To determine the spatial and temporal dynamics of influenza A virus during a single epidemic, we examined whole-genome sequences of 284 A/H1N1 and 69 A/H3N2 viruses collected across the continental United States during the 2006–2007 influenza season, representing the largest study of its kind undertaken to date. A phylogenetic analysis revealed that multiple clades of both A/H1N1 and A/H3N2 entered and co-circulated in the United States during this season, even in localities that are distant from major metropolitan areas, and with no clear pattern of spatial spread. In addition, co-circulating clades of the same subtype exchanged genome segments through reassortment, producing both a minor clade of A/H3N2 viruses that appears to have re-acquired sensitivity to the adamantane class of antiviral drugs, as well as a likely antigenically distinct A/H1N1 clade that became globally dominant following this season. Overall, the co-circulation of multiple viral clades during the 2006–2007 epidemic season revealed patterns of spatial spread that are far more complex than observed previously, and suggests a major role for both migration and reassortment in shaping the epidemiological dynamics of human influenza A virus. This study is the first of its kind to reconstruct the spread of an epidemic of influenza A virus across a single country, in this case the United States. In contrast to a single viral lineage spreading across this country, a phylogenetic analysis of the whole-genome sequences of more than 300 influenza A viruses of the A/H1N1 and A/H3N2 subtypes sampled from the 2006–2007 epidemic season reveals that multiple phenotypically and antigenically distinct viral lineages of entered and co-circulated in the US during this time. Furthermore, the widespread co-circulation of multiple lineages, even in geographically remote localities, allowed for frequent reassortment between influenza A viruses of the same subtype. Through reassortment, a minor lineage of A/H3N2 viruses surprisingly re-acquired sensitivity to the adamantane class of antiviral drugs, and a new A/H1N1 antigenic variant emerged that later became globally dominant. In sum, these results highlight the complexity of the spread of influenza A virus in time and space, and highlight the need for intensified global surveillance involving whole-genome sequence data. Intensive study of the molecular evolution of influenza A virus has provided important insights into its seasonal genesis and spread in human populations Although antigenic drift is clearly a key determinant of influenza A virus evolution, this process has rarely been observed in a single locality over a single epidemic season Every winter, epidemics of human influenza recur in the United States, and are associated with an annual average of 226,000 hospitalizations and 36,000 deaths, mainly caused by secondary bacterial pneumonia in the elderly and young children Our goal here is to determine the spatial-temporal dynamics of influenza A virus during a single epidemic season (2006-2007) in the United States through the phylogenetic analysis of whole-genome sequence data. Since the 1968 pandemic, A/H3N2 viruses typically dominate most influenza seasons, including 16 of the past 20 US epidemics , defined by both high bootstrap values and long branch lengths, are evident on the trees of each genome segment, as exemplified by the HA phylogeny . The phyClade A was the most geographically and temporally pervasive of the eight clades, circulating in 24/30 localities and 14/15 weeks studied, although allowall clades were sampled over wide temporal and geographic scales , 4. NotaThe number of isolates collected from different US localities varied widely  We inferred the antigenic characteristics of these eight clades based on their phylogenetic relationships and the number of amino acid differences at antigenic sites in the HA from vaccine reference strains of known antigenicity. Accordingly, set 1 clades A, B, C, D, and E are likely to be New Caledonia-like in antigenicity, given that (a) 90% of A/H1N1 viruses from this US epidemic were New Caledonia-like  were available for study due to the dominance of the A/H1N1 subtype during the 2006–2007 season, abundant genetic diversity is evident on all eight segment phylogenies, as exemplified by the HA tree  of the A/H3N2 isolates from the 2006–2007 US epidemic were members of a major clade (denoted clade ‘a’). On both the HA and NA phylogenies, clade a contains the antigenically novel A/Brisbane/10/2007 isolate selected for the 2008–2009 vaccine, whereas only three 2006–2007 singleton isolates  belong to the clade that contains the 2006–2007 influenza vaccine strain A/Wisconsin/67/2005, confirming prior observations of a vaccine mismatch  [30]. Th7.0% of tIn addition to the major clade a, a minor clade of five A/H3N2 viruses, denoted clade b, also circulated during the 2005–2006 season . AlthougFour singleton A/H3N2 viruses  also circulated during this season . IsolateMajor topological differences between the eight phylogenies of the A/H3N2 virus genome strongly suggest that several reassortment events took place involving multiple clades from the 2006–2007 US epidemic. Whereas the adamantane-resistant clade a and the sensitive clade b both appear to derive from the N-lineage on the trees for the PB2, PA, HA, NA and NS segments, clade b instead derives from the adamantane sensitive clades from 2004–2005 on the M and PB1 trees , 8. ThisNo clear signal of the geographical spread of A/H3N2 influenza viruses could be detected due to our small sample size. All clades were geographically widespread and a secondary parsimony character mapping analysis again revealed strong population subdivision and weak migration . The major clade a was present in all thirteen localities in which A/H3N2 viruses were collected, and the five isolates contained in minor clade b were geographically dispersed across both urban and remote areas spanning four of five US regions: Los Angeles, California; Chicago, Illinois; Hopkinsville, Kentucky; Madison, Alabama; New York City, New York; and Houston, Texas . New YorThis study utilized whole-genome sequence data from a surveillance initiative of unprecedented scope and scale that sampled both A/H1N1 and A/H3N2 influenza viruses across the US over the course of a single season through the Influenza Genomics Sequencing Project Substantial antigenic diversity was also observed during the 2006–2007 season in the US, as at least five antigenically distinct types of influenza A virus co-circulated: three antigenically distinct variants of A/H1N1 viruses , and at least two antigenically different types of A/H3N2 virus (A/Wisconsin/67/2005-like and A/Brisbane/10/2007-like), while clade b and isolate s4 also may represent additional antigenic variants of A/H3N2 virus . HoweverAlthough A/Solomon Islands/3/2006-like viruses and A/Brisbane/59/2007-like A/H1N1 viruses were represented only by minor clades during the 2006–2007 season , Solomon Islands-like viruses achieved global A/H1N1 dominance by the start of the 2007–2008 season, and the reassortant clade of Brisbane-like viruses rose to dominance later during the 2007–2008 season The extensive genetic diversity present in both A/H1N1 and A/H3N2 viruses suggests that multiple introductions of virus have taken place during the 2006–2007 season, particularly as our method of collecting viruses clearly underrepresented areas that are major ports of international travel. As a case in point, further sampling in the Los Angeles and New York City regions, where our study still detected significant diversity even at very low sampling levels, would likely augment the total number of viral lineages detected, including those imported from South-East Asia. By sampling in both metropolitan and relatively isolated areas, our study yielded important information on the geographic distribution of viral genetic variation: namely, that extensive viral diversity, including multiple antigenically distinguishable lineages, disseminated widely across the entire United States during the epidemic, even into relatively remote areas, so that it was not confined to the major cities where the virus is thought to enter. As a particular case in point, even relatively low-density areas or those distant from major metropolitan areas, such as Hopkinsville, Kentucky , harbor significant amounts of both genetic and antigenic diversity, suggesting that influenza viruses of multiple antigenic (and other phenotypic) types extensively infiltrate the United States over the course of a single season. However, it is important to note that our analyses cannot exclude that a single co-infected individual could have introduced multiple clades of influenza virus into the United States, as the frequency of co-infection among patients in this study is unknown and represents a key area for further research.Importantly, it is also possible that the 2006–2007 US epidemic was particularly difficult to reconstruct due to the unusual complexity of its evolutionary dynamics, which likely relates to the incomplete dominance of either the A/H1N1 or A/H3N2 subtype. The dynamics of influenza virus epidemics vary greatly on an annual basis, and influenza epidemics that are dominated by the A/H3N2 virus have been associated