plant membranes. Remarkably, the toxic MpNEP2 [Pya onto the non-toxic HaNLP3 scaffold resulted in slightly increased toxicity upon infiltration into tobacco leaves and enhanced binding capacity towards multilamellar vesicles containing GIPCs compared to wild-type HaNLP3. Consistent with these data, MD simulations of these mutants partially recover the functional plasticity of the loops observed in NLPPya, clearly pointing to L2, L3/Lc1 as key switch elements of toxicity. Interestingly, HaNLP3 variants are almost equivalent to NLPPya in terms of binding to multilamellar vesicles, however, they differ significantly in terms of their toxic effects as shown by infiltration assays and conductivity measurements  ammonium sulfate and centrifuged at 24,523 g and 4°C for 20 min. The precipitate was resuspended in deionized distilled water and loaded onto Q Sepharose FF ion-exchange column, equilibrated in 20 mM Tris buffer pH 9. Protein was eluted with a 0 to 0.5 M NaCl gradient in 20 mM Tris buffer pH 9. Fractions containing HaNLP3 protein were pooled, concentrated and subjected to a HiLoad 26/600 Superdex 75 column equilibrated in 20 mM Tris buffer pH 7.5, 150 mM NaCl and 5% (v/v) glycerol. Protein-containing fractions were pooled and dialyzed against deionized distilled water. The protein was concentrated and stored at -80°C. All the purification steps were followed by SDS-PAGE analysis. HaNLP3 variants were expressed in Escherichia coli BL21(DE3) in TB medium. Cells were grown at 37°C until absorbance at 600 nm reached value of 1, followed by an overnight induction with 0.5 mM isopropyl 1-thio-β-D-galactopyranoside at 20°C. Cells were then harvested by centrifugation at 4°C and 5,647 g for 10 min and resuspended in lysis buffer  glycerol). Cell debris was removed by centrifugation at 50,000 g, 4°C for 1 h after sonication. The supernatant was filtered through 0.22 μm filter and loaded to a Ni-NTA column (Qiagen), equilibrated with 50 mM Tris pH 7.5, 250 mM NaCl and 10 mM imidazole. The bound fraction was washed with 50 mM Tris pH 7.5, 250 mM NaCl and 20 mM imidazole and eluted within the gradient of imidazole from 20 to 500 mM. Fractions that contained HaNLP3 variants were pooled and dialysed against 20 mM Tris pH 7.5 and 150 mM NaCl, concentrated and stored at -80°C until used. E. coli production of NLPPya and its mutants was performed as reported in Lenarčič 2017 [The HaNLP3 protein was expressed and purified as described previously  with thečič 2017 .Deglycosylated HaNLP3 protein was prepared using recombinant N-glycosidase F enzyme according to the Roche kit instructions (3U enzyme/10 μg protein).Pya and its mutants were recorded on a Chirascan spectrometer (Applied Photophysics) using 0.08 mg/ml protein in 20 mM MES pH 5.8 in a 0.1-cm path length quartz cuvette. Spectra were measured in a 200–250 nm wavelength range with a step size of 0.5 nm and integration time of 0.5 s at 20°C. Each spectrum represents the average of five scans. Spectra were processed, baseline corrected, smoothed and converted with the Chirascan software. Spectral units were expressed as the mean molar ellipticity per residue.Far UV CD spectra of NLPPya and its mutants was measured by differential scanning fluorimetry (thermofluor) assay using 2x SYPRO Orange dye (Thermo Fisher Scientific). Protein concentration was 0.1 mg/ml. Thermal stability of proteins was tested in 20 mM MES pH 5.8 and 150 mM NaCl. Samples were subjected to a temperature gradient from 25°C to 85°C at a 1°C/min step. Temperature melting profiles were acquired with LightCycler 480 (Roche). Melting temperatures Tm were determined by using Boltzmann function in Origin 8.1 (Origin Lab). Each Tm value represents the average of six repeats from three independent experiments.The thermal stability of NLPThe amino acid sequences of non-toxic HaNLP3 and toxic NLPs without signal peptide sequences were aligned using Clustal Omega .2 or 10 mM MgCl2 for 15 min. The level of leaf chlorosis/necrosis induced by HaNLP3 variants was determined using ImageJ software by calculating the ratio of the chlorotic area to the whole infiltrated area, both measured in squared pixels. Each value represents the average of fifteen infiltrations.The infiltration assay was performed on tobacco plants. 50 or 100 μl of proteins (diluted in deionized distilled water) with a concentration 0.2 μM, 1 μM or 10 μM were infiltrated into tobacco leaves using a syringe without a needle. Lesion formations were examined 3–7 days post-infiltration. To test the effect of metal ion presence on HaNLP3 toxicity, 1 μM HaNLP3 was preincubated with 10 mM CaClPya mutants) and 10 μM (HaNLP3 variants) proteins. After 10 min incubation, 8 leaf disks were punched out (Ø 6 mm) and transferred to 10 ml of deionized distilled water. After 30 min of shaking at 50 rpm, leaf disks were transferred to 4 ml of fresh deionized distilled water. Conductivity was measured after 2 h using 712 Conductometer (Metrohm). All values were normalized to mock treated samples (leaves infiltrated with deionized distilled water). Each value represents the average of five replicates.Leaves of tobacco were infiltrated with 0.5 μM  were prepared by hydration of POPC or POPC:GIPCs thin lipid films in warm 20 mM MES, 150 mM NaCl, pH 5.8 and vortexing of the lipid suspensions in the presence of glass beads. Small unilamellar vesicles (SUVs) were obtained by sonication of MLVs on ice for a total of 30 min, with 10 s on-off cycles at 40% amplitude using a Vibracell Ultrasonic Disintegrator VCX 750 .GIPC-containing liposomes were prepared by mixing POPC  and GIPCs at the appropriate mass ratios, i.e. 1:3, 1:5 or 1:6. Different mass ratios of POPC Pya and HaNLP3 proteins and their mutants (0.06 mg/ml) were incubated with multilamellar vesicles (4.5 mg/ml) composed of POPC or POPC:GIPCs in 20 mM MES, 150 mM NaCl, pH 5.8, for 30 min and 600 rpm at room temperature. To test the effect of metal ion presence on HaNLP3—GIPC interaction, 10 mM CaCl2 or MgCl2 were added to the buffer. The mixture was centrifuged, and the liposomes were washed twice with the same buffer and subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. Protein bands were statistically analyzed after densitometric quantification using image analyser system . Each value represents the average of three independent experiments.Liposome sedimentation assay was performed as previously described . BrieflyBinding analysis was conducted using Biacore T100  docked with Series S sensor chip L1 . The system was first primed with SPR running buffer . After regenerating the chip, POPC SUVs (200 × diluted in running buffer) were injected over the first flow cell while POPC:GIPCs SUVs were captured on the second flow cell (10 min injection at 2 μl/min). The surface was then conditioned with a 120 s injection of 0.1 mg/ml BSA at 10 μl/min. The proteins were injected in a single cycle mode in concentrations 62.5, 125, 250, 500 and 1000 nM.Pya and HaNLP3 were immobilized covalently via amine groups to the separate flow cells on the sensor surface with final immobilization level of 3800 RU for NLPPya and 4500 RU for HaNLP3. The binding of 10 μM lipids was monitored for 180 s followed by dissociation time of 180 s using SPR running buffer at a 10 μl/min flow rate. The removal of bound lipids was achieved using two short  pulses of 0.07% SDS.The binding of lipids to proteins was performed using Series