Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H + natural killer (NK) cells. The NK cell activation receptor Ly49H receptor targets certain genotypes of the viral protein, m157. During multi-strain infection, strains of MCMV encoding an m157 capable of binding Ly49H are excluded from the salivary gland and the saliva of C57BL/6 mice, allowing for the shedding of only non-Ly49H binding strains of MCMV in the saliva. This within-host competition could therefore have significant impacts on the circulation of MCMV strains, as only the most virulent MCMV strains were present in the saliva.Infection of the host with multiple strains of a pathogen is common and occurs with the herpesvirus, human cytomegalovirus (HCMV). However the effects of multi-strain infection on the host and the pathogen remain poorly studied. Here we show, in a mouse model, that infection of C57BL/6 mice with multiple strains of murine CMV (MCMV) results in profound within-host competition. Competition between the strains of MCMV is dependent on Ly49H It is becoming increasingly clear that many infections are caused by multiple distinct strains of the infecting pathogen. A recent review documented 51 infections of humans in which there is definitive evidence of such multi-strain infection HCMV, a member of the betaherpesvirus subfamily, is a large double stranded DNA virus with a worldwide prevalence of 55–100% depending on socioeconomic status and geographical location. HCMV infection is life-long but is generally asymptomatic in the immunocompetent host. However in the immunocompromised individual HCMV can cause significant morbidity and mortality. Despite the advent of antiretroviral therapy, AIDS patients remain at risk of HCMV induced retinitis, uveitis and vitritis Infection with multiple strains of HCMV was first reported in immunocompromised, HIV infected, individuals more than 25 years ago Multi-strain HCMV infection can also be found in normal healthy individuals Theoretical and empirical studies suggest individual strains of pathogen can participate in either positive (complementary) or negative (competitive) interactions when co-infecting the same host. These interactions can affect a range of medically relevant outcomes such as: strain selection post vaccination, the spread of drug resistance, genetic exchange and the evolution of virulence  Ly49H− strains of MCMV but resistant to m157Ly49H+ strains In order to investigate empirically the outcome of multi-strain CMV infection we have developed a novel mouse model using naturally circulating strains of MCMV. As with HCMV in humans, multi-strain MCMV infection of free-living mice is common + NK cells. Competition was also noted between K181 (m157Ly49H+) and C4C (m157Ly49H−), but not between K181Δm157 (m157Ly49Hnull) and C4C (m157Ly49H−), confirming a requirement for an m157 protein capable of binding Ly49H in the competition described here. Within-host competition was complete and prevented the shedding of m157Ly49H+ strains of MCMV into the saliva during co-infection with m157Ly49H− strains. Within-host competition was evident as early as day three p.i. suggesting that NK cells prevented the dissemination of m157Ly49H+ to the salivary glands. Therefore these studies demonstrate that NK cells are capable of mediating competition between strains of MCMV and have the potential to shape dissemination patterns of this virus.We noted no evidence of within-host complementation, with no enhancement of viral titers in mice receiving the mixed inoculum compared to mice receiving a single strain infection. In marked contrast profound, MCMV strain specific, within-host competition was observed in B6 mice. During single strain infection all four viral strains, C4A, C4B, C4C and C4D disseminated to and replicated within the salivary glands of mice. However, during multi-strain infection, both C4A and C4B were excluded from the salivary glands, while C4C and C4D replicated normally at this site. Functional studies demonstrated that the m157 protein produced by both C4A and C4B ligated Ly49H, whereas C4C and C4D m157 proteins failed to ligate this receptor. Subsequent studies using Ly49H congenic mice, Ly49H blocking antibodies and NK cell depletion studies demonstrated that within-host competition was dependent on Ly49HExperiments were performed with the approval of the Animal Ethics and Experimentation Committee at the University of Western Australia and in accordance with the guidelines of the National Health and Medical Council of Australia.rCmv1 and B6.BALB.TC1 , were bred at the Animal Services Facility, at the University of Western Australia. All mice were infected at seven to eight weeks of age and were maintained under specific pathogen free conditions prior to and during experimentation.Highly inbred, female BALB/cArc (BALB/c) and C57BL/6JArc (B6) mice were obtained from the Animal Resource Centre . The congenic mouse strains, BALB.B6-3 BALB/c derived MEF were added to each well of a flat bottom 96 well tray in 1×MEM supplemented with 2% NCS plus antibiotics and incubated overnight at 37°C in 5% CO2. MEF were infected with MCMV at a multiplicity of infection (MOI) of 0.5 by centrifugal enhancement. BWZ-HD12 reporter cells were added to infected MEFs 24–48 hours later when cytopathic effect in the infected MEF approached 100%. Infected MEF and reporter cells were incubated overnight at 37°C in 5% CO2, with the β-galactosidase production assessed eight hours after the addition of 0.15 M chlorophenol red-ß-D-galactopyranoside (CPRG). Chlorophenol red production was measured at a wavelength of 570 nm, with 630 nm used as a reference wavelength. Positive controls were 0.05 µg/ml PMA and 0.5 µg/ml ionomycin. K181 infected MEF were used as an additional positive control, and uninfected MEF served as a negative control.Primary mouse embryonic fibroblasts (MEF) were grown in minimal essential media  supplemented with 8% newborn calf serum  and antibiotics ). The BWZ-HD12 reporter cells  were grown in RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum , antibiotics, 2 mM glutamine, 1 mM pyruvate, 50 µM 2-mercaptoethanol and 200 µg/ml hygromycin. Reporter assays to measure the Ly49H binding capacity of individual MCMV strains were performed as previously described The capacity of the MCMV strains used in this study to replicate in MEF was assessed by multi-step growth curves analysis as previously described Mus musculus domesticus, collected from a location near (C)anberra, Australian Capital Territory, Australia and have been previously described Perth (hereafter referred to as K181) has been described previously in vitro on BALB/c MEF and were maintained as low passage stocks.The MCMV strains C4A, C4B, C4C, and C4D were isolated from a single free-living wild mouse, in vivo experiments were performed with secondary salivary gland derived virus stocks. These stocks were raised by two passages of virus in weanling (three week old) female, B6 mice for inoculation of B6 background mice, or in BALB/c mice for inoculation of BALB/c background mice. Control, single strain infected mice, were inoculated with 1×104 pfu of virus via the intraperitoneal (i.p.) route. Multi-strain infections were also given via the i.p. route and comprised a mixed inoculum of up to four different strains of MCMV, with each strain being an equal fraction of the total 1×104 pfu inoculum.All Δm157 was produced by recreating the 104 bp spontaneous loss-of-function mutation in m157 found upon serial in vivo passage of K181 through Ly49H+ congenic mice Δm157 was performed by ET recombination as described CGTCGTGGAATGCCTTCGAATTCTCTGGGACACACAAAGGATCTGCGTACGATATGCATGTCTGTTTTTGGGAA and delta m157 reverse ACAAGGACGACGACGACAAGTAACCCGAACGTTGCTTTTTATCATATAGGTACCAATTTCTTTTGCGTCGGAA. PCR primers contain sequences specific for the plasmid pFLRTKn (underlined) and sequence homologous to nt 216554–216603 and 216400–216449, respectively in K181 (accession number AM886412). The K181 bacterial artificial chromosome (BAC), pARK25, used for mutagenesis has been described m157 in K181Δm157 was confirmed by sequence analysis (data not shown). K181Δm157 replicated like wild type virus in vitro and failed to stimulate the Ly49H reporter cell line BWZ-HD12 (data not shown).K1812 to allow for plaque formation. Plaque assays were stopped after five days with addition of methylene blue stain containing 5% formaldehyde. After 24 hours the plates were washed, air-dried and plaques counted by light microscopy, all titers are expressed as plaque forming units (pfu) per gram of tissue.Viral titers in mice were determined by standard plaque assay 5′-ACAGGACGACCGAGTTCCCCG-3′) and reverse (5′-GTTGYACATCTAAGATCGAGAAACA-3′)  were used to amplify the MCMV gene, m144. Strain-specific oligonucleotide probes were: C4A (FAM-ATTCGAACCTGTTCACTGGCG-BHQ1), C4B (HEX-CTTAGCTTCTTTTGACAGTCACGGTC-BHQ1) (both obtained from GeneWorks), C4C (LC610-CAAAGCGGCGCAGCAACATAAC-BHQ1), C4D (LC670-TCCAAACCCCAAACCAAAACCACGC-BHQ1)  and K181 (FAM-CCTCAAACGTCAAAACAACACCACGA-BHQ1) (GeneWorks). Reaction mix comprised 10 µl Roche  2×Probes Master Mix and 500 nM primers. Probes were used at a concentration of 250 nM for C4A, K181, C4C and C4D and at 375 nM for C4B. A total of 100 ng genomic DNA was added to each reaction mix. Cycling conditions were as follows: 10 minutes 95°C, followed by 45 cycles each consisting of 30 seconds at 95°C and 2 minutes at 66°C, and a final cooling step to 50°C for 10 seconds. Concentrations of viral DNA were calculated relative to an internal standard control using the fit-points method on the LC480 software.Viral genomes in the tissues of infected mice were quantified by multiplex PCR. DNA was extracted from 10 mg of spleen, liver or salivary gland using the Wizard Genomic DNA kit according to the manufacturer specifications . Primers, forward  alone. Efficacy of NK cell depletion was assessed at day 18 post infection (p.i.) by flow cytometry of splenocytes using the pan NK cell marker CD49b . PK136 treatment resulted in a 38% reduction in NK cells relative to PBS treated mice. Ly49H blocking studies were performed with the antibody 3D10 (anti-Ly49H), which was provided by Professor Wayne Yokoyama (Washington University). Mice were injected with 200 µg of 3D10 on day −2 and day 0 and then every four days. On day 0 mice were infected as above with a mixed inoculum of C4A and C4C. Control mice co-infected with C4A and C4C were treated with isotype control antibody, 9E10 (anti-cMyc). All antibodies were supplied by WAIMR monoclonal antibody facility  as a protein G affinity purified product.To