with higher disease transmission and more rapid spread than milder A/H1N1-dominated seasons, as well as stronger synchrony in timing across the United States Additional sequencing of influenza viruses in areas outside the United States is also essential to understand the global context of the diversity that enters the US during a given epidemic. From this, and previous studies Our findings also suggest that the genetic diversity of the A/H3N2 virus is substantial even when A/H3N2 is not the dominant subtype, as was the case for most of the 2006–2007 epidemic. A major clade (a), a minor clade (b), a reassortant singleton (s4), and three singletons  that appear to be descendents of the N-lineage Finally, the existence of the adamantane-sensitive clade b (A/H3N2) during this epidemic was surprising, given that global resistance to adamantanes among influenza A/H3N2 viruses has increased dramatically in recent years, with more than 95% of A/H3N2 influenza viruses classified as resistant in the previous 2005–2006 season in the US All viruses were collected as part of a larger 2006–2007 US surveillance effort conducted by Surveillance Data Inc., in which a total of 610 influenza virus specimens of both type A and type B were obtained from nasal and nasopharyngeal swabs from patients seen with influenza-like illness. At the time of this study, 353 type A influenza virus genomes had been sequenced and were available for study. Fifty-six participating physicians, primarily located at family practices, were recruited from 21 states that were geographically distributed across the US . Doctors swabbed all patients ≥ one year of age who presented with fever and upper respiratory symptoms from December 1, 2006 to April 1, 2007. An in-office immunoassay rapid test (Quidel QuickVue Influenza A+B Test) was used to identify positive influenza samples (A or B), and positive swabs were sent to a reference laboratory in Rochester, New York, to be typed as AH1N1, A/H3N2, or influenza B, following growth in Primary Rhesus Monkey Kidney Cells (RhMK) culture. RNA was extracted from viruses via automated nucleic acid extraction using the Roche MagNA Pure instrument and was shipped to the J. Craig Venter Institute in Rockville, Maryland, for whole virus sequencing . However, as A/H3N2 replaced A/H1N1 as the dominant subtype towards the end of the 2006–2007 season in March, the majority  of A/H3N2 isolates used in this study were collected between February 25, 2007–March 10, 2007. These 69 A/H3N2 isolates came from 13 states representing all six US regions. Within these 13 states, isolates were sampled from 20 localities : Columbiana, AL , Madison, AL; Hacienda Heights, CA ; Louisville, CO ; St. Petersburg, FL ; Bloomingdale, IL , Lake Zurich, IL , Naperville, IL ; Florence, KY , Hopkinsville, KY; Bronx, NY , Glendale, NY; Akron, OH, Washington, OH; Newberg, OR ; Conroe, TX ; Dale City, VA , Richmond, VA; Bennington, VT ; Woodinville, WA .Genome sequences were downloaded from the NCBI Influenza Virus Resource For the A/H3N2 viruses, phylogenetic trees were inferred using the maximum likelihood method available in PAUP* 4) model, with the frequency of each substitution type, proportion of invariant sites (I), and the gamma distribution of among-site rate variation with four rate categories (Γ4) estimated from the empirical data . In all cases tree bisection-reconnection (TBR) branch-swapping was utilized to determine the globally optimal tree. To assess the robustness of each node on the phylogenetic tree, a bootstrap resampling process  using the neighbor-joining (NJ) method was used, incorporating the ML substitution model. Clades of related isolates were identified by high bootstrap values (>70) and exceptionally long branch lengths.In each case, the best-fit model of nucleotide substitution was identified by MODELTEST 1, Ca2) The parsimony-based MacClade program Figure S1Phylogenetic relationships of the PB2 gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Colored rectangles (labeled A–H) represent eight clades of related viral isolates from the 2006–2007 U.S. season that are present on phylogenies for all genome segments , S5, S6.(0.50 MB EPS)Click here for additional data file.Figure S2Phylogenetic relationships of the PA gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Color schemes and rooting are same as in (0.45 MB EPS)Click here for additional data file.Figure S3Phylogenetic relationships of the NP gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Color schemes and rooting are same as in (0.49 MB EPS)Click here for additional data file.Figure S4Phylogenetic relationships of the NA gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Color schemes and rooting are same as in (0.49 MB EPS)Click here for additional data file.Figure S5Phylogenetic relationships of the M gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Color schemes and rooting are same as in (0.44 MB EPS)Click here for additional data file.Figure S6Phylogenetic relationships of the NS gene segment of A/H1N1 influenza viruses sampled from the United States during the 2006–2007 influenza season and globally from 2001–2006, estimated using an ML method. Color schemes and rooting are same as in (0.45 MB EPS)Click here for additional data file.Figure S7Phylogenetic relationships of the PB2 gene segment of 69 A/H3N2 influenza viruses sampled from the United States during the 2006–2007 influenza season and 104 background global A/H3N2 influenza viruses sampled from 2003–2006. Isolates that are adamantane sensitive (have a serine at position 31 of the M2 gene) are shaded in blue, while isolates that are adamantane resistant are shaded in red. Clades of related viral isolates are denoted by rectangles. Clades from the 2006–2007 U.S. epidemic are labeled as follows: major clade (a), minor clade (b), and four singleton isolates . Clades from previous global epidemics include the ‘N-lineage’ (adamantane-resistant isolates from 2005–2006). Bootstrap values (>70%) are shown for key nodes. The tree is mid-point rooted for purposes of clarity only, and all horizontal branch lengths are drawn to scale.(0.43 MB EPS)Click here for additional data file.Figure S8Phylogenetic relationships of the PA gene segment of 69 A/H3N2 influenza A viruses sampled from the United States during the 2006–2007 influenza season and 104 viruses sampled globally from 2003–2006. Labeling, color scheme, and rooting are identical as (0.43 MB EPS)Click here for additional data file.Figure S9Phylogenetic relationships of the NA gene segment of 69 A/H3N2 influenza A viruses sampled from the United States during the 2006–2007 influenza season and 104 viruses sampled globally from 2003–2006. Labeling, color scheme, and rooting are identical as (0.47 MB EPS)Click here for additional data file.Figure S10Phylogenetic relationships of the NS1/2 gene segment of 69 A/H3N2 influenza A viruses sampled from the United States during the 2006–2007 influenza season and 104 viruses sampled globally from 2003–2006. Labeling, color scheme, and rooting are identical as (0.46 MB EPS)Click here for additional data file.Table S1Amino acids at variable sites of the HA gene segment of A/H1N1 influenza viruses from clades A–H, the A/New Caledonia/20/1999(H1N1) and A/Solomon Islands/3/2006(H1N1) vaccine strains, and isolates from 2002/2003 and 2005 , with di(0.10 MB DOC)Click here for additional data file.Table S2http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html). Clade membership corresponds to the HA phylogeny (Influenza A viruses used in hylogeny .(0.49 MB DOC)Click here for additional data file.Table S3http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Influenza A viruses used in (0.09 MB DOC)Click here for additional data file.Table S4http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html), and accession numbers starting with ISD are from the Los Alamos National Laboratory's Influenza Sequence Database (www.flu.lanl.gov).Influenza A viruses used in (0.05 MB DOC)Click here for additional data file.Table S5http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Influenza A viruses used in (0.04 MB DOC)Click here for additional data file.Table S6http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Influenza A viruses used in (0.16 MB DOC)Click here for additional data file.Table S7http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).Influenza A viruses used in (0.17 MB DOC)Click here for additional data file.Table S8http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html).A/H3N2 influenza viruses used in the 2006–2007/2007–2008 (A/Wisconsin/67/2005) and 2008–2009 (A/Brisbane/10/2007) influenza vaccines, the HA and NA gene segments of which are included in (0.03 MB DOC)Click here for additional data file.Table S9Number of amino acid differences in the NA gene between clades A–H of A/H1N1 influenza virus from the 2006–2007 U.S. season. As comparison, isolates used as the H1N1 component of the influenza vaccine in 2006–2007  (NC99)) and 2007–2008 (A/Solomon Islands/3/2006(H1N1) (SI06)) are included. Amino acid differences between clades that cluster phylogenetically into set 1 (clades A–E and NC99) and clades that cluster phylogenetically into set 2 (clades F–H and SI06) are in bold.(0.05 MB DOC)Click here for additional data file.