S sensor chip CM5. Firstly, NLPPyaP41A, D44N, N48E (25 mg/ml) proteins were crystallized in the presence of 0.2 M di-ammonium hydrogen phosphate and 20% (w/v) PEG 3350 by sitting drop vapor diffusion, and 22% (w/v) PEG 4000, 0.3 M MgCl2, 0.1 M Tris pH 9, 10% (v/v) glycerol and 5% (v/v) methanol by hanging drop vapor diffusion, respectively, using a 1:1 protein:reservoir to crystallization buffer ratio at 20°C. Crystals were frozen in liquid nitrogen, and diffraction data were collected at 100 K and at the wavelength of 1.0 Å at Elettra Synchotron . The diffraction data for HaNLP3 was processed to 2.0 Å resolution and to 1.95 Å resolution for NLPPyaP41A, D44N, N48E. Data were processed with XDS [12121 for HaNLP3 and P41212 for NLPPyaP41A, D44N, N48E by molecular replacement using the NLPPya structure (PDB ID 3GNU) in PHASER [PyaP41A, D44N, N48E: 6QBD).HaNLP3 (20 mg/ml) and NLPwith XDS . The cryn PHASER  as a sean PHASER  and refin PHASER  and phenn PHASER . StructuPya (PDB ID 3GNU) and MpNEP2 (PDB ID 3ST1) together with those of HaNLP3 (PDB ID 6QBE) and NLPPyaP41A, D44N, N48E (PDB ID 6QBD) solved in this work. The HaNLP3 variants 1, 2 and 3 models were built by introducing the mutations into the crystal structure of HaNLP3 using the t-leap module of Ambertools 16 [Starting points of our MD simulations were the crystal structures of NLPtools 16 .Pya and HaNLP3 . It is likely that ion binding will similarly affect the dynamical properties of all investigated systems.The protonation states of ionizable residues were assigned on the basis of the Propka program . All proPya, 33113 for HaNLP3, 33335 atoms of HaNLP3 variant 1, 26888 atoms for MpNEp2 and 31641 for NLPPyaP41A, D44N, N48E. Na+ ions were added to achieve charge neutrality using ion parameters [The topologies were built with Amber parm99-SB force field  using thrameters .Pya, NLPPyaP41A, D44N, N48E, MpNEP2, HaNLP3 and its variants 1, 2, and 3 were equilibrated for 1 μs each, leading to a cumulative simulation time of 7 μs. The initial 200 ns of each simulation were not considered for the analysis.The topologies were later converted in a GROMACS format using the software acpype. MD simulations were performed with GROMACS 5.0.4 . An inteα atoms of the protein was initially built from the atoms position vectors. A RMS-fit to the starting configuration of the MD simulation was performed to remove the rotational and translational motions. Once diagonalized, the covariance matrix leads to a set of orthogonal eigenvectors  and their corresponding eigenvalues. The eigenvectors with the largest eigenvalues correspond to the most relevant motions. Thus, the first principal component (PC1) is representative of the largest amplitude motion of the protein and this is usually referred to as “essential dynamics”. The Normal Mode Wizard plugin of the Visual Molecular Dynamics (VMD) molecular visualization program has been used to visualize the essential dynamics and to draw arrows highlighting the motion [Principal component analysis (PCA) was employed to extract the ‘essential movements’ of the proteins from the MD simulation trajectory. In this analysis, the mass-weighted covariance matrix of the Ce motion .The correlation matrices, based on the Pearson’s correlation coefficients, were then employed to identify the coupling of the motions between each couple of residues along the MD trajectory. The values of the coefficients range from a value of -1, which indicates a totally anti-correlated motion, to a value of +1, which represents a linearly correlated lockstep motion , 50.cpptraj module of Ambertools 16 [Root mean square fluctuations (RMSF), H-bond analysis, cross correlation matrices and PCA of the MD simulations trajectories were performed with the tools 16 .S1 FigThe model of protein is shown in sticks.(PDF)Click here for additional data file.S2 FigPya A127 and HaNLP3 W153 in Lc1 loop; red, L3 loop; blue, NLPPya D158, involved in sugar binding. Amino acids that were swapped between HaNLP3 and NLPPya are marked in bold.Pink, region between conserved disulfide bond type 1 NLPs; yellow, non-conserved disulfide bond in HaNLP3; cyan, L2 loop; green, NLP(PDF)Click here for additional data file.S3 FigPya mutants upon infiltration of proteins in tobacco leaf . (B) Circular dichroism of NLPPya mutants. (C) Representative melting curves of NLPPya mutants, measured in differential scanning fluorimetry assay. (D) Thermal stability of NLPPya mutants measured in thermofluor assays. Values are shown as means ± SD (n = 6).(A) Detection of necrosis-inducing ability of NLP(PDF)Click here for additional data file.S4 FigPyaP41A, D44N, N48E polypeptide chain in the region between Ala69 and Gly76. (B) Overall crystal structure of NLPPyaP41A, D44N, N48E  in comparison to apo-NLPPya (white). Mg2+ ions are displayed as purple spheres: (1), position of Mg2+ in apo-NLPPya; (3), position of Mg2+ in NLPPyaP41A, D44N, N48E. Inset: Triple mutation site P41A, D44N and N48E. Amino acid residues are displayed as sticks. (c) Differences in positions of Mg2+ ion in apo-NLPPya (white), glucosamine-NLPPya (pink), polypeptide chain of NLPPya without bound hexose (light red) in the same asymmetric unit as glucosamine-NLPPya complex, and in chains A (cyan) and B (yellow) of NLPPyaP41A, D44N, N48E crystal.(A) Stereo image of representative 2Fo-Fc  and Fo-Fc  electron densities of NLP(PDF)Click here for additional data file.S5 FigFirst column depicts graphical representation of root mean square fluctuation (RMSF) per residue projected on the most representative cluster of the protein (shown as tube) as extracted from the molecular dynamics (MD) trajectories. Colors from red to blue along with a decreasing diameter of the tube indicate a progressive RMSF decrease. In the second column we report the cross-correlation matrices. Correlation scores ranges from +1 (red) to -1 (blue). A positive sign of the map (blue color) indicates that residues move in a correlated manner (lockstep), otherwise, a negative value (red color) points to an anti-correlated motion between residues (opposite directions). White color shows that residue displacements are independent of each other. Third column shows essential dynamics of the protein. The latter is represented as cyan new cartoon and blue arrows indicate the direction and the amplitude of the motion.(PDF)Click here for additional data file.S6 FigLeft panel: a graphical representation of root mean square fluctuation (RMSF) per residue projected on the most representative cluster of the protein (shown as tube) as extracted from the MD trajectories. Colors from red to blue along with a decreasing diameter of the tube indicate a progressive RMSF decrease. Central panel: the cross-correlation matrix. Correlation scores range from +1 (red) to -1 (blue). A positive sign of the map (blue color) indicates that residues move in a correlated manner (lockstep), otherwise, a negative value (red color) points to an anti-correlated motion between residues (opposite directions). White color shows that residue displacements are independent of each other. Right panel: the essential dynamics of the protein. The latter is represented as a blue cartoon and blue arrows indicate the direction and the amplitude of the motion.