deplete NK cells, mice were treated at day −1, 0 and then every five days with 150 µg of the antibody PK136 (anti-NK1.1). On day 0, mice were co-infected with 0.5×102 punch was collected from air-dried, saliva impregnated paper. The paper punch was then washed in 100 mM Tris, 0.1% SDS for 30 minutes, then 5 M guanidine thiocyanate for 10 minutes, followed by three washes in sterile water for 10 minutes with a final 10 minute wash in 70% ethanol. Punches were air dried and then loaded directly into PCR wells for analysis by multiplex qPCR.Shedding of virus into the saliva was measured by collecting saliva onto FTA paper  One way analysis of variance (ANOVA) followed by Tukey's comparison was used to determine statistical significance between multiple groups. Unpaired two tailed T tests were used to compare between data from two groups. All statistical analyses were performed using GraphPad Prism 5.0  with the values are expressed as mean ± SEM.This study was designed to explore two potential consequences of multi-strain CMV infection. These were, inter-strain complementation leading to enhanced viral loads in the tissues of infected mice and within-host interactions, either complementary or competitive, that affected the replication capacity of individual strains. These outcomes were assessed by plaque assay and by measuring strain prevalence using strain specific quantitative PCR, respectively in B6 mice infected with up to four strains of MCMV. The strains used, C4A, C4B, C4C and C4D were all isolated from a single wild caught mouse and have been confirmed as distinct strains by full genome sequence analysis 4 pfu of C4A, C4B, C4C or C4D or with an equal mix of all four strains via the peritoneal route. Viral titers were assessed 3 days later in the spleen and the liver. Infectious virus was undetectable in the spleens of C4A inoculated mice 3 days after infection, while titers of virus in C4B infected mice were just above the limit of detection  strains of MCMV is due to the recognition of specific genotypes of the viral m157 glycoprotein by host NK cells via the activation receptor Ly49H Ly49H+ strains of MCMV replicate poorly in the spleen during acute infection Ly49H−) replicate well during acute infection but are more rapidly cleared from the salivary glands of B6 mice m157 gene sequences from the C4 strains of MCMV suggested a possible mechanism behind viral competition. The m157 genes of C4A and C4B co-segregated with genotypes of m157 known to ligate Ly49H. In contrast, the m157 genes of C4C and C4D co-segregated with m157 genes, shown by others Resistance of B6 mice to particular  monoclonal antibody prior to and during co-infection with C4A and C4C . Figure rCmv1 mice (Cmv1r) possesses the NKC from B6 mice on the BALB/c background + (B6 and Cmv1r) but not in Ly49H− (BALB/c and TC1) mice.To determine if Ly49H was required for within-host competition, multi-strain infection was assessed in mice congenic for the natural killer cell complex (NKC). BALB.B6-+ Cmv1r mice during single strain infection .Ly49H mediated competition should be mediated by its cognate ligand, virally encoded m157. The MCMV strain K181 expresses an m157 gene product known to ligate the product of the B6 allele of In vivo, titers of K181Δm157 in the salivary glands of B6 mice 18 days p.i. were higher than those of parental K181 infected B6 mice and similar to those observed in C4C infected mice (Δm157 (n = 10) lost significantly more weight than those infected with K181 (n = 10) (p<0.001). Values are percentage weight loss from day 0 to day 3 p.i. and are mean ± SD for: C4C 10.1±1.9, K181Δm157 12.5±1.9 and K181 (weight gain) +0.6±4.0.ted mice . Smith sΔm157. In B6 mice infected with C4C and K181, only C4C DNA was detected in the salivary gland 18 days p.i.  from day 12 p.i, and viral DNA quantified by qPCR (− (TC1 and BALB/c) strains of mice (+ (B6 and Cmv1r) mice  mice . SimilarΔm157 or during co-infection with C4C and K181Δm157 or C4C and K181. To determine at what stage of infection competition was first evident, shedding of virus into the saliva was measured from day five p.i. During single strain infection all three viruses were shed into the saliva  were not significantly different to those in mice given an inoculum of 1×104 pfu of K181 (5.4×107±1.8×107 g/tissue). Moreover similar data were obtained in a repeat experiment where single strain infected mice were inoculated with 5×103 pfu of either C4C, K181 or K181Δm157 and multi-strain infected mice were inoculated with a total of 1×104 pfu of an equal combination of either C4C and K181 or C4C and K181Δm157. In this instance single and multi-strain infected mice received identical inoculums of each individual MCMV strain. Despite this, competition was once again seen in C4C and K181 co-infected mice but not in C4C and K181Δm157 co-infected mice (data not shown). Hence competition is apparent in the spleens of multi-strain infected mice before the maturation of acquired immunity and prior to dissemination to the salivary glands.Viral titres in the spleen are more sensitive to inoculum size than in the salivary glands, and the absence of K181 during multi-strain infection could simply reflect the two-fold reduction in K181 inoculated into multi-strain infected mice. However, K181 DNA levels in the spleens of B6 mice inoculated with 5×10Δm157 were present during multi-strain infection in the liver  resulted in the establishment of competition, indicating that the genetics of the co-infecting viral strains was important. NK cell depletion using PK136 (anti-NK1.1) suggested a role for NK cells in mediating within-host competition, however NK1.1 is also expressed on natural killer T cells. NKC congenic mice were used to demonstrate that competition among MCMV strains occurred only in mice that naturally expressed or were congenic for Ly49H. Antibody blocking studies supported an absolute requirement for Ly49H, an NK cell specific receptor Ly49H+ strain of MCMV, removed competition during co-infection with the m157Ly49H− strain C4C, indicating a requirement for m157 binding of Ly49H. Finally, competition was rapid, prior to the maturation of acquired immunity, and exactly mirrored the tissue distribution of Ly49H immunosurveillance, with competition seen in the spleen but not the liver The Ly49H/m157 axis has provided many insights into the interaction of mice with MCMV and also with more fundamental aspects of immunology such as the potential of NK cells to exhibit memory responses Ly49H+ strains of MCMV were excluded from the salivary glands suggests that NK cells mediate this competition by preventing the dissemination of MCMV to the salivary glands. This hypothesis is consistent with the rapid competition observed in the spleen but appears at odds with the lack of competition seen in the liver. Presumably virus from the liver (and perhaps other organs) could serve as a reservoir for viral dissemination to the salivary gland. However, the origin of MCMV colonizing the salivary glands is unclear. The established paradigm is that infecting virus replicates at the site of entry and then later in secondary organs before final dissemination to the target organ Ly49H+ strains of MCMV during multi-strain infection.The rapidity at which m157Plasmodium chabaudiStreptococcus pneumonia during co-infection with Haemeopilus influenzaeThe “apparent competition” (competition mediated by host immunity) seen in this model has been predicted to be important in shaping interactions amongst co-infecting pathogen strains Ly49H+ and m157Ly49H− MCMV strains resulted in the exclusion of m157Ly49H+ strains from the saliva. Avoidance of Ly49H mediated immunosurveillance leads to enhanced virulence of MCMV in B6 mice as measured by higher viral titers and enhanced morbidity  loss of these genotypes without inactivation of the m157 gene.Mutation of m157 following passage in Ly49H+ NK cells, and these cells proliferate in response to MCMV infection as is seen for Ly49H+ NK cells in B6 mice + NK cells specifically respond to MCMV encoded m04 + NK cells The capacity of NK cells to shape the population dynamics of CMV infection may not be limited to the effects of Ly49H and m157, as NK cell targeting of specific viral proteins is not confined to the Ly49H/m157 axis. In BALB.K mice resistance to MCMV is due to Ly49LLy49H+ strains of MCMV only during multi-strain infection remain unclear. However it is possible that multi-strain infection alters the amplitude or duration of NK cell responses. Perhaps, during multi-strain infection type I interferons or cytokines such as IL-12 and IL-15 are maintained at higher levels in response to the continued presence of m157Ly49H− strains of MCMV. This could result in either an enhanced expansion of the Ly49H+ NK cell pool or a delay in the contraction phase + NK cells following repeated stimulation through this receptor The mechanisms through which Ly49H mediated immunosurveillance drives such profound competition and control of m157Ly49H+ binding strains of MCMV from a population without the need for mutational inactivation of the m157 gene.In summary our data demonstrate that NK cells have the strain specific recognition capacity to meditate within-host competition between strains of MCMV. This competition has the capacity to shape the dynamics of viral shedding, and to select for the more virulent strains of MCMV that avoid Ly49H mediated immunosurveillance. This within-host competition provides a potential mechanism for the rapid removal of m157Figure S1Phylogenetic analysis of published m157 sequences. Sequences of m157 were extracted from DNA databases or produced in this study and used to construct a maximum likelihood phylogeny. Shown in red are m157 genotypes that have been shown to encode products capable of ligating Ly49H. In green are genotypes that produce products that do not ligate Ly49H. Genotypes in black have not been tested. Functional data was obtained from publications 48 and 50 and data described herein.(EPS)Click here for additional data file.Figure S2Surface expression of m157 on infected MEF. MEF were infected with high MOI (>10) and stained for surface expression of m157 using the monoclonal antibody 6H121. 6H121 was raised against the Smith/K181 variant of m157. Uninfected cells (grey shading) were used as controls. Note m157 was only detectable on the surface of MEF infected with K181, solid black line – last histogram. Surface expression of m157 was not detectable on C4A, C4B, C4C or C4D (grey lines – order is front to back) infected cells.(EPS)Click here for additional data file.