APOBEC3 (A3) proteins constitute a family of cytidine deaminases that provide intracellular resistance to retrovirus replication and to transposition of endogenous retroelements. A3A has significant homology to the C-terminus of A3G but has only a single cytidine deaminase active site (CDA), unlike A3G, which has a second N-terminal CDA previously found to be important for Vif sensitivity and virus encapsidation. A3A is packaged into HIV-1 virions but, unlike A3G, does not have antiviral properties. Here, we investigated the reason for the lack of A3A antiviral activity.Sequence alignment of A3G and A3A revealed significant homology of A3A to the C-terminal region of A3G. However, while A3G co-purified with detergent-resistant viral nucleoprotein complexes (NPC), virus-associated A3A was highly detergent-sensitive leading us to speculate that the ability to assemble into NPC may be a property conveyed by the A3G N-terminus. To test this model, we constructed an A3G-3A chimeric protein, in which the N-terminal half of A3G was fused to A3A. Interestingly, the A3G-3A chimera was packaged into HIV-1 particles and, unlike A3A, associated with the viral NPC. Furthermore, the A3G-3A chimera displayed strong antiviral activity against HIV-1 and was sensitive to inhibition by HIV-1 Vif.Our results suggest that the A3G N-terminal domain carries determinants important for targeting the protein to viral NPCs. Transfer of this domain to A3A results in A3A targeting to viral NPCs and confers antiviral activity. In c (HIV-1) -23.23–28PCX2–4C (where X is any amino acid) while A3A contains only a single CDA domain [vif-defective HIV-1 proviruses [A3G contains two copies of the cytidine deaminase active site (CDA) HXEXA domain . The cysA domain . Initialoviruses -8,25-30.oviruses -34. In aoviruses -23.In this study we wanted to investigate why A3A lacks antiviral activity against HIV-1. We observed that A3A was packaged into HIV-1 virions but did not associate with the viral nucleoprotein complex (NPC) and had no antiviral activity. In contrast, we previously reported that A3G, which exhibits strong antiviral activity, was packaged into viral NPC . Sequencvif-defective HIV-1 and pcDNA-A3A together with either pNL-A1  does not have antiviral activity towards HIV-1 irrespective of the presence or absence of Vif -22,25,27-A1 Fig. , lane 1 It is well documented that APOBEC3G (A3G) has antiviral activity and is sensitive to inhibition by HIV-1 Vif . Moreovevif-defective HIV-1 DNA along with increasing amounts of pcDNA-A3G-3A  [RI and Hind III sites of pcDNA3.1(-). pcDNA-A3G-3A was constructed by cloning a BamHI and HindIII fragment from pcDNA-A3A into BamHI and HindIII digested pcDNA-Apo3G. This strategy resulted in the in-frame fusion of A3G residues 1–197 to residues 14 to 199 of A3A   was used-3Vif(-) . Constru-3Vif(-) . pBluesc-3Vif(-)  and was see Fig. .2 flasks to about 80% confluency. Cells were transfected using LipofectAMINE PLUS™  following the manufacturer's recommendations. A total of 5–6 μg of plasmid DNA per 25 cm2 flask was generally used. Where appropriate, empty vector DNA (pcDNA3.1(-)MycHis (Invitrogen)) or vif-defective vector DNA (pNL-A1vif(-)) was used to adjust total DNA amounts. Cells were harvested 24 h post-transfection.HeLa cells were propagated in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum. LuSIV cells are derived from CEMx174 cells and contain a luciferase indicator gene under the control of the SIVmac239 LTR. These cells were obtained from Janice Clements through the NIH AIDS Research and Reference Reagent Program (Cat. no. 5460) and were maintained in complete RPMI 1640 medium supplemented with 10% FBS and hygromycin B (300 μg/ml). For transfection of HeLa cells, cells were grown in 25 cmA peptide antibody to human A3G was prepared by immunizing rabbits with KLH-coupled peptides corresponding to residues 367 to 384 of human A3G. A goat anti-NC(p7) polyclonal antibody was a gift of Robert Gorelick. Viral matrix (MA) protein was identified by a mouse monoclonal anti-MA(p17) antibody . A monoclonal antibody to Vif (MAb #319) was used for all immunoblot analyses and was obtained from Michael Malim through the NIH AIDS Research and Reference Reagent Program. An HIV-positive patient serum was used for the identification of HIV-1 capsid (CA) protein.For immunoblot analysis of intracellular proteins, whole cell lysates were prepared as follows: Cells were washed once with PBS, suspended in PBS and mixed with an equal volume of sample buffer . To analyze virus-associated proteins, cell-free filtered supernatants from transfected HeLa cells (5–6 ml) were pelleted  through a 20% sucrose cushion (4 ml) in an SW41 rotor. The concentrated virus pellet was suspended in PBS and mixed with an equal volume of sample buffer. Proteins were solubilized by heating 10 to 15 min at 95°C. Cell and virus lysates were subjected to SDSPAGE; proteins were transferred to PVDF membranes and reacted with appropriate antibodies as described in the text. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies  and proteins were visualized by enhanced chemiluminescence .Virus stocks were prepared by transfection of HeLa cells with appropriate plasmid DNAs. Virus-containing supernatants were harvested 24 h after transfection. Cellular debris was removed by centrifugation  and clarified supernatants were filtered (0.45 μM) to remove residual cellular contaminations. Filtered virus stocks were further purified and concentrated by pelleting through 20% sucrose .5 LuSIV cells [To determine viral infectivity, virus stocks were normalized for equal reverse transcriptase activity and used to infect 5 × 10IV cells  in a 24-HeLa cells were transfected as indicated in the text. Transfected cells were trypsinized and single-cell suspensions were distributed into 12 well plates containing 0.13 mm cover slips. Cells were grown for 15 h at 37°C in DMEM containing 10% FBS. Cells were fixed at -20°C in precooled methanol (-20°C) for 10 minutes followed by two washes in PBS. For antibody staining, coverslips were incubated in a humid chamber at 37°C for 1 hr with primary antibodies at appropriate dilutions in 1% BSA in PBS. Coverslips were washed once in PBS  and incubated with Cy2-conjugated secondary antibodies (diluted in 1% BSA in PBS) for 30 min at 37°C in a humid chamber. Coverslips were then washed twice with PBS and mounted onto microscope slides with glycerol gelatin  containing 0.1 M N-propyl gallate (Sigma) to prevent photo bleaching. For confocal microscopy, a Zeiss LSM410 inverted laser scanning microscope equipped with a krypton/argon mixed-gas laser was employed. Images were acquired with a Plan-Apochromat 63x/1.4 oil immersion objective (Zeiss). Image quality was enhanced during data acquisition using the LSM line average feature (8x). Post-acquisition digital image enhancement was performed using the LSM software.Sucrose step gradients were prepared as follows: 2.0 ml of a 60% sucrose solution (in PBS) was placed into the bottom of SW55 centrifuge tubes and overlaid with 2.1 ml of a 20% sucrose solution. Immediately prior to addition of concentrated virus stocks (500 μl), the step gradients were overlaid with 100 μl of either PBS or 1% Triton X-100. This procedure minimized the time of detergent exposure of the virus. Samples were then centrifuged in a SW55Ti rotor (Beckman) for 60 min at 35,000 rpm and 4°C. Three fractions  of 1.1 ml each were collected from the top. Aliquots of each fraction of step gradients were subsequently processed for immunoblotting.The author(s) declare that they have no competing interests.RG conceived the study, was leading the execution of the experiments, and participated in the writing of the manuscript. KS coordinated and supervised the study and was involved in the writing of the manuscript. MK, EM, and SK participated in virus production and sample preparation and provided critical comments on the manuscript.