(PDF)Click here for additional data file.S7 FigDynamic properties of HaNLP3 (A) variant 1, (B) variant 2, and (C) variant 3. Left panel depicts a graphical representation of root mean square fluctuation (RMSF) per residue projected on the most representative cluster of the protein (shown as tube) as extracted from the molecular dynamics (MD) trajectories. Colors from red to blue along with a decreasing diameter of the tube indicate a progressive RMSF decrease. In the middle panel we report the cross-correlation matrix. Correlation scores ranges from +1 (red) to -1 (blue). A positive sign of the map (blue color) indicates that residues move in a correlated manner (lockstep), otherwise, a negative value (red color) points to an anti-correlated motion between residues (opposite directions). White color shows that residue displacements are independent of each other. On the right panel, the essential dynamics of the three proteins. These are represented as magenta new cartoons and blue arrows indicate the direction and the amplitude of the motion.(PDF)Click here for additional data file.S8 Fig(PDF)Click here for additional data file.S1 Movie(AVI)Click here for additional data file.S2 Movie(AVI)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableValues are shown as means ± SD (n = 6).(PDF)Click here for additional data file.S3 TableResidues from 114 to 128 belong to L2, from 177 to 187 belong to L3, from 151 to 157 to Lc1, from 54 to 62 to Lc3. Persistency (%) refers to the time the H-bond is established with respect to the length of the trajectory.(PDF)Click here for additional data file.S4 TableResidues from 90 to 104 belong to L2, from 150 to 162 belong to L3, from 126 to 131 to Lc1, from 31 to 39 to Lc3. Persistency (%) refers to the time the H-bond is established with respect to the length of the trajectory.(PDF)Click here for additional data file.S5 Tablea) NLPPya, (b) HaNLP3, (c) MpNEP2, (d) mutant NLPPyaP41A, D44N, N48E and (e) mutant HaNLP3-variant 1. Persistency (%) refers to the time H-bond is established with respect to the length of the trajectory.Hydrogen bonds analysis of a wider region of loops Lc3 and L1 as extracted from the MD trajectories of ((PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.


Faecalimonas umbilicata JCM 30896T, an acetate-producing bacterium isolated from human feces. The genomic analysis reveals genes for acetate and vitamin B12 synthesis and will facilitate the study of the role of this strain in the human gut.Here, we report the draft genome sequence of  Faecalimonas umbilicata JCM 30896T, an acetate-producing bacterium isolated from human feces. The genomic analysis reveals genes for acetate and vitamin B12 synthesis and will facilitate the study of the role of this strain in the human gut.Here, we report the draft genome sequence of  Faecalibacterium prausnitzii and Roseburia spp., can be net utilizers of acetate  supplemented with 5% (vol/vol) horse blood for 4 days at 37°C under a H2-CO2-N2  gas mixture. Total genomic DNA was extracted from F. umbilicata JCM 30896T using a Genomic-tip 100/G kit (Qiagen). Labiase  was used to lyse bacterial cells. A whole-genome shotgun library was constructed using a SMRTbell template prep kit 1.0 (Pacific Biosciences), followed by single-molecule real-time (SMRT) sequencing conducted on the PacBio RS II sequencing system (Pacific Biosciences) by Takara. A total of 97,745 reads (311-fold coverage) with an average length of 13,372 bp were assembled de novo using Hierarchical Genome Assembly Process version 3.0 (HGAP3.0) in SMRT Analysis version 2.3.0  , phosphotransacetylase (EC 2.3.1.8), and acetate kinase (EC 2.7.2.1). In addition, F. umbilicata JCM 30896T was predicted to possess a vitamin B12 biosynthesis pathway. Vitamin B12 is an important cofactor for a variety of enzymes. In the systematic genome assessment of B vitamin biosynthesis, vitamin B12 biosynthesis was present in 42% of the genomes of 256 common human gut bacteria  were predicted to synthesize vitamin B12 (12 was observed between the mucin-degrading bacterium Akkermansia muciniphila and the butyrate-producing bacterium Eubacterium hallii (12 and acetate by F. umbilicata might be beneficial for growth with other bacteria lacking the vitamin B12 biosynthesis pathway, such as A. muciniphila (F. prausnitzii.The genome of ase EC 2..2.1. In The genome sequence will facilitate further studies of the beneficial role of this strain in the human gut.BHEO00000000. The version described in this paper is the first version, BHEO01000000.This whole-genome shotgun project has been deposited in DDBJ/ENA/GenBank under the accession no.


An atypical porcine pestivirus (APPV) causing congenital tremor type A-II in piglets was identified in China in 2016. An increased number of cases of APPV have been reported in various countries all over the world since 2015. This study aimed to develop an effective subunit vaccine against APPV based on the E2 protein, which is the main immunogenicity protein of APPV. In this study, E2 protein was successfully expressed by the baculovirus expression system. E2 protein was confirmed by Western blot assay, which showed that E2 protein possesses N-linked glycosylation sites. The immunogenicity of E2 subunit vaccine was evaluated in mice. The E2 protein emulsified with ISA 201VG adjuvant induced significantly higher levels of APPV-specific antibodies and elicited stronger lymphocyte proliferative responses and higher interleukin-10 secretion than those of the E2 protein emulsified with IMS 1313VG adjuvant. This observation indicates that the E2 subunit vaccine induces a Th2-type immune response. Our results showed that E2 protein can be developed as a safe and effective subunit vaccine for the control of APPV infection. Porcine congenital tremor (CT) is a disease of newborn piglets. It is characterized by serious pathological damage to the brain and spinal cord and manifests as tremors of the head and limbs ,2. KinslAPPV features a single-stranded positive-sense RNA genome of approximately 11 kb and a member of the Pestivirus genus . APPV coCT type A-II outbreaks have been reported worldwide ,12,13,14In the present study, a recombinant baculovirus expressing APPV E2 protein was constructed to evaluate the immune responses of the E2 protein of APPV in mice. The E2 protein emulsified with ISA 201VG adjuvant induced significant humoral and cellular immune responses. Hence, the E2 subunit vaccine is a promising candidate vaccine against APPV.Plasmid pEASY-BLUNT-APPV E2 (GenBank No.KY652092.1) containing the honeybee melittin signal peptide at the N terminus and 6 × His tag at the C terminus without endogenous transmembrane domain was constructed by overlap polymerase chain reaction and maintained in our laboratory. DNA fragments, as templates, were amplified and subcloned into the pFBD vector [® CRL-1711™) were seeded at a concentration of 1 × 106 cells/well into 6-well tissue culture plates and were infected with the recombinant baculovirus (Ac-3-APPV-E2) at a multiplicity of infection (MOI) of 1. After cultivation at 27 °C for 60 h, the supernatant and cell lysate were collected and analyzed by Western blot. The samples were separated using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. The membrane was blocked with 5% skim milk in phosphate buffer saline with Tween (PBST)  and incubated with His monoclonal antibodies and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG . After washing with PBST, protein bands were detected using the enhanced chemiluminescence system and analyzed