Novel effective anti-influenza agent that tolerates influenza virus antigenic variation is needed. Highly conserved influenza virus M2 protein has multiple pivotal functions including ion channel activity for vRNP uncoating, anti-autophagy and virus assembly, morphogenesis and release. Thus, M2 is an attractive target of anti-influenza agents including small molecular drugs and specific antibodies.huscfv-phagemid transformed E. coli clones selected from a HuScFv phage display library using recombinant M2 of clade 1 A/H5N1 as panning antigen. The HuScFv were tested for their ability to inhibit replication of A/H5N1 of both homologous and heterologous clades. M2 domains bound by HuScFv of individual E. coli clones were identified by phage mimotope searching and computerized molecular docking.Fully human monoclonal single chain antibodies (HuScFv) specific to recombinant and native M2 proteins of A/H5N1 virus were produced from huscfv-phagemid transformed E. coli clones  showed different amino acid sequences particularly at the CDRs. Cells infected with A/H5N1 influenza viruses (both adamantane sensitive and resistant) that had been exposed to the HuScFv had reduced virus release and intracellular virus. Phage peptide mimotope search and multiple alignments revealed that conformational epitopes of HuScFv2 located at the residues important for ion channel activity, anti-autophagy and M1 binding; epitopic residues of HuScFv19 located at the M2 amphipathic helix and cytoplasmic tail important for anti-autophagy, virus assembly, morphogenesis and release; epitope of HuScFv23 involved residues important for the M2 activities similar to HuScFv2 and also amphipathic helix residues for viral budding and release while HuScFv27 epitope spanned ectodomain, ion channel and anti-autophagy residues. Results of computerized homology modelling and molecular docking conformed to the epitope identification by phages.HuScFv derived from four HuScFv that bound to highly conserved epitopes across influenza A subtypes and human pathogenic H5N1clades located on different functional domains of M2 were produced. The HuScFv reduced viral release and intracellular virus of infected cells. While the molecular mechanisms of the HuScFv await experimental validation, the small human antibody fragments have high potential for developing further as a safe, novel and mutation tolerable anti-influenza agent especially against drug resistant variants. Influenza virus matrix-2 (M2) is a type III transmembrane protein of a class IA viroporin family . Each M2Presently, there are two families of anti-influenza drugs: adamantanes that inhibit M2 ion channel activity and neuraminidase inhibitors . These dAntibodies have been used with success for influenza treatment -19. ConvE. coli carrying pET-20b(+) vector with full length M2 cDNA insert. The protein was verified by SDS-PAGE and Western blotting   containing the respective inhibitors/controls, i.e., 400 ng HuScFv, 10 μg PAb, 0.8 μM rimantadine or medium alone). After incubation for 15 h, the spent culture fluid of each well was collected and the cells were washed with PBS and harvested. Quantitative real-time RT-PCR was used for enumeration of the viruses in the culture fluids and inside the cells. Plaque (foci) assay was also performed for comparing intracellular viruses among treatments. Three independent experiments were done.Influenza viruses were either added directly (experiment 1) or mixed with purified HuScFv . The qPCR condition was: reverse transcription at 42°C for 1 h, initial denaturation at 95°C for 10 min and 40 cycles at 95°C for 1 min, 58°C for 1 min and 72°C for 1 min. The thermal profile used for dissociation curve analysis was: 95°C for 1 min then ramped down to 55°C (0.5°C/s) and ramped up to 95°C. A standard curve was constructed from Ct of ten-fold dilutions of the pET20b(+) carrying gene sequence coding for M1 (ranged from 1000 to 1 ng or 2.07 × 1011 to 2.07 × 108 copies). Ct of each sample was expressed as log2 of RNA copies calculated from the standard curve.Viral RNA was extracted from 500 μL of each culture supernatant by using viral nucleic acid extraction kit II . The RNA was dissolved with 20 μL of DEPC-treated water. Total RNA was extracted from the virus infected cells by using total RNA purification kit . The amount of total RNA in each preparation was measured by using NanoDrop ND-1000 Spectrophotometer  and kept at -70°C until use. In each qPCR reaction, either 2 μL of extracted RNA from the culture supernatant or 200 ng of total RNA from infected cells were subjected to viral RNA quantification using 1-step Brilliant II SYBR green qRT-PCR master mix kit . Each PCR master mix (12.5 μL) was prepared on ice with 2 × Brilliant II SYBR® Green QRT-PCR Master Mix, RT/RNase block enzyme, 200 nM of each primer specific to gene sequence coding for influenza A virus Influenza virus plaques (foci) in the infected cells were revealed as described previously . InfectePh.D.-12™ phage display peptide library  was used to determine HuScFv bound phage mimotopes as described previously . MimotopE. coli and the titers of the amplified phages were determined according to manufacturer’s instruction . Various amounts  of the phages (50 μl) were mixed individually with fixed amount of HuScFv (5 μg in 50 μl) and incubated at 37°C for 1 h. The HuScFv mixed with irrelevant phage mimotope served as background inhibition controls and HuScFv in buffer were negative inhibition controls . After adding individual mixtures to the rM2 coated wells and incubated, all wells were washed and mouse monoclonal anti-E tag antibody, goat anti-mouse immunoglobulin-HRP conjugate and ABTS substrate were added, respectively, with washing between steps. OD405nm of the content of each wells were determined against the background binding controls. The % ELISA inhibition was calculated: Phage clones displaying the mimotopes were tested in competitive ELISA for determining their capacity in blocking the binding HuScFv to rM2 as described previously . MimotopAmino acid sequences similar to the rM2 and the HuScFv were identified by using a Discovery studio (DS) database. Three dimensional (3D) structures of the sequences that provided maximum identities were used as templates for homology modelling. The modelled structures were validated by PROCHECK analysis . Complexhttp://www.ncbi.nlm.nih.gov/protein). For the multiple sequence alignments, the program MAFFT version 7 was used. The alignments were verified following the algorithm of semi-homology. The verification concerned the possible genetic relationship between compared positions which were possible replacements by single transition/transversion.The amino acid sequences of influenza A viruses including H1N1, H3N2, H5N1 , H7N3, H7N9 and H9N2 were retrieved from the NCBI database  of logThe authors declare that they have no competing interests.TP did most experiments, SM performed phage biopanning and HuScFv expressions, KT, prepared recombinant M2 and NP, PS supervised TP on qPCR, FD and JT, mimotope searching, TS isolated the H5N1 viruses and propagation, PT supervised TP on immunology methods, KB performed homology modeling and molecular docking. WC conceived the project and wrote the manuscript. All authors read and approved the final manuscript.Binding of HuScFv of clones no. 2, 19, 23 and 27 and PAb to M2 in the MDCK cells infected with amantadine sensitive (NP-172)  and resistant (KU08)  H5N1 viruses. Non-infected cells reacted with HuScFv and PAb are shown in respective blocks of the left column.Click here for fileAppearances of influenza virus foci in infected MDCK cells treated with HuScFv, PAb to M2 and rimantadine. Adamantane sensitive [A/chicken/Thailand/NP-172/2006 (H5N1 clade 2)] and resistant [A/dog/Thailand/KU-08/2004 (H5N1 clade 1) viruses were incubated with M2 specific-HuScFv before adding to MDCK cell monolayer. Viruses mixed with rimantadine and PAb to M2 were used as positive inhibition controls while viruses in culture medium served as negative inhibition (infected cell) controls. The cells were cultured in the medium containing respective HuScFv, rimantadine, PAb to M2 and medium alone for 15 h. Extracellular viruses were removed and the cells were washed before subjecting to immune-staining for virus plaques (foci). A and I: Uninfected MDCK cell monolayer; B and J: negative inhibition controls (MDCK cells infected with the viruses); C and K: MDCK cells infected with viruses exposed to PAb to M2; D and L: MDCK cells infected with rimantadine exposed viruses; E and M, F and N, G and O and H and P: MDCK cells infected with viruses exposed to HuScFv2, HuScFv19, HuScFv23 and HuScFv27, respectively.Click here for fileMultiple alignments of the M2 amino acid sequences of various influenza A subtypes and clades of subtype H5N1. The peptides of M2 proteins which were binding sites of the HuScFv of clones no. 2, 19, 23 and 27 are highly conserved across A subtypes and H5N1 clades.Click here for file


The histone variant H2A.Z has been implicated in nucleosome exchange, transcriptional activation and Polycomb repression. However, the relationships among these seemingly disparate functions remain obscure.We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers, and correlates strongly with H3K4 methylation. Accordingly, H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation, but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z, including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.Our findings associate H2A.Z with functionally distinct genomic elements, and suggest that post-translational modifications may reconcile its contrasting locations and roles. Pluripotent embryonic stem (ES) cells are characterized by a plastic epigenome conducive to self-renewal and broad differentiation potential. Histones and chromatin proteins in ES cells are subject to relatively rapid turnover -3. This Pluripotent chromatin is distinguished by characteristic post-translational histone modifications. Bivalent domains that contain 'active' H3 lysine 4 trimethylation (H3K4me3) and 'repressive' H3 lysine 27 trimethylation (H3K27me3) are prevalent in ES cells. Bivalent domains and associated Polycomb repressive complexes 1 and 2 (PRC1 and PRC2) silence developmental loci while maintaining their potential for future activation . During Replication-independent histone deposition is of particular interest as it is targeted to DNA sequences under active regulation ,9. RapidIn addition to its pervasive roles at TSSs and active regulatory elements, H2A.Z has also been linked to Polycomb regulation. A microarray-based chromatin immunoprecipitation (ChIP-chip) analysis in ES cells found that H2A.Z associates exclusively with silent promoters bound by PRC2 . Upon diTo clarify the distribution and potential functions of H2A.Z in ES cells, we used ChIP coupled with high-throughput sequencing (ChIP-Seq) to query the localization of this variant in mouse and human ES cells, and in lineage-restricted progenitors. We found that H2A.Z is ubiquitously deposited at promoters, putative enhancers and other intergenic regulatory elements marked by H3K4 methylation. H2A.Z is also deposited at K27me3 regions/PRC2 binding sites, but it is restricted to those sites that have coexisting H3K4 methylation, and thus constitute bivalent domains. Notably, we found that bivalent chromatin is enriched for a novel population of H2A.Z simultaneously modified by N-terminal acetylation and C-terminal ubiquitination. We propose that distinct modification states enable H2A.Z to facilitate regulation of bivalent PRC2 targets as well as to act at a diversity of other histone exchanging elements in mammalian genomes.We acquired genome-wide maps of H2A.Z in mouse ES (mES) cells using ChIP-Seq. We observed H2A.Z enrichment at a substantial majority of TSSs, with a bimodal distribution spanning the promoters and the 5' end of TSSs specifically Figure . This isWe used the same ChIP-Seq procedures and antibody to profile H2A.Z in human ES (hES) cells Figure ; an idenFurther evidence for correlation between H2A.Z and H3K4 methylation emerges from genome-wide analyses of H3K4 monomethylation me1), dimethylation (me2) and trimethylation (me3). Respectively, 51% of H3K4me1-, 74% of H3K4me2- and 78% of H3K4me3-enriched regions overlap with H2A.Z sites Figure . H2A.Z e, dimethyThese observations led us to consider the basis for the presence of H2A.Z and associated nucleosome replacement with histone variants at bivalent PRC2 target sites in ES cells. We therefore acquired and analyzed ChIP-Seq maps for the pre-initiation complex component TFIID (transcription Factor II D), hypo-phosphorylated RNAPII and pan-histone H3. We also incorporated published RNA-Seq data for mES cells ,30. InteThe presence of H2A.Z at varied classes of genomic elements is consistent with its diverse functions in transcriptional initiation and induction; regulation of DNA methylation; and Polycomb repression ,29,33. HH2A.Z has been shown to be subject to C-terminal ubiquitination by the PRC1 component Ring1B . WesternWe next explored the relationship between C-terminal ubiquitination and N-terminal acetylation on H2A.Z. Although prior studies have linked H2A.Z acetylation to transcriptional activity -37, westTo decipher further H2A.Z acetylation patterns in relation to ubiquitination status, we used MS to measure site-specific acetylation levels for a population of H2A.Z isolated from mES cells. We characterized the levels and numbers of lysine residues  that are acetylated per molecule Figure . The mosWe sought to confirm that the ubiquitinated and dually modified species also localize to bivalent domains. However, we were unable to acquire high-quality ChIP-Seq maps using an antibody against H2A.Zub1. We therefore implemented an alternative approach in which we immunoprecipitated mononucleosomes from mES cells using an antibody against H3K27me3, and then used western blots to evaluate H2A.Z. We found that both H2A.Zub0 and H2A.Zub1 are enriched, and that the level of H2A.Zub1 relative to H2A.Zub0 is significantly higher in the H3K27me3 pull-down relative to control input fractions Figure . We alsoFinally, we examined the genomic localization of the acH2A.Z species in mES and mNP cells using ChIP-Seq. We observed clear enrichment for the modified species at bivalent promoters, in addition to actively transcribed TSSs in both mES and mNP cells Figure . SimilarAcetylated histones have long been known to be a marker of active transcription and are thought to act in part by neutralizing charge interactions to open chromatin and allow access for transcription machinery -37. To eThrough comprehensive analyses of