A role for variant histone H2A.Z in gene expression is now well established but little is known about the mechanisms by which it operates. Using a combination of ChIP–chip, knockdown and expression profiling experiments, we show that upon gene induction, human H2A.Z associates with gene promoters and helps in recruiting the transcriptional machinery. Surprisingly, we also found that H2A.Z is randomly incorporated in the genome at low levels and that active transcription antagonizes this incorporation in transcribed regions. After cessation of transcription, random H2A.Z quickly reappears on genes, demonstrating that this incorporation utilizes an active mechanism. Within facultative heterochromatin, we observe a hyper accumulation of the variant histone, which might be due to the lack of transcription in these regions. These results show how chromatin structure and transcription can antagonize each other, therefore shaping chromatin and controlling gene expression. DNA in living cells is packaged into chromatin by histones and non-histone proteins. This packaging is very dynamic, allowing the controlled access of regulatory proteins such as transcription factors to DNA. Most chromatin is packaged with so-called canonical histones; namely H2A, H2B, H3, and H4. In some regions, however, variant histones replace canonical histones, creating special chromatin regions. Here we show that the variant histone H2A.Z is dynamically recruited to promoter regions where it helps in the recruitment of RNA polymerase II, the enzyme responsible for the first step of gene expression. In addition, we show that H2A.Z also associates randomly in the genome, but these molecules are removed during the passage of RNA polymerase II. In non-transcribed regions, H2A.Z accumulates in large domains called heterochromatin. We propose that a battle between random H2A.Z deposition and RNAPII-dependent H2A.Z eviction shapes the chromatin landscape. Chromatin dynamics is now well recognized as key in the regulation of nuclear processes such as gene expression, DNA replication and DNA repair, therefore impinging on biological phenomena such as cellular differentiation as well as normal and cancer development. Cells have developed strategies to locally reconfigure chromatin structure, including i) the use of ATP-dependent chromatin remodeling complexes to either modify the topology, slide or remove nucleosomes; ii) the covalent modification of histone and; iii) the incorporation of variant histones within nucleosomes.Xenopus, Drosophila and mouse Incorporation of variant histones within chromatin regions is emerging as a way for cells to create specialized chromatin domains in order to mediate various cellular functions Drosophila, chicken, plants and mammals H2A.Z occupies one or two nucleosomes in most promoters, a phenomenon first described in yeast Here we provide evidence for a global role of H2A.Z in gene expression in human cells. We also demonstrate that H2A.Z regulates early steps in transcription by helping in the recruitment or RNA polymerase II (RNAPII) to promoters. Surprisingly, we also show that a mechanism allows for the non-targeted incorporation of H2A.Z molecules in the genome, and that transcription results in the depletion of these randomly incorporated H2A.Z molecules from genes. The battle between these two phenomena shapes the chromatin landscape and may have implications in heterochromatin function.Drosophila by others In order to gain some understanding about the function of H2A.Z in human cells, we performed ChIP-chip experiments in U2OS cells. The H2A.Z-enriched material was hybridized on tiling arrays covering the non-repetitive portion of chromosome 19 and 22 at an average of 250 base pairs resolution. Acetylated-H2A.Z, RNAPII, P-Ser2 RNAPII as well as various histone modifications were also profiled in the same conditions (summarized in It has recently been observed that a large fraction of metazoan genes are associated with RNAPII molecules paused at their 5′ end IL8 and CCL2 as two genes transiently induced upon daunorubicin (dauno) treatment, a DNA damaging agent known to induce p21 expression via the p53 pathway IL8 and CCL2 upon treatment of U2OS cells with dauno. A detailed time course looking at H2A.Z  and RNAPII recruitment at IL8 and CCL2 showed that H2A.Z is recruited to the promoter just prior to RNAPII and leaves the promoter as the polymerase gets recruited  assists in the recruitment of RNAPII and its associated co-factors In order to test for a role of H2A.Z in RNAPII recruitment, we made use of small hairpin-mediated RNA (shRNA) interference. Knockdown of H2A.Z crippled both the induction of IL8  and the IL8  upon indClose inspection of A prediction of the “transcription-dependent eviction model” proposed here is that genes that have not been transcribed for a long time should accumulate “abnormally” high levels of H2A.Z on their transcribed region. The best cases for such long-term-repressed genes lie within heterochromatin. Quite strikingly, and in agreement with the model, high levels of H2A.Z are associated with heterochromatic regions . Figure Previous studies have demonstrated the importance of H2A.Z in transcription, DNA repair and genome stability. Here we provide a strong case for a rather global role of H2A.Z in mediating gene expression and demonstrate that the variant histone acts early during transcription, assisting in RNAPII recruitment. In addition, we provide evidence that H2A.Z is randomly incorporated into the genome at low level and that transcription has an antagonizing effect on this random H2A.Z incorporation. This battle between random H2A.Z incorporation and transcription-dependent H2A.Z depletion helps shaping the euchromatin and heterochromatin landscapes. This is summarized in a model shown in Our genomic data shows that human H2A.Z associates with promoters occupied by RNAPII. Time course experiments, however, showed that H2A.Z and RNAPII do not co-occupy the promoter since H2A.Z is recruited prior to RNAPII and is evicted as the polymerase is loaded. Human H2A.Z is therefore recruited to promoters as part of the activation process; which is different from what is observed in yeast, where the association of Htz1 to promoters does not require any activation cues. Htz1 is rather pre-bound to most promoters and it has been proposed that its loading actually occurs during transcriptional repression in order to prime the promoter for the next activation cycle IL8 and CCL2, we also observe large amount of H2A.Z on promoters carrying paused RNAPII but no evidence for transcription. At these promoters, the ultimate activation cue (the one that would stimulate the release RNAPII from the pause site and its entry into elongation) is not present. Hence, the presence of H2A.Z at promoters does not require full transcriptional activity. We rather propose that H2A.Z is recruited upon transacting signals that triggers RNAPII recruitment. At some promoters, such a signal is sufficient to activate transcription. This is the case at IL8, where we observe H2A.Z-dependent recruitment of RNAPII and active transcription upon dauno treatment. At CCL2, however, a significant amount of RNAPII is pre-bound to the promoter in the absence of dauno. Upon dauno treatment, we observe release of RNAPII into elongation and subsequent association of H2A.Z followed by re-association of RNAPII to the promoter. We therefore conclude that transient