using the Image Lab software 4.0.1. At the same time, the samples were digested with peptide-N-glycosidase F (PNGase F) , which was used to detect whether the E2 protein was modified by N-glycosylation. Then, the treated samples were analyzed by Western blot.The Sf9 cells  in accordance with the manufacturer’s instructions. Then, the purified E2 protein was confirmed by 12% SDS-PAGE and quantified using a BCA protein assay kit.w/o/w) or IMS 1313VG adjuvant  at a ratio of 1:1 (w/w) in accordance with the manufacturer’s instructions. Female BALB/c SPF mice aged 6-weeks old were purchased from Huazhong Agricultural University Experimental Animal Center and randomly divided into three groups of eight mice. Group A, the negative control group, was immunized with 200 µL PBS. Group B was intramuscularly vaccinated with 200 µL of the solution containing 40 µg of APPV E2 protein emulsified with ISA 201VG adjuvant. Group C was intramuscularly vaccinated with 200 µL of the solution containing 40 µg of APPV E2 protein emulsified with IMS 1313VG adjuvant. All mice were injected with the same dose of vaccine as booster immunization at 14 days post-primary immunization (dpi). Serum samples were collected at 0, 14, 28, and 42 dpi and stored at −20 °C until use.Animal experiments were performed in accordance with the protocols approved by the animal ethical and welfare committee of Huazhong Agricultural University . The purified APPV E2 protein was emulsified with ISA 201VG adjuvant  at a ratio of 1:1 (Anti-E2 IgG and IgG isotyping in the serum samples was performed by indirect enzyme-linked immunosorbent assay (ELISA). Ninety-six-well flat-bottomed plates were coated with purified E2 protein (100 ng/well) diluted in 0.1 M carbonate/bicarbonate buffer (pH 9.6). The plate was washed thrice and blocked with PBST  at 37 °C for 1 h. After washing with PBST thrice, the serum samples diluted with PBST (1:400) were added to the wells and incubated at 37 °C for 1 h. The plate was washed again and incubated with HRP-conjugated goat anti-mouse IgG , IgG1 , IgG2a (1:5000), and IgG3 (1:5000) . Finally, total IgG and IgG isotyping titers were determined at 630 nm.6 cells/mL) were seeded into a 96-well plate with 100 µL Roswell Park Memorial Institute (RPMI) 1640 containing 10% fetal bovine serum (FBS) and stimulated with concanavalin A (10 µg/mL), purified E2 protein (10 µg/mL), and 100 µL RPMI 1640 containing 10% FBS. After culturing at 37 °C for 72 h, 20 µL MTS (5 mg/mL in PBS) was added to every well, and culturing was resumed at 37 °C for 4 h. The stimulation index (SI) was calculated in accordance with the formula: SI =  / .To evaluate T lymphocyte proliferation, the splenic lymphocytes were isolated from immunized mice using the lymphocyte isolation reagent . Lymphocytes -γ, interleukin (IL)-2, interleukin (IL)-4, and interleukin (IL)-10 ELISA kit in accordance with the manufacturer’s protocol. The concentrations of different cytokines were determined by the standard curve.Splenic lymphocytes were isolated and resuspended in RPMI 1640 medium supplemented with 10% FBS. Cell densities were adjusted to 4 × 10p < 0.05 was considered statistically significant.Statistical analysis was performed by one-way ANOVA through GraphPad Prism software . The supernatant and cell lysate were derived from sf9 cells infected with Ac-3-APPV-E2 and analyzed by Western blot. A single protein band with a molecular mass of approximately 36 kDa A can be To obtain a purified APPV E2 glycoprotein, we infected High Five cells with Ac-3-APPV-E2. At 120 h after infection, the extant E2 glycoprotein in the supernatant was harvested by centrifugation. Then, the APPV E2 protein was purified by Ni-NTA chromatography. On the basis of the UV absorption peak value of the AKTA protein purification apparatus, recombinant E2 protein was eluted with five column volumes of 80 mM imidazole .As shown in Subtype IgG levels  were measured by indirect ELISA to analyze the immune response profiles at 0, 14, 28, and 42 dpi. As shown in p < 0.05). However, no significant difference in SI was observed between E2-IMS 1313VG group and E2-ISA 201VG group (p > 0.05). These results indicate that immunization of APPV E2 protein in mice can strengthen proliferative response and T cell activation.To evaluate T lymphocyte proliferation, we detected APPV E2-specific lymphocyte proliferative responses at 42 dpi. As shown in p > 0.05). The concentrations of IL-4 and -10 (induced by the Th2 cellular response) in Groups B and C were significantly higher than those of the negative control group (p < 0.05). These results indicate that immunizing E2 protein can induce Th2-dominant cellular immune responses.To characterize cellular immune response in the mice immunized with APPV E2 protein, we used a commercially available mouse IFN-γ, IL-2, IL-4, and IL-10 ELISA kit to determine the concentrations of Th1- and Th2-type cytokines in the medium supernatant of splenic lymphocytes stimulated with E2 protein. As shown in ol group C,D. SpecNumerous CT cases have widely emerged in pig industries worldwide ,12,13,14In our previous research, we demonstrated that the CSFV E2 subunit vaccine developed using baculovirus confers complete protection against CSFV . On the To obtain a secreted APPV E2 protein, we used an E2 gene containing the honeybee melittin signal peptide at the N terminus. Western blot analysis suggested that the expression product can be secreted into the culture supernatant. Then, the E2 protein was secreted in culture supernatant purified by the AKTA protein purification apparatus . The recombinant E2 protein was immunogenic in mice. Results of animal experiments showed that the APPV E2 protein emulsified with adjuvant can induce high levels of specific IgG antibody in mouse. The E2-ISA201VG subunit vaccine produced significantly higher antibody titers than the E2-IMS1313VG subunit vaccine. The high levels of the IgG1 antibody were induced by the E2-ISA201VG and E2-IMS1313VG subunit vaccines. The levels of IgG1 antibody were significantly higher than those of IgG2a and IgG3. The IgG1 antibody is a representative of Th2-type immune response. These results suggest that the APPV E2 subunit vaccine induces Th2 type-dominant immune response in mice.Cellular immune response can play an important role in protecting against viral disease. The APPV E2-specific T lymphocyte proliferative response was detected in Groups B and C. The E2-ISA201VG subunit vaccine and E2-IMS1313VG subunit vaccine can yield a high SI value. The E2-ISA201VG and E2-IMS1313VG subunit vaccines can induce high levels of production of IFN-γ, IL-2, IL-4, and IL-10, especially production of IL-10, which is markedly higher than those of other cytokines. Results indicate that the Th2 type-dominant cellular immune response was induced in mice. This result is consistent with the subtype IgG results.In conclusion, the recombinant baculovirus Ac-3-APPV-E2 was successfully constructed. Our findings demonstrate that the APPV E2 protein is a glycoprotein. The APPV E2 glycoprotein can induce a robust humoral immune response and cellular immune response in mice and Th2-type immune response. Our results show that the APPV E2 glycoprotein can be developed as a safe and effective subunit vaccine for the control of APPV infection. Further studies will be conducted to evaluate the immunogenicity of the APPV E2 subunit vaccine in pigs.


NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2. DNA damage encountered by the replication fork causes fork stalling and is a major source of mutations when not adequately repaired. Fork stalling can lead to fork collapse, that is, a state of the fork in which normal DNA synthesis cannot be resumed at the site of stalling. Collapsed forks must be rescued by replication forks initiated nearby, but little is known about the rescue mechanism by which an active fork merges with a collapsed fork. We used an inhibitor of DNA replication to generate collapsed replication forks and then studied genetic control of collapsed-fork rescue. We found that NSMCE2, which is a gene product that is known to regulate repair responses to replication stress, is required for cells to effectively rescue collapsed replication forks in order to complete DNA synthesis. DNA double strand breaks that are associated with normal collapsed-fork rescue do not accumulate in cells that are deficient for NSMCE2, suggesting that DNA breakage is part of the rescue and repair mechanism. Failure to rescue collapsed forks leads to DNA damage in mitosis and DNA damage in the following cell cycle. Our work highlights a unique role for NSMCE2 in rescue of collapsed replication forks. Replication-associated DNA damage is common in human cells and can lead to the development of somatic mutations. DNA damage during replication can be induced by DNA lesion-producing chemicals, proteins bound to the DNA, DNA polymerase inhibitors, or nucleotide limitation . HydroxyThe DNA helicase mutated in Bloom’s syndrome BLM possesses multiple functions in DNA replication fork stabilization and homologous recombination (HR), which is a mechanism that operates in the repair of replication-associated DSBs . RecruitNSMCE2 homolog MMS21 is lethal; however, sumoylation-deficient hypomorphs are viable and have defects in replication-specific DNA repair [The E3 SUMO ligase NSMCE2 is a component of the SMC5/6 complex, which is present at stalled replication forks and a key component of the stalled fork proteome . In buddA repair . These cA repair , 19. DurA repair , 21. Oncmms21 mutant cells, we hypothesized that NSMCE2 may be a critical regulator of RAD51 function at collapsed replication forks.In human cells, forks adopt a RAD51-dependent structure during stalling, which resembles a Holliday junction . RAD51 iHere we studied the role of NSMCE2 in repair and rescue of collapsed replication forks. We found that NSMCE2 is essential for formation of DSBs during collapsed-fork rescue. Interestingly, lack of DSBs during collapsed-fork rescue is associated with hyper-accumulation of RAD51 and impaired sister chromatid recombination. Defects in the rescue of collapsed replication forks in NSMCE2-deficient cells lead to DNA damage in mitosis.NSMCE2-/- clones of HEK293T cells in which we targeted a single site in exon 2 of NSMCE2 and isolated two clones with different frameshift mutations containing no detectable NSMCE2 protein  after prolonged fork stalling by 24-hour treatment with HU. The SCE assay measures crossovers generated after resumption of DNA synthesis that can be detected in the subsequent mitosis. NSMCE2-deficient cells had a 45% reduction in the number of HU-induced SCEs/metaphase compared to control cells . Thus, tBecause HU-induced SCEs were suppressed in NSMCE2-deficient cells, we hypothesized that the excess RAD51 leads to a defect in DSB formation during rescue of collapsed forks. To test this possibility, we measured DSB accumulation in control and NSMCE2-deficient cells after prolonged exposure to HU. In HeLa cells exposed to control siRNA, a 16-hour treatment with HU did not induce DSBs; however, a 48-hour treatment with HU led to an accumulation of DSBs detectable by pulsed-field gel electrophoresis (PFGE) . InteresNSMCE2 . We founNSMCE2-deficient cells maintained normal cell-cycle progression in the absence of HU treatment ; howeverTo investigate further the nature of the mitotic damage invoked in HU-treated, NSMCE2-deficient cells, we measured the frequency of ultra-fine bridge (UFB) formation in cells undergoing mitosis. In order to obtain a sufficient number of cells in anaphase, cells were pretreated or not with HU for 24 hours; they were then blocked in G2 with the CDK1 inhibitor RO-3306 at 7.5 μM for 15 hours and then finally released into metaphase for 1 hour prior to fixation . Flow cyWe then tested whether mitotic DNA damage originated from collapsed forks in the previous S phase. As a marker for damaged DNA and repair in anaphase cells, we counted the number of FANCD2 foci and found a 1.8 fold increase in NSMCE2-deficient cells treated with HU compared to control cells . In ordeIn order to measure DNA damage in metaphase cells, we measured the levels of γ-H2AX associated with metaphase chromosomes and the levels of chromosome aberrations detectable at metaphase. For the analysis of γ-H2AX levels, normal HEK293T and NSMCE2 null cells were treated with 2 mM HU for 24 hours, released into medium with RO-3306 to block them in G2, then released into normal medium and prepared for analysis of γ-H2AX levels by immunofluorescence . HU-treaNSMCE2 is epistatic to BLM during rescue of collapsed forks. In these experiments, we used siRNAs to deplete BLM in NSMCE2 null and control cells . We therefore cannot rule out the possibility that NSMCE2 plays other roles during the unperturbed cell cycle or in situations where template switching can bypass DNA damage during replication, such as in cells treated with methyl methanesulfonate or UV irradiation. We observed higher levels of DSBs in untreated NSMCE2 null cells , but obsThe importance of mechanisms that regulate RAD51 protein levels is underscored by studies that have identified increased RAD51 protein levels as a negative predictor of patient outcome in several cancer types . The preAntibodies for immunofluorescence and western blots were obtained as follows: anti-RPA2 , anti-RAD51 , anti-NSMCE2 , anti-BLM [siNSMCE2-2: 5'-rUrUrArCrArUrArArUrGrGrUrUrUrArGrUrUrGrCrCrGrArUrCrCrA-3'5'-rGrArUrCrGrGrCrArArCrUrArArArCrCrArUrUrArUrGrUdAdA-3'siNSMCE2-6: 5'-rUrArUrArUrUrCrArCrUrArCrUrCrArCrUrUrCrArGrUrCrUrGrArC-3'5'-rCrArGrArCrUrGrArArGrUrGrArGrUrArGrUrGrArArUrAdTdA-3'siBLM: 5’-rArUrUrCrUrUrGrArGrArGrCrArGrUrArUrCrCrCrGrGrGrArUrU-3’5’-rUrCrCrCrGrGrGrArUrArCrUrGrCrUrCrUrCrArArGrAdAdAdT3’siRAD51: 5’-rGrArGrCrUrUrGrArCrArArArCrUrArCrUrU-3’5’-rCrArCrCrUrUrGrArArGrUrArGrUrUrUrGrU-3’Genome editing was carried out with Integrated DNA Technologies (IDT) ALT-R CRISPR-Cas9 system with crRNA guide (GTCCATACCAGAGTTGATAC) targeting the first coding exon. Ribonucleoprotein particles were introduced into HEK293T cells using FuGENE HD (Promega). After 48 hours, single cells were deposited in a 96-well plate by flow cytometry. After four to six weeks, clones were analyzed by the T7 endonuclease assay (New England Biolabs), and clones that scored positively were PCR sequenced. >50 clones were analyzed in the first screen and a single heterozygous NSMCE2 null mutant was obtained. This clone was genome edited again to obtain two NSMCE2 null mutants.Cells were lysed in RIPA buffer  supplemented with 5 mM EDTA, 1 mM EGTA, 25 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethane sulfonyl fluoride (PMSF) in 1x EDTA-free Halt protease inhibitor (Thermo Scientific). Protein concentration was measured using Pierce BCA Protein Assay. 