ChIP-Seq, MS and biochemical data, we document the existence of a novel, dually modified H2A.Z species that preferentially localize to bivalent chromatin domains in ES cells. Our results also clarify that, in both mouse and human ES cells, H2A.Z promiscuously co-localizes to genomic loci enriched for H3K4 methylation, including both bivalent PRC2 targets and as active TSSs as well as at distal enhancer elements. We demonstrate a strong association between H2A.Z acetylation and transcriptional activity. The co-occurrence on the same histone molecule of acetylation, previously linked to gene activity and induction, and ubiquitination events, downstream of Polycomb repressors, echoes the duality of the bivalent H3K4me3 and H3K27me3 combination. We propose that the modified variant contributes to the transcriptional dynamics and epigenomic plasticity of pluripotent ES cells by maintaining dynamic chromatin at key loci poised for alternate developmental fates. This study provides a framework for future studies into the nature of bivalent chromatin functions, and opens up new avenues for decoding the interplay between chromatin-modifying enzymes and modifications of variant histones.129SVJae × C57BL6) were cultured using standard procedures and reagents .2: magnesium chloride; mNPs: mouse neural progenitors; MS: mass spectrometry; MTLs: multiple transcription factor binding loci; NaCl: sodium chloride; PRC: Polycomb repressive complexes; RNAPII: RNA polymerase II; TF: transcription factor; TFIID: transcription Factor II D; TSSs: transcription start sites.acH2A.Z: acetylated H2A.Z; acH2A.Zub0: acetylated and non-ubiquitinated H2A.Z; acH2A.Zub1: acetylated and monoubiquitinated H2A.Z; bp: base pair; ChIP-chip: chromatin immunoprecipitation coupled with microarray; ChIP-Seq: chromatin immunoprecipitation coupled with high-throughput sequencing; EGTA: ethylene glycol tetraacetic acid; ES: embryonic stem; H2A.Zub0: non-ubiquitinated H2A.Z; H2A.Zub1: monoubiquitinated H2A.Z; H3: histone H3; H3K4me3: histone 3 lysine 4 trimethylation; H3K27me3: histone 3 lysine 27 trimethylation; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; hES: human embryonic stem; HPLC: high performance liquid chromatography; K: lysine; kb: kilobases; KCl: potassium chloride; LC: liquid chromatography; me1: monomethylation; me2: dimethylation; me3: trimethylation; mES: mouse embryonic stem; MgClThe authors declare that they have no competing interests.MK conceived the study, performed the experiments, analyzed the data and wrote the manuscript. JJ performed and analyzed the mass spectrometry experiments. RK and ER analyzed data. ME and HK generated the Ring1B knockout ES cells. SC coordinated mass spectrometry experiments. BB conceived the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.Composite plots show H3.3 ChIP-Seq signal enriches at K4me3-only and bivalent promoters (PRC1-positive (+) and PRC1-negative (-)), but is depleted in no-mark promoters in mES cells.Click here for fileComposite plots show H3 distribution across K4me3-only, bivalent (PRC1-positive and PRC1-negative) or no-mark TSSs (±2.5 kb). Nucleosome-deficient regions are observed at K4me3-only and bivalent promoters, but not at no-mark promoters.Click here for fileSummary of ChIP-Seq data sets.Click here for fileSummary of antibody information.Click here for file


NIR was identified as an inhibitor of histone acetyltransferase and it represses transcriptional activation of p53. NIR is predominantly localized in the nucleolus and known as Noc2p, which is involved in the maturation of the 60S ribosomal subunit. However, how NIR functions in the nucleolus remains undetermined. In the nucleolus, a 47S ribosomal RNA precursor (pre-rRNA) is transcribed and processed to produce 18S, 5.8S and 28S rRNAs. The 18S rRNA is incorporated into the 40S ribosomal subunit, whereas the 28S and 5.8S rRNAs are incorporated into the 60S subunit. U3 small nucleolar RNA (snoRNA) directs 18S rRNA processing and U8 snoRNA mediates processing of 28S and 5.8 S rRNAs. Functional disruption of nucleolus often causes p53 activation to inhibit cell proliferation.Western blotting showed that NIR is ubiquitously expressed in different human cell lines. Knock-down of NIR by siRNA led to inhibition of the 18S, 28S and 5.8S rRNAs evaluated by pulse-chase experiment. Pre-rRNA particles (pre-rRNPs) were fractionated from the nucleus by sucrose gradient centrifugation and analysis of the pre-RNPs components showed that NIR existed in the pre-RNPs of both the 60S and 40S subunits and co-fractionated with 32S and 12S pre-rRNAs in the 60S pre-rRNP. Protein-RNA binding experiments demonstrated that NIR is associated with the 32S pre-rRNA and U8 snoRNA. In addition, NIR bound U3 snoRNA. It is a novel finding that depletion of NIR did not affect p53 protein level but de-repressed acetylation of p53 and activated p21.We provide the first evidence for a transcriptional repressor to function in the rRNA biogenesis of both the 40S and 60S subunits. Our findings also suggested that a nucleolar protein may alternatively signal to p53 by affecting the p53 modification rather than affecting p53 protein level. In the nucleolus of mammalian cells RNA polymerase I transcribes a 47S ribosomal RNA precursor (pre-rRNA) which contains a 5′ external transcribed spacer (5′-ETS), followed by the 18S rRNA, internal transcribed spacer 1 (ITS1), 5.8S rRNA, internal transcribed spacer 2 (ITS2), 28S rRNA and the 3′ external transcribed spacer (3′-ETS). Upon synthesis, the 47S pre-rRNA transcript is modified by ribose methylation and pseudouridine conversion and cleaved at specific sites to generate a series of intermediates and consequently produce matured 18S, 28S, and 5.8S rRNAs. Several cleavage pathways have been described for processing of the pre-rRNA to produce the matured rRNAs and at least two cleavage pathways have been described in mammalian cells . The 18SXenopus homologues of the LSm (like Sm) proteins including LSm2, -3, -4, -6, -7, and -8 have been identified as U8 binding proteins and the presence of LSm8 was considered to be consistent with the nuclear localization of U8 Xenopus 29 kDa protein (X29) binds U8 RNA 227G cap from U8 RNA, which may lead to degradation of U8 RNA resulting in an inhibition of pre-rRNA processing Modifications and cleavages of pre-rRNA are directed by small nucleolar RNAs (snoRNAs) myc including Bop1, Pes1 and WDR12 have been identified to play key roles in the processing of 28S and 5.8S rRNAs in mammalian cells. Bop1 was the first identified mammalian protein being involved in the processing of 28S and 5.8S rRNAs and functioning in cell proliferation For the 60S ribosome subunit biogenesis, three down-stream genes of onco-protein Ribosome biogenesis governs protein synthesis and cell proliferation, is thus tightly controlled. Disruptions of nucleolar function caused by chemical reagents or deficiency of nucleolar proteins have been shown to generate nucleolar stress signaling to p53. For instance, low concentrations (5–10 nM) of actinomycin D selectively inhibit RNA pol I dependent transcription and stimulates stabilization of p53 In unstressed cells, the p53 protein level remains low through regulation of its protein stability by a number of negative regulators. MDM2 serves as a key negative feedback regulator for p53 and various stresses activate distinct cellular signaling pathways leading to the suppression of MDM2 activity and activation of p53 In vitro study found that Aurora B phosphorylates multiple sites in the p53 DNA-binding domain Our previous study identified 1A6/DRIM as the human UTP20 which functions in the pol I transcription and 18S rRNA processing In the present study, we showed that NIR is required for 18S, 28S and 5.8S rRNA processing and further investigated the mechanisms by which NIR functions in the rRNA processing. We also found an alternative pathway for p53 activation caused by depletion of NIR.To evaluate the function of NIR protein and to detect endogenous NIR expression, we analyzed the expression of NIR in various human cell lines. Cellular fractions were prepared and proteins from the fractions were subjected to Western blotting. 3H] uridine and total RNA extracted at different time periods was resolved on a 10% polyacrylamide/7.5 M urea gel. As shown in To evaluate whether NIR is required for rRNA processing, a NIR-specific siRNA  was transfected into U2OS cells to knock down NIR expression and newly synthesized rRNA was analyzed with pulse-chase experiment. Schematic representation of rRNA processing pathways is shown in Eukaryotic rRNA processing occurs coordinately with the assembly of pre-ribosomal particles (pre-rRNP) in the nucleolus. To further confirm the participation of NIR in the rRNA processing, pre-rRNP particles were fractionized by applying nuclear extracts on sucrose gradient centrifugation. As shown in in vivo.To investigate if NIR is associated with the 32S rRNA and 12S rRNA, cell lysates was prepared from U2OS cells and immunoprecipitation was performed with anti-NIR antibody. Proteins from one half of the immunoprecipitation were subjected to Western blotting for evaluation of NIR. RNA extracted from the remaining half of the immunoprecipitation was analyzed by Northern blotting probed with an ITS-2 probe. The results showed that both of 32S and 12S rRNAs existed in the NIR-specific immunoprecipitates . This anOur above findings prompted us to investigate if NIR interacts with U8 snoRNA, which is known to bind 32S rRNA and facilitate 28S and 5.8S rRNA processing. To address this issue, immunoprecipitation was performed with anti-NIR antibody on the cell lysates extracted from U2OS cells. Proteins from one half of the immunoprecipitation were subjected to Western blotting for evaluation of NIR. RNA extracted from the remaining half of the immunoprecipitate was subjected to Northern blotting hybridized with a biotin-labeled U8 snoRNA-specific probe. As shown in in vivo. What we next wanted to know was if depletion of NIR affected U3 snoRNA level. NIR specific siRNA was transfected into U2OS cell and total RNA was extracted and subjected to Northern Blotting hybridized with a U3 snoRNA probe. The same amount of RNA was resolved on a 1% agarose gel and stained with EB  together with a control siRNA 5′-CGU ACGCGGAAUACUUCGA-3′) were chemically synthesized . The synthesized siRNA was transfected into cells at a concentration of 100 nM with Lipofectamine 2000™ (Invitrogen) according to the manufacturer's instructions.HeLa, H1299, U2OS, HEK293, MCF10A, MCF-7, T47D, MDA-MB231, HepG2, A549 and K562 cells were obtained from the American Type Culture Collection (ATCC) and were grown according to the instructions provided by the ATCC. Cells were incubated in a humidified atmosphere with 5% COGAATTC5′-AGCT (EcoR I)ATACTTACCTGGCAGGG-3′, reverse primer: AAGCTT5′-AGCT(Hind III)GGAAA GCGCGAACGCAG-3′. The PCR product was cloned into p-GEM-T-easy vector (Promega). The generated plasmid was verified by DNA sequencing. Anti NIR polyclonal antibody was described previously pGEM-T-U3, p-GEM-T-U8 and pCMX-Flag-NIR are described previously 2, layered over a cushion of 0.35 M sucrose/0.5 mM MgCl2 and centrifuged. The resulting nuclear pellet was resuspended in 0.35 M sucrose/0.5 mM MgCl2 and sonicated to disrupt nuclei and release nucleoli. The sonicate was layered over a cushion of 0.88 M sucrose/0.5 mM MgCl2 and centrifuged to pellet nucleoli and the supernatant was retained as the nucleoplasmic fraction. Nucleoli were washed by resuspending in 0.5 ml of 0.35 M sucrose/0.5 mM MgCl2 and centrifugation. The nucleolar pellet was resuspended in high salt RIPA buffer containing DNase, and then sonicated and centrifuged after incubation, the supernatant was retained as the nucleolar extract and the NaCl concentration adjusted to 150 mM by adding “no salt” RIPA buffer.Cellular fractions were obtained as previously described Immunoprecipitation was performed as described previously Proteins were separated on SDS-PAGE and transferred onto PVDF membranes (Amersham Biosciences). Blots were probed sequentially with corresponding antibodies and HRP-conjugated secondary antibodies. Immunocomplexes were detected with ECL Western blot Detection Reagent  before exposure to X-ray film.5′-GCGGCGGCAAGAGGAGGGCGGACGCCGCCGGGTCTGCGCTTAGGGGGA-3′ was biotin-labeled with Biotin 3′ End DNA Labeling Kit (PIERCE) and used to probe 32S rRNA precursor. Hybridization was performed as described previously For detection of 32S rRNA, Northern blotting was performed as described previously To visualize U3 snoRNA, U8 snoRNA or U1 snRNA, Northern blotting was performed as described previously p21 mRNA level was determined by RT-PCR as previously described methyl-3H] methionine (PerkinElmer Life Sciences) for 30 min. Cells were then changed to medium containing 15 µg/ml of non-radioactive methionine. Total RNA was isolated with Trizol reagent (Invitrogen) at different time points. The [methyl-3H] methionine labeled RNAs were resolved on a 1% agarose–formaldehyde gel and detected by fluorography. For 5.8S rRNA labeling, pulse-chase experiment was performed as described previously 3H] uridine (PerkinElmer Life Sciences) for 30 min. After two brief rinses, cells were changed to nonradioactive medium and total RNA was isolated with Trizol reagent at different time points. Equal amount of RNA was resolved on a 10% polyacrylamide/7.5 M urea gel and newly synthesized 5.8S rRNA was detected by fluorography.For 18S and 28S rRNA processing analysis, pulse-chase labeling was performed as described previously g for 5 min. The nuclear lysate were extracted in 25 mM Tris (pH 7.5), 100 mM KCl, 1 mM DTT, 2 mM EDTA, 0.1% NP-40, 1 mM NaF, 40 mg/ml of phenylmethylsulfonyl fluoride, 10 mg/ml of protease inhibitor cocktail, 0.1 U/ml of RNasin (Promega) and sonicated. The nuclear lysate was overlaid on 10 to 40% (wt/wt) sucrose gradients in 25 mM Tris (pH 7.5), 100 mM KCl, 1 mM DTT, 2 mM EDTA and centrifuged at 36,000 rpm for 3 h at 5°C in a Beckman SW41Ti rotor. The gradients were collected downward and the absorbance at 254 nm was measured Nuclear extracts were fractionated as described previously Cells were plated on coverslips in 6-well plates one day before harvest. Double indirect immunofluorescence was performed with the monoclonal anti-1A6/DRIM antibody 6D9 and a polyclonal antibody directed against NIR after cells were fixed. 1A6/DRIM specific immunocomplexes were detected with TRITC-conjugated goat anti-mouse IgG and NIR specific immunocomplexes were detected with FITC-conjugated goat anti-rabbit IgG. Immunofluorescence signals were recorded by confocal laser scanning microscopy (Leica TCS-ST2).