association of H2A.Z with promoters always precedes (and favors) RNAPII recruitment; and that this may lead to transactivation or promoter poising depending on the promoter context. Interestingly, H2A.Z reappears on the promoter after the first wave of RNAPII has left the promoter, suggesting that H2A.Z is not only recruited during the initial round of transcription but is reloaded prior to each new round. This suggests that the presence of H2A.Z on the promoter may play an active role beyond that of creating unstable promoter chromatin.While we see transient recruitment of H2A.Z upon activation of inducible genes such as ArabidopsisOur data show that the transient association of H2A.Z with human promoters helps with the recruitment of RNAPII. We observed the same in yeast Perhaps the most striking finding in this study is the fact that transcription results in the removal H2A.Z from transcribed regions. Transcriptional elongation was known to perturb chromatin structure but, to our knowledge, this study represents the first evidence that the elongation complex can discriminate between canonical histones and their variants. Most likely, elongation disrupts both H2A- and H2A.Z-containing dimers but a transcription-coupled mechanism would prevent H2A.Z-dimers to re-associate in the wake of RNAPII. What allows elongation complexes to discriminate between histone molecules is unknown but may be linked to chromatin assembly/disassembly complexes as well as the various histone chaperones that interact with the elongating polymerase. Activities such as FACT, Asf1, Rtt106, Spt6, Spt2, Chd1 and others have been shown or proposed to be important for the co-transcription chromatin assembly The question of why cells have evolved mechanisms to remove H2A.Z during elongation will require additional work, but we envision that it may play a role in repressing cryptic initiation. Indeed, the fact that the presence of H2A.Z within promoters helps recruiting RNAPII suggests that removing H2A.Z molecules from transcribed region may be important to suppress cryptic initiation inside genes. Alternatively, removing H2A.Z from transcribed region may somehow help with subsequent transcription rounds, a model compatible with previous work suggesting that this variant histone affects elongation Also striking is the fact that H2A.Z molecules quickly re-appear on genes after transcription has ceased. Indeed, the level of H2A.Z molecule in recently transcribed genes reaches background level within 2 hours after cessation of transcription. This implies that random H2A.Z incorporation occurs via an active mechanism. Whether this non-targeted incorporation proceeds via SWR1 (in yeast), p400 and SRCAP  like for promoter-targeted incorporation will require additional experiments.The H2A.Z molecules associated with promoters are acetylated while the ones that accumulate in heterochromatin are under-acetylated. Elegant immunostaining work by the Chueng laboratory showed that a ubiquitinylated form of H2A.Z associates with the inactive X chromosome de novo formation of heterochromatin rather than its maintenance. Finally, it remains possible that H2A.Z plays a role in other aspects of heterochromatin function. All these possibilities will require further experimentations but the data shown here clearly establishes the accumulation of hypo-acetylated H2A.Z in large domains as a landmark of facultative heterochromatin.It is tempting to speculate that the accumulation of under-acetylated H2A.Z in facultative heterochromatin plays a role in gene silencing. So far, H2A.Z was shown to be important for the formation of pericentromeric heterochromatin In yeast, Htz1 was shown to restrict the spreading of Sir2-dependent silencing The work presented here provides clear evidence that H2A.Z is recruited to chromatin via at least two distinct mechanisms. First, a very dynamic recruitment of H2A.Z at promoters plays a role in the recruitment of RNAPII. Second, a non-targeted mechanism allows for the random incorporation of some H2A.Z molecules in the genome. Finally, we show that a process linked to transcriptional elongation allows for the depletion of the variant histone from active genes. This battle between active H2A.Z incorporation and transcription-dependent H2A.Z depletion shapes the euchromatin and heterochromatin landscape of mammalian cells. Because heterochromatin formation plays a key role in normal development and in tumorigenesis, the results presented here shall open the way to experiments aiming at better understanding the role of H2A.Z in these important aspects of biology.2. For time course experiments, cells were serum-deprived (by changing the medium to 0.5% FBS) for 24 hours prior to addition of 250 nM dauno for various periods of time as indicated in figure legends. ShRNA-mediated knockdowns were performed as described previously U2OS cells were grown in McCoy's medium in the presence of 10% FBS, 0.2 mM L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin at 37°C in the presence of 5% CO8 U2OS cells. For yeast cells, chromatin immunoprecipitations were performed as described previously For mammalian cells, chromatin immunoprecipitations were performed as described previously DNA from mammalian or yeast ChIP and controls samples were labeled using a double or single amplification protocol respectively as described previously The data were normalized using limma's loess function To interpolate between probes, a standard Gaussian filter (SD = 200 bp) was applied twice to the data as described previously  To ident To identhttp://www.yeastgenome.org/) in a window excluding the first and last 250 pb to avoid contamination by intergenic signal (genes<500 bp were then discarded). The dataFigure S1The average signal of most histone marks and proteins tested in this study on the 2439 unique TSS of chr19. H2A.Z/H2B (red), RNAPII/input (green), P-Ser2 RNAPII/input (grey), H3K36me3/H4 (gold), H3K4me3/H4 (dark blue), H3K4me1/H4 (light blue), and H4/input (yellow).(0.39 MB PDF)Click here for additional data file.Figure S2H2A.Z occupies distal regulatory elements. (A) Distal regulatory elements were defined as statistically enriched H3K4me1regions in intergenic regions based on  that are(0.48 MB PDF)Click here for additional data file.Figure S3A complement to (1.61 MB PDF)Click here for additional data file.Figure S4Same analysis as for (1.12 MB PDF)Click here for additional data file.Figure S5A complement to (0.73 MB PDF)Click here for additional data file.Figure S6IL8 and CCL2 genes are indicated.Gene expression profiling of daunorubicine treatment. U2OS cells were treated with dauno as described in (0.29 MB PDF)Click here for additional data file.Figure S7Additional replicates to the experiment shown in (0.29 MB PDF)Click here for additional data file.Figure S8A complement to (0.26 MB PDF)Click here for additional data file.Figure S9A complement to (0.41 MB PDF)Click here for additional data file.Figure S10H2A.Z is depleted from transcribed open reading frames in yeast. Yeast H2A.Z ChIP-chip data from (0.23 MB PDF)Click here for additional data file.Figure S11H2A.Z covers the entire length of heterochromatic genes except for genes that escape silencing. (A) Genome browser screenshots of two heterochromatic regions on chromosome 19 that contain one gene that escapes silencing (red arrows). (B–G) The average ChIP-chip signal is shown over all unique TSS inside heterochromatic regions , euchromatic genes , and heterochromatic genes that escaped silencing . The genes that escape silencing are defined as genes with low H3K9me2 and high H3K36me3 that are contained within a heterochromatic region. The data are shown for H2A.Z/H2B (B), acetylated H2A.Z/H2B (C), RNAPII/input (D), H3K36me3/H4 (E), H3K4me3/H4 (F), and H3K9me2/H4 (G).