30–50 μg of total protein from cell lysates was separated by electrophoresis through 4–20% gradient polyacrylamide gels and transferred onto Hybond nitrocellulose membranes by semi-dry transfer. Before addition of primary antibodies, membranes were probed with Ponceau S (Sigma) for 7 min, imaged, and washed with 1% glacial acetic acid in water. Membranes were blocked for 1 hour in Tris-buffered saline with 0.1% polysorbate 20 (TBST) containing 5% Bio-Rad Blotting-Grade Blocker, then incubated with primary antibody in 3% BSA in TBST overnight at 4°C.U2OS cells that were stably transfected with a His-tagged SUMO2 were kindly provided by Dr. Michael Matunis at Johns Hopkins University, who obtained them from Dr. Mary Dasso’s lab at NIH. The cells were reverse-transfected with siRNAs using LifeTechnologies’ Lipofectamine RNAiMAX. Cells were treated with 2 mM HU for 16 hours. His-SUMO2 conjugates were purified as described by Tatham et al. . Briefly7 cells per 200 μl of Buffer A . Triton X-100 was added to a concentration of 0.05%, and the cells were incubated for 5 min on ice. Nuclei were collected by low-speed centrifugation  and the supernatant was reserved as the cytoplasmic fraction. The nuclei were washed once in Buffer A, and then lysed in 100 μl Buffer B . Nucleoplasmic proteins were separated from chromatin-bound proteins by centrifugation . Nucleoplasmic fractions were collected in the supernatant. The chromatin pellet was resuspended in 250 μl Laemmli buffer and the material was sonicated. The cytoplasmic and nucleoplasmic fractions were clarified by high-speed centrifugation . The proteins in the fractions were analyzed by Western blot. Initially, cytoplasmic and nucleoplasmic fractions were analyzed separately; however, because the cytoplasmic fraction contained varying amounts of different RPA components, for comparisons of the amounts of RPA70 we combined equal parts of the cytoplasmic and nucleoplasmic fractions.The protocol from Mendez and Stillman for chromatin isolation by small-scale fractionation was followed . HEK293T2) and nucleoplasm was extracted for 90 seconds with cold extraction buffer  prior to 30-min fixation in 4% formaldehyde at room temperature. Cells were washed twice with cold PBS then treated with 0.5% Triton X-100 at room temperature before staining. Cells were blocked at room temperature for 1 hour using sterile filtered 3% BSA in PBS, then probed using primary antibodies for 1 hour at room temperature. Secondary antibodies (Alexa Fluor 488 and 546) were used at 1:1000 for 45 min and nuclei were stained using Molecular Probes NucBlue reagent (R37606). For 5-ethynyl-2´-deoxyuridine (EdU) labeling, cells were incubated in 10 μM EdU for 20 min. EdU labeling and detection was performed using Life technologies Click-iT EdU Alexa Fluor 647 imaging kit according to manufacturer’s instructions. Cells were mounted in Molecular Probes ProLong Gold Antifade Reagent. Fixed and stained cells were imaged using the Leica SP5-II spectral confocal microscope using the 63x/1.4 NA PL Apo objective. Using the nuclear signal to mask the region of interest enabled accurate measurement of the number and intensity of nuclear foci and the percent of nucleus occupied by signal for each antibody target. No less than 10,000 foci were analyzed per experimental group. Box and whisker plots were used to visualize the distribution of foci. For analysis of EdU labeled forks and ultra-fine bridges (UFBs), z-stacks were created using 100x objective and deconvolved using the GE DeltaVision Elite High Resolution Microscope. Images were analyzed and 3D representations were created using the NIS Elements software.The protocol was adapted from Dimitrova and Gilbert . Cells wCells were harvested with trypsin/EDTA, resuspended in ice cold PBS, and fixed and stained using BioLegend True-Nuclear Transcription Factor Buffer. Cells were then stained for γ-H2AX using directly conjugated antibody and counterstained using 7AAD to monitor cell DNA content. For cell-cycle assays, analysis was performed using the BD Pharmingen FITC BrdU Flow Kit. A minimum of 30,000 events was recorded for each group using the BD FACSCanto II or BD LSR II flow cytometer. Apoptosis analysis was carried out using the BioLegend FITC Annexin V Apoptosis Detection Kit with propidium iodide (PI).For SCE analyses, cells were cultured with 10 μM BrdU (Sigma-Aldrich). After 60 hours, the cells were incubated with 0.02 μg per ml colcemid (Invitrogen) for up to 2 hours, harvested and processed as described earlier . For theFor analysis of micronuclei, normal or NSCME2-depleted HeLa cells were seeded onto coverslips, treated or not with 2 mM HU for 24 hours, and fixed with 4% formaldehyde. The cells were stained with Nuc-Blue (Thermo Fisher) at 2 drops per ml in PBS for 30 min, washed briefly, mounted, and imaged using the GE DeltaVision Elite High Resolution Micro.For analysis of chromosomes at metaphase, normal and NSMCE2 null HEK293T cells were seeded into 60 mm dishes and incubated overnight. For analysis of γ-H2AX labeled chromosomes, cells were treated with 2 mM HU for 24 hours, then washed and incubated in medium with 7.5 μM RO-3306 for 10 hours for HEK293T control or 20 hours for NSMCE2 null cells. Cells were then released into normal medium and harvested at the indicated time points. Cells were fixed with 4% formaldehyde and stained with anti-phospho-histone H3 (serine 10). For analysis of chromosome spreads, cells were treated with 0.02 μg per ml colcemid for 45 min or with 0.6 μg per ml colcemid for 16 hours prior to harvest. Cells from both experiments were harvested and metaphases prepared as described . Metapha5 cells were prepared in disposable plug molds (Bio-Rad Laboratories). In some experiments, cells were released into medium containing 10 μM aphidicolin for 24 hours. Plugs were then incubated in lysis buffer  at 37°C for 16 hours. Plugs were then washed four times in 20 mM Tris-HCl, pH 8.0, and 50 mM EDTA before loading onto an agarose gel. Electrophoresis was performed for 21 hours at 14°C in 0.9% (wt/vol) agarose containing Tris-borate/EDTA buffer in a PFGE apparatus , according to the following protocol: block I: 9 hours, 120° included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 hours, 117° included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 hours, 112° included angle, 4.0 V/cm, 9 to 5-s switch. The gel was then stained with SYBR Gold  and analyzed by the AlphaImager system (ProteinSimple). Relative DSB levels were assessed by comparing DSB signals for each treatment to the background levels observed in untreated conditions using Image J. Data were analyzed with GraphPad Prism software.The procedure was performed as previously described , 14. Sub1 anti-BrdU/IdU , rat anti-BrdU/CldU , and mouse IgG2a anti-ssDNA . Secondary antibodies included Alexa Fluor 488 anti-mouse IgG1, Alexa Fluor 594 anti-rat, and Alexa Fluor 647 anti-mouse IgG2a, respectively (Life Technologies). Images were acquired on Leica DMI6000 epifluorescence microscope using Leica LAS-AF software. Signals were measured using NIH ImageJ software with custom-made modifications and the data analyzed with GraphPad Prism software.maRTA was performed as previously described with some modifications . BrieflyStatistical evaluations of experiments with continuous variables  or discrete variables in which the data was normal were carried out by paired Students t-test. Evaluations