The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently. In April 2009, the swine-origin pandemic AH1N1)2009 virus, A(H1N1)pdm09, emerged, originating from the swine H1 virus in North America and the avian-like swine virus in Europe N12009 vi. Since iThe use of monoclonal antibodies (MAbs) is an established laboratory strategy for characterization of virus strains and their antigenicity ,9. In adA hybridoma method for HuMAbs was developed previously in our laboratory by fusion of the peripheral blood mononuclear cells (PBMCs) of influenza-vaccinated healthy volunteers with the fusion partner cell line, SPYMEG . In the HuMAbs were prepared as described previously . BrieflyThe following virus strains were used: A/New Caledonia/20/1999 , A/Brisbane/59/2007 , A/Hiroshima/52/2005 (H3N2), A/Uruguay/716/2007 (H3N2), B/Florida/4/2006, and B/Malaysia/2506/2004. Viruses were propagated in Mardin-Darby canine kidney (MDCK) cells or in 9-day-old embryonated chicken eggs.TM-I (Invitrogen) supplemented with 0.4% bovine serum albumin, antibiotics, and 5 µg/ml acetylated trypsin. Amplified viruses were titrated by a focus-formation assay with MDCK cells. The swab samples collected in December 2009 and January 2011 yielded 30 and 28 clinical isolates, respectively pdm09 were kindly provided by Baba Pediatric Clinic and Medical Association of Higashinari-ku, Osaka. For viral isolation, MDCK cells were infected using the nasal swabs in a BD Universal Viral Transport  and cultured in DMEM/F-12+GlutaMAXectively  and S2. IFA was performed as previously described . BrieflyThe focus-formation assay was performed as previously described , but witTaq High Fidelity; Invitrogen) using the following HA primer set: forward, 5’-CGACGGAATTCATGAAGGCAATACTAGTAG-3’, and reverse, 5’-CAGCTCTCGAGTTAAATACATATTCTACACTGTAG-3’. The PCR products were purified using the QIAquick Gel Extraction kit  and inserted into the pGEM-T Easy Vector . The plasmid was subcloned into the expression vector pCAGGS/MCSII. The expression plasmids were then transfected into human embryonic kidney 293T cells with lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Expression was confirmed by IFA using an anti-HA MuMAb .As previously described ,25, the 2SO4 solution. Color development was read at 450 nm in an ELISA Photometer . All samples were run in triplicate.IgG isotyping was carried out as described previously . ELISA mFirst, viral titers were determined by a hemagglutination assay. Briefly, the viruses were serially 2-fold diluted with PBS and the dilutions were mixed with 0.7% (v/v) guinea pig red blood cells . After incubation at room temperature for 1 hour, hemagglutination units (HAUs) were estimated. Next, HI titration was performed as follows. Purified HuMAb 5E4 at a concentration of 100 µg/ml  were serially 2-fold diluted and mixed with 8 HAU per 50 µl of viral sample. After incubation at 37°C for 1 hour, the mixtures were further incubated with 0.7% (v/v) guinea pig red blood cells for 1 hour at room temperature. The maximal reciprocal MAb dilution that completely inhibited hemagglutination was designated the HI titer. One hundred microliters of ferret anti-A/California/7/2009 serum  was mixed with 300 µl of receptor destroying enzyme  to eliminate any non-specific inhibitors and incubated at 37°C for 18 hours. This step was followed by heat inactivation at 56°C for 30 minutes, dilution in PBS , and removal of nonspecific agglutinator by absorbing with test erythrocytes for 1 hour at room temperature . The HI 50 titer. Ferret anti-A/California/7/2009 serum was prepared as described in “HI assay”.The VN test was carried out as described previously  but withTM-I supplemented with 0.4% bovine serum albumin, antibiotics, and 2 µg/ml acetylated trypsin, the supernatants were collected and subjected to VN and HI assays. Those viral samples that showed more than 4-fold reduction of VN50 and HI titer were subjected to direct sequencing analysis of their entire HA gene.Escape mutants were selected by culturing A/Suita/1/2009 or A/Osaka/63/2009 in the presence of MAb as described previously , with mi5’-ATGGAGTTTGGGCTGAGCTGGGTT-3’ (H-chain-forward)/5’-CTCCCGCGGCTTTGTCTTGGCATTA-3’ (H-chain-reverse) and 5’-ATGGCCTGGRYCYCMYTCYWCCTM-3’ (L-chain-forward)/ 5’-TGGCAGCTGTAGCTTCTGTGGGACT-3’ (L-chain-reverse), respectively. PCR products were ligated into pGEM-T Easy Vector (Promega) and their sequences were determined and analyzed using a BigDye Terminator v3.1 Cycle Sequencing kit and an ABI Prism 3100 Genetic Analyzer . The sequences were then analyzed and used to search the NCBI database using IgBLAST software (http://www.ncbi.nlm.nih.gov/igblast/).Total RNA, extracted from the hybridoma using an RNeasy Mini kit (Qiagen), was subjected to reverse transcription-polymerase chain reaction (RT-PCR) using a PrimeScript RT reagent kit (Takara) with an oligo (dT) primer. The coding regions of the H- and L-chains of HuMAb were amplified by PCR with the following primers: The HA structure was constructed using Molecular Operating Environment software  based on the crystal structure of A/California/4/2009 (PDB ID: 3LZG) .2SO4 solution. Color development was measured at 450 nm in an ELISA Photometer . Next, an AC-ELISA was performed using 5E4. Coating, blocking, incubation with the HRP-conjugated Ab, and color development were performed/measured as described above. Each of viruses was diluted with PBS to apply the same amount of viral antigen. Antigen was detected by HuMAb 5E4, which was added to the wells at concentrations of 10 or 100 µg/ml for 1 hour at 37°C. HuMAb D23-1G7C2 against dengue virus [First, the amount of viral antigen (wild-type viruses and escape mutants of A/Suita/1/2009 and A/Osaka/63/2009) was confirmed. ELISA microplates  were coated with a mouse anti-HA antibody  overnight at 4°C. The wells were then blocked with skim milk (5% in PBS) for 2 hours at 37°C. After washing with PBS, the wells were incubated with viruses  for 1 hour at 37°C. They were then washed five times in PBS and incubated with A(H1N1)pdm09-vaccinated human serum (1:100) for 1 hour at 37°C. After washing further five times in PBS, the wells were incubated with HRP-conjugated goat anti-human IgG  for 1 hour at 37°C, washed five times in PBS, and then incubated with tetramethylbenzidine peroxidase substrate  at room temperature in the dark. After 30 minutes, the reaction was stopped by adding 2N Hue virus  was usedVibrio cholerae RDE (Sigma) for 1 hour at 37°C as described by Hensley et al. [A 10% solution (v/v in PBS) of guinea pig erythrocytes was treated with various concentrations (0.5 to 32 µg/ml) of y et al. . The celTreatment with α2,3-sialidase was performed as previously described . Briefly5’-CGACGATGAAGGCAATACTAGTAG-3’ and reverse primer 5’-CAGCTTTAAATACATATTCTACACTGTAG-3’ [Viral RNA was extracted with the QIAamp Viral RNA Mini Kit (Qiagen) and subjected to one-step RT-PCR  with the HA forward primer TGTAG-3’ . The PCRdN and dS, respectively) were estimated for each 51 bp window (sliding in 3 bp increments) and for each epitope region by the Miyata-Yasunaga method [dN>dS and dN<dS indicate positive and negative selection, respectively [Rates of nonsynonymous and synonymous substitutions per site  were downloaded from the FTP site of Influenza Virus Resource  on AprilThe amino acid diversity at each epitope was estimated according to the Shannon index  using thi is the proportion of the i-th variant of the epitope.in which H is the Shannon index and P50 titers. The Steel-Dwass method was used to analyze the proportion of mutant sequences. A P-value of <0.05 was considered significant.The Mann-Whitney U test was used to analyze the HI and VNhttp://www.ncbi.nlm.nih.gov/GenBank/) accession numbers for the immunoglobulin genes discussed in this paper are as follows: IgG-VH in clone 5E4 (AB777475) and IgG-VL in clone 5E4 (AB777476). The accession numbers analyzed for viruses in this study are as follows: AB675046 to AB675075 and AB777447 to AB777474.The GenBank . Written informed consent was obtained from the participants.H and VL regions of the HuMAb were sequenced and analyzed with respect to the closest germline sequences using IgBLAST software in the NCBI database pdm09, which did not react with seasonal influenza A  or influenza B viruses , according to IFA (data not shown). This HuMAb was shown to recognize the HA protein expressed in transfected 293T cells (data not shown). IgG isotyping revealed that HuMAb 5E4 was IgG1. The Vdatabase . In addidatabase . 