(1.08 MB PDF)Click here for additional data file.Figure S12Removing the ZNF genes from our dataset does not affect the results shown in (0.80 MB PDF)Click here for additional data file.Table S1Table containing the list of all experiments described in this work.(0.05 MB DOC)Click here for additional data file.Dataset S1UCSC Genome browser-ready file containing the log2 enrichment ratios for the RNAPII/input, H2A.Z/H2B, H2A.Z/input and H2A.Z/H4 ChIP-chip experiments. The sum of (8.49 MB GZ)Click here for additional data file.Dataset S2UCSC Genome browser-ready file containing the log2 enrichment ratios of the acH2A.Z/H2B, H2B/H4, and RNAPII P-Ser2/input ChIP-chip experiments described in this work. The sum of (7.82 MB GZ)Click here for additional data file.Dataset S3UCSC Genome browser-ready file containing the log2 enrichment ratios of the H3K36me3/H4 and H4K4me1/H4 ChIP-chip experiments described in this work. The sum of (6.32 MB GZ)Click here for additional data file.Dataset S4UCSC Genome browser-ready file containing the log2 enrichment ratios of the H3K4me2/H4, H4K4me3/H4, and H3K9me2/H4 ChIP-chip experiments described in this work. The sum of (8.50 MB GZ)Click here for additional data file.Dataset S5UCSC Genome browser-ready file containing the log2 enrichment ratios of the H4/input, RNAPII in shH2A.Z, and RNAPII in shControl ChIP-chip experiments described in this work. The sum of (6.99 MB GZ)Click here for additional data file.Dataset S6UCSC Genome browser-ready file containing the log2 enrichment ratios of all yeast ChIP-chip experiments described in this work.(3.91 MB GZ)Click here for additional data file.Dataset S7UCSC Genome browser-ready file containing the heterochromatin regions identified in this study using H3K9me2.(0.00 MB GZ)Click here for additional data file.


EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community.DMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 107 M-1 as determined by enzyme-linked immunosorbent assay (ELISA).In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbThis recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies. According to Global Health Council despite advances in the understanding of cancer biology, malignant neoplasms remain the second leading cause of mortality in United States. Novel therapies are needed and over the past two decades new immunotherapy strategies have emerged as a promising approach. The success of these immunotherapeutic strategies is highly dependent on finding an ideal antigen to target. One such target is the EGF receptor which is over or aberrantly expressed in a variety of human cancers -12. HoweThere are a number of immune approaches that specifically target EGFRvIII. For example, a peptide vaccine targeting this mutant receptor is currently being tested in clinical trials for GBM patients ,24. AnotH and VL chains, by a flexible linker . Th. ThvIII,vIII potentially cross reacted with mLEEK, to eliminate the non-specific 45 kDa band we mutated the tyrosines at H59 and H105 to phenylalanine.The crystal structure of MR1-1 in complex with the EGFRvIII peptide has been done by Landry et al. This shoDMvIII has improved specificity towards EGFRvIII, we conducted another western analysis. We found that RAbDMvIII now specifically recognizes only the EGFRvIII 145 kDa protein band and not the EGFR variant 45 kDa band  . It is ne Figure . This reDMvIII using a concentration of 117 nM, which is lower than its dissociation constant value of 886 nM. The OD values from this ELISA were placed in a Scatchard plot equation where the fraction of bound antibody sites α  is plotted on the X axis and the ratio of the fraction of bound antibody sites α to free antigen sites c, (α/c) is plotted on the Y axis. The slope gives the value of -K, the affinity constant of the antibody, which is 1.7 × 107 M-1 . As a negative recombinant antibody control for EGFR wt and EGFRvIII, we also used RAbCD133, which utilizes the same expression vector as RAbDMvIII but is directed against CD133 and recognizes neither EGFR wt nor EGFRvIII. HC2 cell lysates immunoprecipitated with RAbDMvIII and G100 antibody and then further immunoblotted with E3138 antibody showed the expected 145 kDa band corresponding to EGFRvIII in the pellet whereas RAbCD133 did not show the 145 kDa band in the pellet as it does not bind to EGFRvIII . The presence of the EGFRvIII epitope completely blocked RAbDMvIII from interacting with the EGFRvIII found in the HC2 cells  is another sensitive biochemical test to monitor for the expression of EGFRvIII. To determine if the RAbs Figure  and GBM s Figure . The tumy Figure  &6B. In s Figure . The recl Figure . We extee Figure  &6F. As e Figure  and 6G.DMvIII efficiently detects surface EGFRvIII, we asked if it might be an effective reagent for flow cytometry experiments. Under native conditions we observed that RAbDMvIII binds to surface expressed EGFRvIII very efficiently. Figure DMvIII . The dot blot and histogram in blue represents human-IgG1 isotype control and as low as 0.1 ug of RAbDMvIII shows a significant shift in fluorescence intensity. Higher concentration of RAbDMvIII (1 μg and 2 μg) show the presence of multiple peaks consistent with the immunofluorescence assay  and complement mediated cytotoxicity (CMC) [A distinct advantage is that recombinantly produced antibodies require no animal for production but otherwise have the properties of conventional antibodies but with additional, improved properties. Indeed, the RAbty (CMC) ,36. Studty (CMC) .9 M-1 between antibodies and tumor antigens may actually impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting [7 M-1 was nearly as toxic as a mAb with an affinity of 1.9 × 109 M-1. Our antibody has an affinity of 1.7 × 107 M-1 as shown by saturation ELISA so it could be a reasonable candidate for therapeutic studies.Effective tumor targeting requires that the antibody should have an affinity for its tumor antigen but extremely high affinity may in fact be detrimental . Previouargeting . Fujimorargeting  and Van argeting  have alsargeting . In anotargeting  have shoDMvIII in targeting tumor spheres derived from glioblastoma tumors. These experiments have already shown that this reagent specifically lyse the cells expressing EGFRvIII and spare normal neural stem cells . We performed cytotoxicity assays using a coupled luminescence method on various EGFRvIII transfected cell lines with human NK cells as effectors and found that this reagent significantly induces cell killing of EGFRvIII transfected cells. RAb DMvIII also remarkably reduces the sphere initiating frequency on GBM cells but not in normal cells as it has been shown that EGFRvIII is required for the maintenance of glioma growth in vivo [DMvIII, RAbCD133 or a non-specific human control antibody and orthotopically injected into the cortex of NOD-SCID mice. Tumor formation was analyzed after 26 weeks which revealed a higher incidence of human tumor cells engraftment with RAbCD133 and Hu-IgG1 as compared to RAbDMvIII . In order to provide this reagent to other researchers and for future studies, we are planning to scale up antibody