of experiments with non-normal discrete variables  were analyzed by Mann-Whitney test. All p-values were two-sided.S1 FigNSCME2 that carried a 10-bp deletion in exon 2 of NSMCE2. (Lower panel) Analysis by PCR and sequencing showed that clone 9 and clone 15 each contain the 10-bp deletion and a second clone-specific frameshift mutation. The sequence of the PAM site is denoted in blue and the sequence of the guide RNA is denoted in red. (C) Analysis by hemocytometer-based cell counting of cell proliferation of normal, heterozygous, and null cell clones of NSMCE2. The cell counting experiments indicated that the rate of proliferation of NSMCE2+/+ cells is approximately 20 hours per division and of NSMCE2-/- cells 40 hours per division. (D) Flow cytometric analysis of the cell cycle. Cells were treated or not with 2 mM HU and then released (Rel) into normal medium for 6 hours. Cells were pulsed with 10 μM EdU prior to harvest and processing for flow cytometry. The NSCME2 null cells exhibit a mild G1 delay. Normal cells were pulsed with EdU for 20 min and NSMCE2 null cells were pulsed for 40 min to account for the slower cell cycle.(A) 80% reduction of the levels of NSMCE2 after depletion with siRNA in HeLa cells as measured by Western blot and qPCR analysis. (B-D) Analysis of the construction of NSMCE2 null cells in the HEK293T cell line. (B) (Upper panel) Western blot analysis of normal, heterozygous, and two cell clones (clone 9 and clone 15) that are null for NSMCE2. HSC70 was used as a loading control. The homozygous null cell clones were both derived from the heterozygous mutant of (TIF)Click here for additional data file.S2 Fig(A) Representative Western blots of HeLa cells transfected with control or two different siRNAs against NSMCE2 and treated or not with 2 mM HU for 24 hours. Multiple loading controls (HSP90) are shown for separate gel runs and Westerns of the same cell lysate. (B) Western blot analysis of SMC5. For SMC5 experiments, β-actin was used as a loading control.(TIF)Click here for additional data file.S3 Fig(A) Complementation of accumulation of BLM foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and were treated with 2 mM HU for 24 hours. Box and whisker plots represent distributions of the number of BLM foci per cell. The median values are shown in boxes. At least 10,000 BLM foci were analyzed in each experimental condition. Three independent experiments were performed. (B) A representative image of the colocalization of RPA (red) and RAD51 (green) in HeLa cells exposed to 2 mM HU for 24 hours prior to fixation (upper panel). Quantitation of the area of RAD51 foci (lower panel). Mean and standard error are shown. At least 10,000 RAD51 foci were analyzed in each experimental condition. Three independent experiments were performed. (C) Colocalization of RAD51 and EdU in HU-treated cells. Representative images of control and NSMCE2-depleted HeLa cells exposed to 2 mM HU for 24 hours. EdU was incorporated for 12 min prior to HU treatment. After HU, cells were fixed and stained with RAD51. Images show the merge of EdU (green) and RAD51 (red) channels. (D) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient U2OS cells. Box and whiskers plot represent distributions of the number of RPA foci in cells exposed to control or NSMCE2 siRNA and treated or not with 2 mM HU for 24 hours. The median values are shown in boxes. Three independent experiments were performed. (E) Complementation of accumulation of RPA foci by transfection of siRNA-resistant NSMCE2 cDNA construct. HeLa cells were exposed to control or NSMCE2 siRNAs and treated with 2 mM HU for 24 hours. Box and whiskers plot represent the distributions of the number of RPA foci per cell. The median values are shown in boxes. Three independent experiments were performed. (F) Reduced accumulation of chromatin-bound RPA in HU-treated NSMCE2 null cells compared to HU-treated normal HEK293T cells. Western blot analysis of levels of chromatin-bound RPA (RPA p70 subunit). Cells were treated or not with 2 mM HU for 16 hours. The M fraction contains equal parts of the cytoplasmic and nucleoplasmic fractions. The C fraction contains the chromatin-bound material. The red carets point to the HU-induced chromatin-bound RPA. Four independent experiments were performed. (G) Quantitation of the experiment shown in F. (H) Reduced levels of ssDNA in HU-treated NSMCE2-deficient cells. Quantitation of immunofluorescence analysis of BrdU to measure exposed ssDNA in non-denaturing conditions (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours. The bar represents median values of the numbers of BrdU foci and the error bar represent the SEM values from three independent experiments. Representative images of BrdU foci are shown (right panel). (I) Similar levels of SCEs in normal HEK293T cells and NSMCE2 null cells. Box and whiskers plots represent the numbers of SCEs per metaphase. A minimum of 14 metaphases were scored in two independent experiments. (J) Reduced levels of γ-H2AX in HU-treated, NSMCE2-deficient cells. Flow cytometric analysis of γ-H2AX response in HeLa cells. Mean and standard deviation is shown. To the right of the bar graph are representative histograms showing γ-H2AX induction. Shaded histograms represent the treated cell populations. Three independent experiments were performed. (K) Complementation of accumulation of γ-H2AX foci by transfection of siRNA-resistant NSMCE2 cDNA construct. Quantitative analysis of γ-H2AX foci (upper panel). Box and whisker plots represent distributions of the number of γ-H2AX foci per cell. The median values are shown in boxes. At least 10,000 γ-H2AX foci were analyzed in each experimental condition. Below the bar graph are representative immunofluorescence images. Three independent experiments were performed.(TIF)Click here for additional data file.S4 Fig(A) BLM is retained in PML nuclear bodies in NSMCE2-deficient cells and the numbers of BLM foci induced by HU are reduced in NSMCE2-deficient cells. Merged images of untreated or HU-treated, control- and NSMCE2-depleted HeLa cells stained with antibodies to BLM (red) and PML (green) and counter stained with DAPI. Depletion of NSMCE2 is associated with increased numbers of PML nuclear bodies. In HU-treated, control-depleted cells, BLM moves to stalled replication forks. In HU-treated NSMCE2-depleted cells, BLM remains associated with PML. (B) Reduced accumulation of RPA foci in HU-treated, NSMCE2-deficient cells. Images of HeLa cells stained with phalloidin, DAPI, anti-RPA p32 antibodies. Actin staining also revealed striking morphological changes in NSMCE2-deficient HeLa cells. (C) Over-accumulation of RAD51 foci in HU-treated, NSMCE2-deficient cells. Images of HeLa cells stained with DAPI and anti-RAD51 antibodies. (D) Reduced accumulation of BrdU foci in HU-treated, NSMCE2-deficient cells. Images are of HeLa cells stained with DAPI and anti-BrdU antibodies. Cells were incubated with 10 μM BrdU for 48 hours, treated with HU for 24 hours, then processed for immunofluorescence. (E) Excess accumulation of 53BP1 foci in HU-treated NSMCE2-deficient cells. Images of HeLa cells treated with HU for 24 hours then released for 24 hours and stained with DAPI and anti-53BP1 antibodies.