50 titer compared with the wild-type virus). When we sequenced the entire HA gene from each of these samples, we found that all harbored an A189T substitution (based on H3 numbering) [50 titer compared with the wild-type virus). The entire HA gene was sequenced in 9/15 samples, and all harbored a D190E substitution. Viruses incubated in the absence of 5E4 showed no amino acid substitutions. These substitutions were positioned within the antigenic site Sb (amino acid position 187 to 198) and in close proximity to the receptor binding domain  . Fifteeng domain  [28,41,4To classify these mutations as either antigenic or adsorptive, we performed AC-ELISA using one escape mutant from each of the viruses shown in 50 assay, 5E4 showed peak VN50 titers of 256 in the 2009/10 season. The reactivity of 5E4 decreased significantly in the 2010/11 season and the peak VN50 titers at this time were 64. By contrast, ferret serum showed similar titers in 2,560 to 40,960 in both periods. These results indicated that the antigenic variants in Sb increased significantly in Japan during 2009 and 2011. Six isolates, A/Suita/24/2009, A/Suita/28/2009, A/Suita/15/2011, A/Suita/104/2011, A/Suita/105/2011 and A/Suita/117/2011, which showed various reactivities against 5E4 and ferret serum, were randomly extracted (as indicated by colored dots in The emergence of possible antigenic variants of Sb during the period between 2009 and 2011 was investigated by testing for loss of reactivity with 5E4 in HI and VN tests and comparing this with the reactivity with ferret anti-A(H1N1)pdm09 serum  and S2. dS) and nonsynonymous (dN) substitutions were calculated per site in the HA1 gene against the control vaccine strain, A/California/7/2009, by the Miyata-Yasunaga method [dN>dS within nucleotide positions 210 to 260, 580 to 630, and 920 to 980 in both the 2009/10 and 2010/11 seasons, suggesting that these substitutions are caused by positive selection in 2009. By contrast, positions 380 to 450, 520 to 580, and 630 to 680 showed dN>dS only in the 2010/11 season, indicating that positive selection occurred in these positions after the 2009/10 season. We focused the substitution rates at five antigenic sites pdm09 in Osaka, Japan was investigated. All of the viral isolates used in a method  using Sqic sites . Sa and To examine the genetic evolution of newly emerged A(H1N1)pdm09 in response to selective pressure, 7,415 HA genes in A(H1N1)pdm09 were extracted globally between April 2009 and January 2013 from the GenBank database. As shown in In this study, characterization of viral isolates of A(H1N1)pdm09 using a neutralizing HuMAb, 5E4, revealed an unusually high rate of introduction of variants against the antigenic site Sb from 2009 to 2011 in Osaka, Japan. Also, 2010/11 isolates showed an increased rate of genetic mutation at Sb. In agreement with these findings, our global characterization of amino acid sequences at Sb derived from the GenBank database exhibited the same results. These results indicate that antigenic variants against Sb have emerged and have increased in prevalence. These findings may be of considerable utility for developing a future vaccine against A(H1N1)pdm09. Classical seasonal H1N1 has five antigenic sites in the globular head of the HA protein: Sa, Sb, Ca1, Ca2 and Cb. It has not been fully elucidated whether these antigenic sites are equally important for A(H1N1)pdm09. However, a similar antigenic structure to that of seasonal H1N1 has been predicted for A(H1N1)pdm09 by homology modeling . MoreoveGenetically distinct variants containing several significant amino acid changes in both the HA and NA genes have emerged in 2010. Ding et al. reported that T206 and E/G225 had been genetically positively selected by April 2010. Notably, both amino acid residues are located in the highly variable epitope regions of HA1 . First iWe showed a high level of genetic variation in Ca2 and Sb in 2010/11 samples in Japan . The emeIn influenza surveillance, detailed antigenic characterization of isolates is usually performed with polyclonal ferret sera against influenza virus. However, while standard infected ferret sera react well, MAb reactivity with viral isolates can be heterogeneous . In thisFigure S1AC-ELISA using wild-type viruses (open bars) and escape mutants (solid bars) of A/Suita/1/2009 (left panel) and A/Osaka/63/2009 (right panel). MuMAb C179 and HuMAb D23-1G7C2 were used as the coating and detecting antibodies, respectively. All data are represented as the means ± s.d. of three independent experiments.(PDF)Click here for additional data file.Figure S250 titers in 30 and 28 viral isolates obtained in 2009/10 and 2010/11, respectively, in Osaka, Japan using HuMAb 5E4 and ferret anti-H1N1pdm serum.The distribution of HI and VN The X- and Y-axises show the reciprocal antibody dilutions. Colored dots indicate the individual viral isolates for which data are shown in (PDF)Click here for additional data file.Table S150 and HI titers by HuMAb 5E4 and ferret serum against viral isolates in 2009/2010.VN(PDF)Click here for additional data file.Table S250 and HI titers by HuMAb 5E4 and ferret serum against viral isolates in 2010/2011.VN(PDF)Click here for additional data file.Table S3The diversity of the amino acid residues in the antigenic site Ca1 in Periods 1 to 6.(PDF)Click here for additional data file.Table S4The diversity of the amino acid residues in the antigenic site Ca2 in Periods 1 to 6.(PDF)Click here for additional data file.Table S5The diversity of the amino acid residues in the antigenic site Cb in Periods 1 to 6.(PDF)Click here for additional data file.Table S6The diversity of the amino acid residues in the antigenic site Sa in Periods 1 to 6.(PDF)Click here for additional data file.Table S7The diversity of the amino acid residues in the antigenic site Sb in Periods 1 to 6.(PDF)Click here for additional data file.


Amplification of innate immune responses by endogenous danger-associated molecular patterns (DAMPs) promotes inflammation. The involvement of S100A8 and S100A9, DAMPs belonging to the S100 calgranulin family, in the pathogenesis of cardiovascular disease is attracting an increasing amount of interest. S100A8 and S100A9  preferentially form the S100A8/A9 heterodimer  and are constitutively expressed in myeloid cells. The levels of circulating S100A8/A9 in humans strongly correlate to blood neutrophil counts and are increased by traditional cardiovascular risk factors such as smoking, obesity, hyperglycemia, and dyslipidemia. S100A8/A9 is an endogenous ligand of toll-like receptor 4 (TLR4) and of the receptor for advanced glycation end products (RAGE) and has been shown to promote atherogenesis in mice. In humans, S100A8/A9 correlates with the extent of coronary and carotid atherosclerosis and with a vulnerable plaque phenotype. S100A8/A9 is locally released following myocardial infarction and amplifies the inflammatory responses associated with myocardial ischemia/reperfusion injury. Elevated plasma levels of S100A8/A9 are associated with increased risk of future coronary events in healthy individuals and in myocardial infarction survivors. Thus, S100A8/A9 might represent a useful biomarker and therapeutic target in cardiovascular disease. Importantly, S100A8/A9 blockers have been developed and are approved for clinical testing.  Inflammation plays a central role in the development of atherosclerosis and in plaque vulnerability . The chrThe S100 proteins form a calcium-binding cytosolic protein family defined by their common ability to dissolve in 100% ammonium sulphate . Several++CD16− monocytes compared to their nonclassical CD14+CD16++ counterparts , 8349, 8The demonstrated associations between S100A8/A9 and the incidence of acute CV events have prompted further research into the role of S100A8/A9 as potential disease mediator and prognostic biomarker in coronary artery disease. Plasma S100A8/A9 was found to be highly increased during the ischemic event in acute coronary syndrome patients compared with stable angina or with individuals with angiographically assessed normal coronary artery morphology  90]. As. As90]. α and IL-6 [Compared with cardiac troponin, which is acutely released from necrotic cardiomyocytes and peaks within hours after the ischemic injury, S100A8/A9 peaks after 3–5 days and continues to be elevated for several weeks after the event . S100A8/and IL-6 , 95. Monand IL-6 . In a moand IL-6 . Thus, Sand IL-6 .To date, the only study assessing the value of S100A8/A9 as a potential prognostic biomarker in the immediate post-ACS period has been performed by Morrow et al. in 237 case-control pairs selected from the PROVE IT-TIMI 22 statin therapy trial cohort . S100A8/2+ and Zn2+ dependent manner and blocks their interaction with RAGE and TLR4 [in vivo, as ABR-215757 inhibits TNFα production in response to LPS challenge in a mouse model, to a similar extent as a Fab antibody fragment specific for the interaction site of S100A9 with its receptors [−/− mice demonstrated that ABR-215757 treatment reduced atherosclerotic plaque size, inflammation, and vulnerability features [Due to its potential involvement in atherogenesis, plaque vulnerability, ischemia-associated myocardial inflammation, and heart failure, S100A8/A9 represents an attractive target in CVD. Quinoline-3-carboxamides are orally active chemical compounds with potent anti-inflammatory properties in various models of autoimmune disease such as SLE, experimental autoimmune encephalomyelitis, and collagen arthritis –102. Theand TLR4 . This efeceptors . Additioeceptors . Testingeceptors , juvenileceptors , SLE [99eceptors . ABR-215features .The experimental and clinical studies presented in the present review have demonstrated a promising potential for S100A8/A9 as a clinical biomarker and treatment target in CVD. As biomarker, S100A8/A9 correlates with the extent of subclinical carotid and coronary artery disease, increases rapidly in plasma during myocardial ischemia and necrosis, and is associated with unfavorable prognosis in MI and heart failure patients and in patients undergoing carotid arterectomy . HoweverThe main obstacle related to the use of S100A8/A9 as a therapeutic target in CVD is the relative abundance of this protein in human circulation, with median values of approximately 5 mg/L in healthy individuals, rising up to 15 mg/L in ACS patients , 97. HowTo conclude, S100A8/A9 seems to play a central role in the complex interactions between innate immunity, traditional CV risk factors, and CVD. Activated neutrophils and monocytes are the main sources of extracellular S100A8/A9 and diabetes, dyslipidemia, obesity, and smoking are associated with elevated circulating protein levels. S100A8/A9 seems to be involved in atherogenesis, plaque vulnerability, and post-ischemic myocardial damage. Pending further investigation, S100A8/A9 might serve as a clinical biomarker and therapeutic target in CVD, with S100A8/A9 blockers readily available and approved for clinical testing.