production using hollow fiber bioreactor system. Hence this antibody could be of great significance in the field of brain tumor research.In separate studies, we have already begun to explore the effectiveness of the RAb in vivo . To furtDMvIII has shown high specificity towards EGFRvIII and thus holds great potential to be further investigated for therapeutic purposes.Despite significant advances in the field of neuro-oncology and immunology, there is no widely available EGFRvIII antibody for scientific or clinical use. Furthermore, there is no EGFRvIII antibody clinically proven to be used on patients because of cross reactions and other high risk allergic reactions due to murine Fc portion of the monoclonal antibodies. RAbU87-MG, HEK293, A431, and HC2 cell lines were obtained from the American Type Culture Collection (ATCC). U87-MGvIII, a human glioma cell line stably transfected with EGFRvIII, was generously donated by Dr Donald O'Rourke. Cell lines were grown in Dulbecco's modified Eagle's medium (DMEM) (glucose at 4.5 g/L) supplemented with either 10% fetal bovine serum or calf serum as per cell line requirement, 100 units/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml kanamycin sulfate. The U87-MGvIII cell lines were maintained in Geneticin (G418) selection at 500 μg/mL.6 U87-MGvIII or A431 cells in the right hind flank of athymic mice. After fourteen days, the mice were sacrificed and the flank tumors were dissected out and fixed in 10% formalin. Four micron tissue sections of both A431 and U87-MGvIII tumors were cut and mounted on the same slide. Cut and mounted primary human glioblastoma tumor sections were obtained from Stanford University brain bank under IRB approved protocols.NOD-SCID mice were injected subcutaneously with either 1 × 10U76382).The pBudCE4:Her2/neu:CD16 bispecific minibody was a kind gift from Dr. Louis Weiner . This coThe plasmid construct pBudCE4:Her2/neu: CD16 was digested with Not I and Bgl II to remove the Her2/neu scFv. The artificially synthesized MR1-1 was amplified using primers AAGGAAAAAAGCGGCCGCACCATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGGTGACGACTACAAGGACGACGATGACAAGCAGGTGAAACTGCAGCAGTCT (scFvEGFRvIII-Not1-ATG/Igk (F)) and GGAAGATCTACCGCCACTGCCACCTCCGCCTGAACCGCCTCCACCACTCGAGCCACCTTTGATTTCCAGCTTGGTGC (scFvEGFRvIII-Linker/BglII(R)) and ligated into the scFv Her2/neu position. The resulting recombinant antibody was then digested with Sfil and Xba1 to remove the scFv for CD16. Primers ACATTGGTGCCGAAACCTATTCACTGCTTACTCGCGGCCCAGCCGGCCCAGGTGAAACTGCAGCAGTCT (scFvLEEK-Sfil(F)) and GCTCTAGAACCGCCACTGCCACCTCCGCCTGAACCGCCTCCACCACTCGAGCCACCTTTGATTTCCAGCTTGGTGC (scFvLEEK-XbI/linker(R)) were used to amplify the second EGFRvIII scFv and ligated into the scFv CD16 position. The plasmid pBudCE4(scfvEGFRvIII) has the CH3 gene with "knobs-into-holes" configuration . AdditioDMvIII whereas the antibody produced by pBudCE4(scfvEGFRvIII) and pBudCE4(scfvCD133) are called RAbvIII and RAbCD133 respectively.Site directed mutagenesis was used to change tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti EGFRvIII scFv sequence inserts . Primer CTATTCTCCTTACTCTTTTGCTATGGACTACTGGG (F) and primer CCCAGTAGTCCATAGCAAAAGAGTAAGGAGAATAG (R) were used for the H105 mutation while primer ACTGGCGGTTATAATACCTTCTATTCAGACAATGTAAAG (F) and primer CTTTACATTGTCTGAATAGAAGGTATTATAACCGCCAGT (R) were used for the H59 mutation. The resulting mutated vector is called pBudCE4(DMscfvEGFRvIII) and the antibody produced by this construct is called RAbThe pBudCE4scfvEGFRvIII, pBudCE4scfvCD133 and pBudCE4DMscfvEGFRvIII plasmids were stably transfected into HEK293 cells using calcium phosphate, and the positive clones were selected using zeocin at 400 μg/ml. Media was collected from the stably transfected cells and the His-tagged antibodies were isolated and purified using Ni-NTA agarose  according to the manufacturer's recommended protocol. The antibodies were eluted using 500 mM imidazole. The fractions containing the antibodies were identified through Coomassie staining of SDS PAGE. The fractions containing the antibody protein were combined and dialyzed against phosphate buffer solution (PBS). The protein concentration was determined by a Bio-Rad Dc protein assay.2HPO4, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.2% sodium azide, 0.004% sodium fluoride, 1 mM NaVO4, 25 mM β-glycerophosphoric acid, 100 μg of phenylmethanesulfonyl fluoride/ml, 10 μg of aprotinin/ml, and 10 μg of leupeptin/ml (pH 7.35). Cell lysates were centrifuged at 12,000 × g for 10 min at 4°C to clear out cell debris, and protein concentration was determined by Bio-Rad DC protein assay. For gel electrophoresis under non-reducing conditions, forty micrograms of cell lysate was prepared in 6x Laemmli Sample Buffer lacking β-mercaptoethanol and the samples were not boiled. The proteins were resolved on 4-20% Tris-glycine SDS-PAGE gels (Invitrogen) and transferred to nitrocellulose membrane for immunoblot analysis. The membranes were blocked with blocking buffer 5% Blotto  and incubated with the following antibodies: RAbvIII, RAbDMvIII, mouse anti-actin , and mouse anti-EGFR . Horse radish peroxidase (HRP) affinity pure Donkey anti-Human IgG  was used as the secondary for the recombinant antibodies. Proteins were detected with ECL reagents .Confluent HC2 and A431 cells were washed with ice-cold PBS and lysed using a buffer containing 10 mM NaCD133 and RAbDMvIII for 1 hr at 4°C. Forty μls of washed 50% slurry of protein Gammabind G Sepharose  was added to each lysate/antibody mix and incubated overnight at 4C. Immunoprecipitates were washed three times with the same lysis buffer and resuspended in sample buffer containing β-mercaptoethanol. Immunocomplexes were resolved on 4-20% Tris-glycine SDS-PAGE gels under normal reducing conditions and transferred to nitrocellulose membranes for immunoblot analysis by mouse anti-EGFR antibody .HC2 (EGFRvIII transfected) and A431 (EGFR wt transfected) cell lysates were isolated as described in the western blot section and 300 μl of lysate was incubated with 12 μg of G100 antibody , RAbDMvIII, mouse anti-EGFR (EGFR (528): sc-120, Santa Cruz Biotechnology). FITC goat anti-human  and FITC goat anti-mouse  were used as secondary antibodies. Images were obtained using a Leica SP2 AOBS Confocal Laser Scanning Microscope. For peptide competition, ~20 times higher molar concentration of EGFRvIII peptide  and scrambled peptide  was incubated with the primary antibody at 37°C for 1 hour and the mixture was centrifuged at 14000 × g for 5 min. The supernatant was used at the primary antibody incubation step of immunofluorescence protocol.HC2 and A431 cells were fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X, and blocked with 5% goat serum . The following antibodies were used for IF: RAbCD133 (recombinant negative control) and RAbDMvIII. Each slide was then incubated with the appropriate secondary and/or tertiary antibody. The secondary antibodies used were: HRP-Anti Rabbit , anti-Human IgG rabbit , HRP anti-rabbit antibody . Protein expression was detected by betazoid DAB . The slides were also counterstained with hematoxylin. Glioblastoma slides were analyzed in a similar fashion to the A431 and U87-MGvIII slides except that the slides for RAbDMvIII were blocked with equal volume of background sniper , 5% Blotto and normal human serum  and the secondary antibody for RAbDMvIII was rabbit anti-V5  followed by the tertiary antibody, which was HRP-anti-rabbit .Slides were deparaffinized in xylene, and rehydrated in decreasing percentages of ethanol. 