(TIF)Click here for additional data file.S5 Fig(A) DSBs that accumulate after release from HU block are replication-dependent. HeLa cells were treated or not with 2 mM HU or 10 μM aphidicolin for 24 hours. The HU-treated cells were released into normal medium or medium that contained 10 μM aphidicolin for an additional 24 hours. The cells were analyzed by PFGE. Means and SD are shown. Three independent experiments were performed. (B) Similar cell-cycle distributions in HeLa cells transfected with control or NSMCE2 siRNA after release from HU block. Flow cytometric analysis of the cell cycle after HU treatment and release. 24 hours after transfection, cells were treated or not with 2 mM HU for 24 hours prior to release into normal medium for 40 min, 6 hours, or 12 hours. The cells were then pulsed with 20 μM EdU for 20 min prior to harvest and staining with click reagents. A minimum of 10,000 events were recorded in each experimental condition. (C) No induction of DSBs in NSMCE2 null cells treated with HU. Quantitation of PFGE analysis of HEK293T cells treated or not with 2 mM HU for 24 hours prior to release into normal medium for 12 hours (upper panel). The bar graph shows the mean fold change values, normalized to the untreated normal HEK293T mean, and SEM values from three independent experiments. A representative gel image of one experiment is shown below the graph (lower panel). (D) Similar levels of apoptosis in HeLa cells exposed to control and NSMCE2 siRNAs after treatment with 2 mM HU for 24 hours and release into normal medium for 24 hours. Analysis of apoptosis staining with propidium iodide and antibodies against AnnexinV. A minimum of 10,000 events were recorded in each experimental condition. (E) Reduced levels of γ-H2AX in HU-treated NSMCE2 null cells. Graph showing fold change in median levels of HU-induced γ-H2AX in normal HEK293T cells, the heterozygous NSMCE2+/- cells, and the two NSMCE2-/- clones 9 and 15. Data were normalized to HU-treated normal HEK293T cells. Two independent experiments were performed.(TIF)Click here for additional data file.S6 Fig(A) Persistence of RAD51 at collapsed replication forks in NSMCE2 null cells. Immunofluorescence analysis of HEK293T cells treated or not 2 mM HU for 24 hours prior to release for 2 hours, 4 hours, or 6 hours before staining, imaging, and quantitation. Each point on the graph represents the median value and the error bar represent SEM values from randomly binned averages of 10 cells from at least 50 cells in each experimental condition. Two independent experiments were performed. (B) Representative images of data shown in (A) showing co-localization of γ-H2AX, RPA, and RAD51 in HEK293T cells after 24 hours treatment with HU and release into normal medium for 2 hours.(TIF)Click here for additional data file.S7 Fig(A) Quantitative analysis of G1 arrest after release of control and NSMCE2-depleted HeLa cells from HU block into normal medium for 24 hours. (B) Quantitative analysis of abnormal anaphases encountered after release from HU block (left panel). HeLa cells were exposed to control or NSMCE2 siRNAs and treated or not with 2 mM HU for 24 hours, then released into fresh media for 16 hours before fixation and staining. The graph plots percent values of normal mitoses (blue), mitoses with anaphase bridges (red), and mitoses with lagging chromosomes (green). The value shown above the graph is the percent of abnormal mitosis scored. Representative images of normal mitosis (B’), mitosis with an anaphase bridge (red caret in B”), and mitosis with a lagging chromosome (red caret in B”‘) are shown (right panel). At least 100 anaphase cells were analyzed in each experimental condition. Three independent experiments were performed. (C) Quantitative analysis of micronuclei formation after release from HU block (left panel). Experiment was performed three times and analyzed by chi square test. A representative image is shown to the right of the graph. Scale bars represent 10 microns. (D) Quantitation of 53BP1 nuclear bodies/cell using bins of 1, 2, and 3 or more bodies/cell. HeLa cells were exposed to control or NSMCE2 siRNAs and treated with 2 mM HU for 24 hours, then released into fresh media for 24 hours before fixation and staining. Each bar represents the mean percent of total cells observed in each class and the error bars represent the SEM values. Flow cytometric analysis showed that the majority of cells are in G1 (see panel A). Cells with large nuclei indicative of being in the G2 phase were excluded from the quantitation. Three independent experiments were performed. (E) Quantitative analysis of area of the nucleus inhabited by 53BP1 signal after release from HU block based on the same data in panel D. Box and whisker plot represents area distribution per cell in at least 250 cells in each experimental condition. (F) Representative images of cells stained for 53BP1 analyzed in panels D and E. (G) Flow cytometric analysis of the effect of the treatment schedule to enrich for mitotic cells used in the experiments shown in (TIF)Click here for additional data file.S8 Fig(A) Quantitation of median fluorescence intensity of γ-H2AX on phospho-histone H3-positive chromosomes. Cells were treated with 2 mM HU for 24 hours, released into medium containing 7.5 μM RO-3306 for 10 hours  or 20 hours (NSMCE2 null) to block cells at the G2/M boundary, and then released into normal medium and harvested at the indicated times for analysis of metaphase chromosomes. Regions of interest were drawn using Image J based on the DAPI signal in phospho-histone H3-positive (serine 10) cells and γ-H2AX signal was quantified. Two independent experiments were performed. (B) Representative images of metaphase chromosomes stained with γ-H2AX, anti-phospho-histone H3 and DAPI.(TIF)Click here for additional data file.S9 Fig(A) Representative images of metaphases prepared from normal HEK293T and NSMCE null cells. (B) Quantitation of chromosome aberrations identified in untreated and HU-treated normal HEK293T and NSMCE2 null cells. Representative chromosome images are shown below the grid of counts of chromosome aberrations. Total indicates the number of chromosomes scored. 1, chromatid break. 2, chromosome break. 3, chromosome gap. 4, telomere fusion. 5, tri-radial. 6, quadriradial. 7, secondary constriction. Approximately 25 metaphases were analyzed from each of two experiments.(TIF)Click here for additional data file.S10 FigAnalysis of HU-induced phenotypes in normal HEK293T and NSMCE2 null cells in which RAD51 levels were reduced or not by siRNA-mediated depletion. (A) Analysis by flow cytometry of the fluorescence intensity of γ-H2AX after treatment with HU for 24 hours followed by release into normal medium for 6 hours (Rel). The error bars represent the SD of median fluorescence intensity from a minimum of 10,000 events in five independent experiments. (B) Analysis by PFGE of DSBs after treatment with HU for 24 hours followed by release into medium for 6 hours (Rel). The bar graph represents the mean fold change in DSBs detected by PFGE normalized to untreated normal HEK293T cells exposed to control siRNA. The error bars represent the SD of three independent experiments. The inset gel shows the results from one experiment. (C) A representative histogram of the median fluorescence intensity of γ-H2AX from one of the experiments shown in A. (D) Western analysis of RAD51 levels from samples prepared for one of the PFGE experiments shown in (B).(TIF)Click here for additional data file.S1 DataThe tabs are labeled F1A for (XLSX)Click here for additional data file.