MYBBP1A is a predominantly nucleolar transcriptional regulator involved in rDNA synthesis and p53 activation via acetylation. However little further information is available as to its function. Here we report that MYBBP1A is developmentally essential in the mouse prior to blastocyst formation. In cell culture, down-regulation of MYBBP1A decreases the growth rate of wild type mouse embryonic stem cells, mouse embryo fibroblasts (MEFs) and of human HeLa cells, where it also promotes apoptosis. HeLa cells either arrest at G2/M or undergo delayed and anomalous mitosis. At mitosis, MYBBP1A is localized to a parachromosomal region and gene-expression profiling shows that its down-regulation affects genes controlling chromosomal segregation and cell cycle. However, MYBBP1A down-regulation increases the growth rate of the immortalized NIH3T3 cells. Such Mybbp1a down-regulated NIH3T3 cells are more susceptible to Ras-induced transformation and cause more potent Ras-driven tumors. We conclude that MYBBP1A is an essential gene with novel roles at the pre-mitotic level and potential tumor suppressor activity. The 160-kDa Myb-binding protein 1a  is a nucleolar protein whose function is mostly unknown. Although predominantly localized in the nucleolus, MYBBP1A is found also in the nucleoplasm S. cerevisiaetrans-activating capacity by competing with p300 Prep1-hypomorphic mice is responsible for the hyper-activation of PGC-1α and increased insulin sensitivity The ubiquitously expressed MYBBP1A was originally identified as an interactor of the c-Myb oncoprotein with homology to the fifth essential DNA polymerase of MYBBP1A is also required for the acetylation and subsequent stabilization of p53 since it prevents Mdm2-dependent degradation of p53 in vivo data are essential to fully evaluate the function of MYBBP1A.These multiple and diverse functions of MYBBP1A suggest that this protein plays an important role in essential biological functions such as cell division, cell proliferation and apoptosis. However nothing is known of its importance relative to other well-known regulators of these pathways, nor about its physiological function during development. Indeed, at least some functional interactors of MYBBP1A, such as p53, are not essential during development. It is clear, therefore that MYBBP1A is essential for early mouse development prior to blastocyst formation. We also report that MYBBP1A down-regulation induces apoptosis and mitotic anomalies in mouse embryonic stem cells, embryonic fibroblasts and human HeLa cells, where it alters the expression of key cell cycle regulators. Moreover, Mybbp1a may act as a tumor suppressor, as its down-regulation facilitates the transformation of NIH3T3 cells by the HRas oncogene.In this paper we report that The mice were housed in the University of Adelaide Medical School Animal House and all mouse work was covered by ethics approval from both the Institute of Medical and Veterinary Science and the University of Adelaide animal ethics committees. This project was covered by University of Adelaide Animal Ethics Committee approvals M6098 and M5201 and IMVS Animal Ethics Committee project no. 69/98.Mybbp1a targeting vector was constructed from sequences subcloned from an E14TG2a ES cell genomic library. A 3.8 kb 5′ genomic fragment containing the promoter region approximately 450 bp upstream from the initiation ATG was generated by PCR amplification using oligos  incorporating NotI and XbaI sites at the 5′ and 3′ ends respectively. A 6.0 kb XbaI/HindIII 3′ genomic fragment encompassing part of exon 14, exons 15–26 and 3′ flanking sequences was cloned downstream of the 5′ fragment. The 1.8 kb pgkNEO expression cassette Mybbp1a genomic fragments in the opposite transcriptional orientation to the Mybbp1a gene . The Mybbp1a targeting vector was electroporated into the W9.5 ES cell line and selected with G418 as described Suppl. Figure S1), a 950 bp fragment located 60 bp upstream of the 5′ end of the targeted region. This probe was generated by PCR amplification using the oligonucleotides: 5′-CTTGGGTCTTCTTGGGGTTCC-3′; 5′-GCCAGCATGGCAGGCTTGG-3′. The targeted deletion was confirmed by further analysis of Southern analysis of selected clones with the 3′ genomic DNA probe . Targeted clones were injected into C57BL/6 blastocysts. Germline transmission was seen for a single clone and chimeric offspring were bred to C57BL/6 mice to derive heterozygous MYBBP1A+/− mice.The Suppl. Figure S1 B, C. Mouse embryos were genotyped by PCR using oligonucleotides KO3 and KO4 to detect the wild type allele (488 bp product) and KO3 and KO5 to detect the targeted allele (570 bp product) as shown in Suppl. Figure S1 D. . DNA from E9.5, E6.5 embryos and from blastocyst outgrowths was prepared by boiling at 100°C for 8–10 min in water∶PBS (1∶1). Blastocysts were cultured in DMEM containing 10% FCS for 4–6 days and outgrowth material was collected for PCR analysis as above.Chimeric mice and their heterozygous progeny were backcrossed for at least six generations onto a C57BL/6 background. Mouse genotyping was performed on tail DNA of weaned mice and E11.5 embryos prepared using DNeasy kits (Qiagen), Southern blot analysis of XbaI-digested DNA and probing with the 5′ probe as described above and shown in Mybbp1a -shRNA or empty vector lentiviral infection and puromycin selection (1 µg/mL), 1.25×105 cells per well were plated in 6-well plates and cultured. Cells were harvested and counted in triplicate on days 1, 2 and 3.Wild-type ES cells were derived from E2.5–3.5 C57Bl/6 embryos using standard procedures MEFs were derived from wt C57BL/6 embryos using standard procedure.2 in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetal bovine serum , 0.2 mg/ml streptomycin (Gibco), 20 U/ml penicillin (Gibco), 2 mM glutamine (Gibco) and 1 mM sodium pyruvate (Gibco). HEK 293T were grown in Iscove's Modified Dulbecco's medium  supplemented with 10% FBS, 0.2 mg/ml streptomycin, 20 U/ml penicillin and 2 mM glutamine.NIH3T3, MEFs, HeLa and Phoenix cells were grown at 37°C, 5% COTo block cells at the G1/S transition, a double-Thymidine block was used. Cells (NIH 3T3 or HeLa) were treated with 2 mM Thymidine (Sigma) for 18 hours (first block), followed by a release of 9 hours and a second pulse with 2 mM Thymidine for 17 h (second block).MYBBP1A in HeLa cells, 2×105 cells/well were plated in triplicate in 6-well plates for 24 hours, transfected using lipofectamine (Invitrogen) with 40 nM siRNA or control oligonucleotides , following the manufacturer protocol. The sequences of the siRNAs are the following:To transiently silence CCAGGCTGGTGAATGTGCTGAAGAT, 52% GC);siRNA1 ;siRNA2 .siRNA3 , 5 µg packaging plasmid (pMDLg/p RRE), 2.5 µg of pRSV-REV and 15 µg of the target plasmid  following a calcium phosphate transfection protocol. The day after transfection the medium was replaced by fresh medium supplemented with 1 mM Sodium Butyrate (Sigma). After 30 h, the viral supernatant was collected and used to infect NIH 3T3 cells. The day after the infection 2 µg/ml Puromycin was added to the medium for selection. The shRNA target sequences are the following:CCGGCCTGCCCTAGAGACTCCTATTCTCGAGAATAGGAGTCTCTAGGGCA); shRNA2 (CCGGCCTGATGAAGTCCGTGCAATTCTCGAGAATTGCACGGACTTCATCA); shRNA3 (CCGGCCGGAGTGTATTTGGTCATATCTCGAGATATGACCAAATACACTC); shRNA4 (CCGGCCCAATGATTCGGAGATGAAACTCGAGTTTCATCTCCGAATCATTG); shRNA5 (CCGGCCAAGCGTAACAGCTCACTTACTCGAGTAAGTGAGCTGTTACGCTT).shRNA1  were used as suggested by the manufacturer.5 cells/well in a 6-well plate and transfecting them with lipofectamine the day after. At 24, 48 and 72 h after transfection the cells were detached and counted with a cell counter .Measurement of HeLa cell proliferation was performed by plating 2×102O) for 20 min. at RT. Cells were washed with dH2O and dried at RT. Stained and dried cells were solubilized with a solution of 10% Acetic Acid for 10 min. at RT and analyzed with a Microplate Reader 680 (Bio-Rad) at 570 nm.Crystal Violet assay was performed plating 3,000 cells/well in a 12-well plate and fixing the cells at the following time points: T0 (8 h after plating) and 2, 4, 6, 8, 10 days after. Cells were washed in PBS, fixed with 11% glutaraldehyde (Sigma) for 15 min. at RT, washed again in PBS and allowed to dry at RT. Cells were stained with Crystal Violet solution  and plated again at 300000 cells per well and counted again the third day. The procedure was repeated 5 times.Cells were washed with PBS 1× and scraped with RIPA buffer . Cells were incubated on ice for 30 min and after centrifugation at 14,000 rpm for 15 min at 4°C, protein concentration was detected with the Bradford reagent  and measurement at 595 nm with Ultraspectrophotometer 2100pro. Protein extracts were resuspended in Laemmli buffer ES cell immunoblotting analysis was performed with anti-Oct-3/4 (C-10) , anti-Cleaved Caspase-3 (Asp175) , anti p160, and anti-Vinculin .Cells were grown on sterile 13 mm glass coverslips  for at least 24 h. Cells were washed with PBS and fixed  for 10 min at RT. After washing with PBS, cells were permeabilized with 0.2% PBS-Triton X-100 for 5 min at RT, washed with PBS and blocked with 1× PBS-1% BSA for 30 min at RT. Coverslips were incubated with the indicated antibodies in blocking solution for 30 min at 37°C. Cells were washed with PBS and incubated with the secondary antibodies (Alexa-488 and Alexa-546 conjugated anti-mouse or anti-rabbit immunoglobulin) and incubated for 30 min at RT protected from light. Cells were washed again with PBS and incubated with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI) nuclear staining (Fluka) for 3 min at RT or Hoechst 33258 . Cells were then washed with PBS and coverslips mounted with Fluorescent Mounting Medium (Dako Cytomation). Images were obtained with a Leica DMIRE2  with a 63× objective. Incubations with matched mouse isotype IgGs, irrelevant rabbit IgGs or secondary antibodies were always negative.After the washing step following the incubation with the secondary antibodies, cells were fixed again in 4% paraformaldehyde, 0.1% Triton X-100 at 4°C for 30 minutes. Cells were then incubated for 30 min at RT with 100 mM Glycine (VWR) and washed once with PBS. FISH was performed with Telomere PNA FISH kit/Cy3 (DAKO) following the manufacturer instructions; PNA probe was labeled with Cy3.5 cells/well in triplicate), transfected as described above with siRNA or High-GC control and blocked at the G1/S transition using a double thymidine block. Starting 8 h after the release from the block and up to 24 hours later, the cells were analyzed with Time Lapse Microscope (OKO VISION). Images were taken in 4 different fields for each sample every 5 min with a 5× objective and analyzed with Image J software.Cells were plated in a 6-well plate (2×10For 5′ethynyl-2′-deoxyuridine (EdU)/7-amino-actinomycin D (7-AAD) double staining, cells were grown and pulsed before the assay with 10 µM EdU for 1 hour, harvested and processed with Click-iT EdU Flow Cytometry Assay kit (Invitrogen) following the manufacturer protocol. Anti-EdU-Fluorescein-isothycyanate (FITC) was used to stain EdU positive cells and 7-AAD was used to stain DNA. Cells were analyzed with FACS CANTO or FACS Calibur (BD Immunocytometry). Results were displayed as bivariate distribution of EdU content versus DNA content. The percent of cells in the S-phase was calculated by gating EdU positive cells using FCS Express V3 (De Novo Software) or CellQuest program (BD).For Annexin V staining, cells were harvested, washed with PBS 1× and the dead and apoptotic cells were detected by Annexin V Phycoerythrin (PE)/7-AAD staining using PE Annexin V Apoptosis Detection kit I (BD Pharmingen), following the manufacturer's instructions. Cells were analyzed with FACS CANTO or Calibur (BD) and results were displayed as bivariate distribution of Annexin V positive cells versus 7-AAD positive cells. The percentage of early apoptotic cells was calculated by gating Annexin V positive cells using FCS Express V3 or CellQuest programs.MYBBP1A ; CDKN1A ; NFKB2 ; GADD45B ; JUN ; TOP2B . The following genes were analysed with Applied Biosystems probe sets: CKS1b (probe Hs01029137_g1); SFN (probes Hs00602835_s1 Hs00968567_s1); TOP2A ; BRCA1 (probe Hs00173233_m1); CDK7 (probe Hs00361486_m1).Total RNA was extracted  and quantified by spectrophotometry . Total RNA (5 µg) was reverse-transcribed using a SuperScript First-Strand kit with random primers (Invitrogen) according to the manufacturer's instructions. For quantitative Real-Time PCR, 20 ng of the reverse-transcribed RNA was amplified with a Light-Cycler 480 (Roche) using a Universal Probe Library Assay (Roche) and following the manufacturer instructions. The PCR conditions were as follow: pre-incubation at 95°C for 5 minutes, 50 cycles of amplification , cooling at 40°C for 10 s. The amount of the different mRNAs was normalized to GAPDH mRNA. The sequences of the primers used with the relative probes are the following: http://www.affymetrix.com/support/technical/manual/expression_manual.affx). The size and the accuracy of quantification of targets were checked by agarose gel electrophoresis of 2 µl aliquots of each sample after fragmentation. External controls (spikes) were used; each eukaryotic GeneChip® probe array contains probe sets for several B. subtilis genes that are absent in the samples analyzed . This Poly-A RNA Control Kit contains in vitro synthesized, polyadenylated transcripts for these B. subtilis genes that are pre-mixed at staggered concentrations to assess the overall success of the assay. Samples were then hybridized as indicated by Affymetrix. A single GeneChip Human Gene 1.0 ST was then hybridized with each biotin-labelled sense target. Hybridizations were performed for 16 h at 45°C in a rotisserie oven. GeneChip cartridges were washed and stained with GeneChip Hybridization, Wash and Stain Kit in the Affymetrix fluidics station following the FS450_0007 standard protocol. Images were scanned using an Affymetrix GeneChip Scanner3000 7G with default parameters. The resulting images were analysed using GeneChip Operating Software v1.2 (GCOS1.2). Raw data were first normalized using RMA method http://david.abcc.ncifcrf.gov/home.jsp) according to RNAs from three technical replicates of HeLa cells transfected with High-GC control or siRNA1, were analyzed with Affimetrix Human Gene 1.0 ST chip (Affimetrix). Biotin-labelled cDNA targets were synthesized starting from 100 ng of total RNA. Double stranded cDNA synthesis and related cRNA was obtained with GeneChip WT cDNA Synthesis and Amplification Kit (Ambion). With the same kit the cDNA sense strand was synthesized, then fragmented and labelled with GeneChip WT Teminal Labeling Kit (Ambion). DNA fragmentation was performed with a combination of uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1), while labeling was completed using terminal deoxynucleotidyl transferase (TdT) in the presence of GeneChip® DNA Labeling Reagent. All steps of the labelling protocol were performed as suggested by Affymetrix -2,5-diphenyltetrazolium bromide  for at least 4 h. After staining, the plate was scanned and the image obtained was analyzed with Image J Software.Mybbp1a shRNA  and selected with puromycin (4 µg/mL). After selection, stable puromycin-resistant pools were subsequently infected with pBABE-RasV12 or empty pBABE vector (as indicated), selected with 50 mg/mL hygromycin and 4×106 cells for each condition were injected in nude mice (6 mice per group). After the injection, mice were monitored daily for 18 days and tumor volume measured every 2 days.NIH 3T3 cells were infected with either an lentivirus empty vector (NT) or a vector expressing specific The following antibodies were used: mono- and polyclonal antibodies against Flag ; polyclonal antibody against p160C ; mouse monoclonal antibody against Prep1 ; mouse monoclonal antibody against Tubulin (Sigma); rabbit polyclonal antibody against Phospho Histone H3 ; human monoclonal antibody against CREST (homemade); mouse monoclonal antibody against Nucleolin (Santa Cruz Biotecnology); mouse monoclonal antibody against Nucleophosmin ; rabbit polyclonal antibody against active Caspase3 and mouse monoclonal antibody against Caspase 9 .Mybbp1a gene is shown in Supplementary Figure S1  including the ATG, exons 1 to 13 and a half of exon 14 . Healthy and fertile heterozygous Mybbp1a+/− mice were obtained. However heterozygous crosses yielded no homozygous knockout mice, indicating that Mybbp1a is essential for mouse development (Mybbp1a−/− embryos at E11.5 (p<0.05), E9.5 (p<0.05) nor at E6.5 (p<0.05). Blastocysts were then isolated from a Mybbp1a+/− heterozygous cross, grown in culture for several days and genotyped. No Mybbp1a −/− blastocysts were detected . Within 72 h of transduction, MYBBP1A shRNA3 retrovirus reduced Mybbp1a protein expression by 82% with respect to non-targeting shRNA  fraction of Mybbp1a in other mouse cells (MEFs and NIH3T3) and in human HeLa cells. Primary MEFs (only two passages in culture) down-regulated for Mybbp1a showed a rapid entry into senescence compared with cells treated with non-targeting vector (NT) in a long term proliferation protocol  was surprising and will be discussed further below.Overall, these results show that both in most of the cell types tested, MYBBP1A is required for normal rates of cell proliferation. The different effect observed in NIH3T3 cells upon MYBBP1A knock-down in HeLa cells. First, time-lapse experiments showed major mitotic abnormalities in MYBBP1A-down regulated HeLa cells. In most control cells the time required for mitosis was about 70 minutes but increased to an average of 92 after MYBBP1A-down regulation  while about 40% died (4% in Control) (We next investigated the basis for cell growth inhibition induced by Control) Fig. 3B.MYBBP1A-down regulated HeLa cells undergo a specific block of the cell cycle at G2/M. Flow cytometry for EdU and 7AAD  and a decrease in the number of cells in S phase, suggesting that the growth of the cells is delayed due to a block prior to cytokinesis. Interestingly  there may be a minor effect at the G1/S checkpoint . No such aberrations were observed in the control cells . These data suggest that MYBBP1A-down regulation causes a block/delay of cells in S/G2/M and that those cells that reached mitosis acquired major abnormalities. Finally, the number of cells in prophase increased at the expense of those in anaphase , respectively, upon siRNA-transfection, compared to control transfection (about 6%) (MYBBP1A down-regulation (50+/−10% at 48 h and 80+/−30% at 72 h) (data not shown). This suggests an even greater effect on cell death.MYBBP1A all point to a possible involvement of MYBBP1A in mitosis. Since nucleoli are disrupted at mitosis we examined the localization of the nucleolar protein MYBBP1A in various phases of mitosis. Suppl. Figure S2B shows that the distribution of Mybbp1a is not due to overexpression, since the same pattern is observed when endogenous Mybbp1a is examined in non-overexpressing cells with an antibody recognizing only the full-length protein.The growth inhibition, extension of mitosis and G2/M block upon down-regulation of Suppl. Fig. S3).At metaphase and anaphase, immunofluorescent staining (IF) showed that Mybbp1a co-localized with phospho-histone H3  where MYBBP1A again assumes a parachromosomal localization. Hence the differential effect of MYBBP1A down-regulation in HeLa v. NIH3T3 cells is not due to a different mitotic localization.The same results were obtained with other cell lines, e.g. HeLa cells (See p<10−4); 71.4% of the genes were down-regulated and 28.6% up-regulated upon MYBBP1A silencing (http://david.abcc.ncifcrf.gov/) shows that 49.8% of these genes fall into categories involved in cell-cycle, mitosis, DNA-damage and stress response, apoptosis, chromosome segregation and chromatin  was carried out on an Affymetrix Gene ST 1.0 Human platform. Of the 28829 genes analyzed, 853 were differentially expressed , in immortalized NIH3T3 and in cancer HeLa cells. In all cases, the deletion or down-regulation of MYBBP1A has had a profound effect on the growth rate of the cells.The present data define an essential role of MYBBP1A in cell cycle and mitosis, as its absence/down-regulation causes chromosomal defects, G2/M arrest, mitotic anomalies and enhances the tumorigenicity of Ras. We have examined the function of MYBBP1A In vivo, MYBBP1A deletion leads to embryonic death at the blastocyst stage. Further analysis is required to determine what is occurring in the mutant embryos and whether cell division defects, as observed in the cell lines tested here, are responsible. Gene expression analyses of human MYBBP1A down-regulated HeLa cells shows altered expression of genes involved in chromosome segregation, kinetochore formation and, in general, mitosis. The involvement of MYBBP1A in mitosis is also directly suggested by its peri-chromosomal localization during metaphase  of genes important in cell cycle regulation. In addition, this analysis revealed, as mentioned above, genes that are important in chromosomal segregation and kinetochore formation. It appears therefore, that MYBBP1A affects two important steps of cell division: the cell cycle and mitosis.Mybbp1a down-regulation in NIH3T3 cells, however, has a different effect - stimulating rather than blocking growth, and enhancing Ha-RasV12 tumorigenesis. In these cells, the shRNA constructs were somewhat less efficient, in general causing about 60% decrease in Mybbp1a levels .MYBBP1A affected the expression of several genes involved in cell cycle control, DNA damage and cell death . Analysis of the genes affected by the MYBBP1A down-regulation  Targeting strategy. The wild type Mybbp1a locus spanning the coding region (18.2 kb) is depicted (top). The Mybbp1a targeting vector  PCR genotyping of mouse embryos from Mybbp1a+/− intercrosses. Controls are in lane 1 (−), which lacks template and contains the 3 primers KO3, KO4 and KO5, and lanes 2 and 3 (C+/−), which is genomic DNA from an adult heterozygous mouse. The remaining lanes are material from blastocyst outgrowths harvested from Mybbp1a+/− intercrosses. The upper band of 570 bp corresponds to the targeted allele and the 488 bp lower band corresponds to the wild type allele. Each allele was detected in a separate reaction. M, DNA size markers.ctor see  was cons(TIF)Click here for additional data file.Figure S2Mybbp1a redistribution at mitosis. A. Representative immunofluorescence confocal images of NIH 3T3 cells stably expressing Mybbp1a-Flag (p160-Flag) in the mitotic phases indicated on the left. Cells were stained with antibodies against Flag , α-Tubulin (green) and DAPI (blue) for DNA. Bar = 10 µm. B. Immunofluorescence staining of endogenous Mybbp1a in NIH3T3 cells compared with the staining of the cells overexpressing Mybbp1a-Flag.(PDF)Click here for additional data file.Figure S3Mybbp1a does not localize at the kinetocore and on telomeres. Representative confocal immunofluorescence of NIH 3T3 cells stably expressing Mybbp1a-Flag in the cell-cycle phases indicated on the left of the panels. The staining was performed against Flag , CREST , PNA  and with DAPI (blue) for DNA. Bar = 5 µm.(TIF)Click here for additional data file.Figure S4Immunofluorescence analysis of MYBBP1A localization at interphase (top) and metaphase (bottom) in HeLa cells. MYBBP1A localizes in the nucleoli at interphase but around the chromosomes at metaphase.(TIF)Click here for additional data file.Figure S5Cell Cycle defects in MYBBP1A-depleted HeLa cells. Four examples of time-lapse microscopic analysis, showing the widespread variation of the time required for mitosis after MYBBP1A-specific downregulation with si1 siRNA in HeLa cells. MYBBP1A-silenced cells have an extended mitotic period. Wide-field Time-Lapse video imaging of HeLa cells transfected with siRNA1 for 48 hours and synchronized with double thymidine block. Images were taken every 5 minutes starting 8 hours after the release from the block (mitosis start) with Oko-Vision Time Lapse microscope for a total of 18 hours. Four different examples are provided.(TIF)Click here for additional data file.Figure S6Representative mitotic alterations caused by MYBBP1A depletion. Confocal microscopy of control (A) and siRNA1 transfected (for 48 hrs) HeLa cells (B). In panel B: 1) multipolar spindle, 2) and 3) altered metaphasic plate, 4) triangular cytodieresis. Cells were stained with anti-Tubulin (green) antibodies to image the mitotic spindle and with DAPI (blue) for DNA. Bar = 12 µm.(TIF)Click here for additional data file.