3% hydrogen peroxide was used to block endogenous peroxidase and the slides were placed in Diva Decloaker  and microwaved for 13 minutes 10 seconds for antigen retrieval. After a cooling down period, the slides were treated with Sniper Universal serum  and then incubated with the following antibodies at 0.5 μg/ml overnight at 4°C: Rabbit anti-EGFR antibody, RAbDMvIII a preliminary ELISA was performed to find the concentration of the antibody. This preliminary ELISA was performed with both RAbDMvIII and RAbvIII antibodies to compare the dissociation constant of both the antibodies. A dilution series of antibodies RAbDMvIII and RAbvIII (20 μg/ml-0.01952 μg/ml) in 5% blotto was used. ELISA plates were first coated with 10 μg/ml of EGFRvIII peptide in coating solution in duplicate for each antibody overnight at 4C. The next day the plates were washed with 1× wash buffer  and 5% blotto was added for 1 hr at RT. Thereafter the respective plates were incubated with different dilutions of RAbDMvIII and RAbvIII ranging from 20 μg/ml to 0.02 μg/ml in 5% blotto for 1 hr at RT. The plates were washed 3 × 10 min each with 1× wash buffer, and then affinity pure Donkey anti-Human IgG  was applied at a 1: 5000 dilution in 5% Blotto for 1 hr at RT, and then the plates were washed 3 × 10 min each. 100 μl of 1× developing reagent was added to each reaction well, and after 10 min., 100 μl of stop solution 1N HCl was added and the absorbance was read at 450 nm. All curves reached a maximum value, which indicated that the reactions proceeded until saturation. The data was plotted as the absorbance at 450 nm versus different antibody concentrations. This gave an equilibrium dissociation constant, Kd, for RAbDMvIII of 886 nM and for RAbvIII of 1.7 μM.To determine the affinity constant of the RAbDMvIII has a lower dissociation constant than RAbvIII as shown by preliminary ELISA we decided to perform indirect ELISA with RAbDMvIII only.Indirect ELISA was performed to determine the affinity constant of the recombinant antibody. This method developed by Friguet et al  uses a sDMvIII ) and final antigen concentration of (40 μg/ml-2.5 μg/ml) for 30 min at RT, and then the antibody-antigen mixture is transferred to the wells of the first antigen coated microtiter plate and incubated for 15 min. After this incubation, the contents are then transferred to the second antigen coated plate and incubated for another 15 min. The plates were washed with wash buffer  and secondary donkey anti-Human antibody from Jackson ImmunoResearch was added for 1 hr incubation at RT. After further washes of 3 × 10 min each, 100 μl of substrate  is applied and 100 μl of stop solution (1 N HCl) is added after 10 min. The absorbance was read at 450 nm in an ELISA plate reader. The data is plotted as the fraction of bound antibody sites versus the ratio of (fraction of bound antibody/concentration of free antigen). The slope gives the value of K, which is the affinity constant.Two ninety-six well ELISA plates  were coated overnight at 4°C with EGFRvIII peptide LEEKKGNYVVTDH-C at a decreasing concentration from 40 ug/ml to 2.5 μg/ml in coating solution . Wells were then blocked with 5% nonfat dry milk for 1 hr at room temperature. In parallel tubes, antibody at 234 nM is incubated with double the concentration of antigen used in the plates that is (80 μg/ml-5 μg/ml) in equal volume for final concentration of antibody to be 117 nM . Cells were resuspended in flow cytometry buffer, consisting of 1× HBSS, pH 7.2, containing 1.55 g/l glucose and 0.1% fraction V of bovine serum albumin . Cells were counted and diluted to a density of 106 cells per milliliter of buffer. 1 μg of RAbDMvIII was used per 106 cells unless otherwise specified. Cells were analyzed on a BD LSR-I flowcytometer (BD Biosciences). Unlabeled cells and cells labeled with isotype control (human IgG1) were used to set gating parameters between positive and negative cell populations. Cell aggregates and small debris were excluded from analysis or isolation on the basis of side scatter (measuring cell granularity) and forward scatter (measuring cell size); dead cells were excluded from analysis on the basis of viability dye fluorescence (propidium Iodide). Fluorescent intensities for cells in the population were point-plotted on two-axis graphs or histogram using FlowJo software (Tree Star Inc.).Flowcytometric analysis was performed using RAbPG performed the majority of the experiments, analyzed the data and wrote the manuscript, SYH constructed the primers for scFv EGFRvIII amplification, MHM designed the conditions to perform PCR for scFv EGFRvIII amplification, SSM performed and analyzed FACS, GL & RTN have been involved in the editing, revising, and formatting of the manuscript. AJW initiated the study, analyzed the data, supervised the overall project and wrote the manuscript. All authors read and approved the final manuscript.


PTPRD) , and serine racemase (SRR) . We also confirmed that variants in KCNQ1 were associated with T2D risk, with the strongest signal at rs2237895 . By identifying two novel genetic susceptibility loci in a Han Chinese population and confirming the involvement of KCNQ1, which was previously reported to be associated with T2D in Japanese and European descent populations, our results may lead to a better understanding of differences in the molecular pathogenesis of T2D among various populations.To investigate the underlying mechanisms of T2D pathogenesis, we looked for diabetes susceptibility genes that increase the risk of type 2 diabetes (T2D) in a Han Chinese population. A two-stage genome-wide association (GWA) study was conducted, in which 995 patients and 894 controls were genotyped using the Illumina HumanHap550-Duo BeadChip for the first genome scan stage. This was further replicated in 1,803 patients and 1,473 controls in stage 2. We found two loci not previously associated with diabetes susceptibility in and around the genes protein tyrosine phosphatase receptor type D ( Type 2 diabetes (T2D) is a complex disease that involves many genes and environmental factors. Genome-wide and candidate-gene association studies have thus far identified at least 19 regions containing genes that may confer a risk for T2D. However, most of these studies were conducted with patients of European descent. We studied Chinese patients with T2D and identified two genes, PTPRD and SRR, that were not previously known to be involved in diabetes and are involved in biological pathways different from those implicated in T2D by previous association reports. PTPRD is a protein tyrosine phosphatase and may affect insulin signaling on its target cells. SRR encodes a serine racemase that synthesizes D-serine from L-serine. Both D-serine (coagonist) and the neurotransmitter glutamate bind to NMDA receptors and trigger excitatory neurotransmission in the brain. Glutamate signaling also regulates insulin and glucagon secretion in pancreatic islets. Thus, SRR and D-serine, in addition to regulating insulin and glucagon secretion, may play a role in the etiology of T2D. Our study suggests that, in different patient populations, different genes may confer risks for diabetes. Our findings may lead to a better understanding of the molecular pathogenesis of T2D. Type 2 diabetes (T2D) affects at least 6% of the world's population; the worldwide prevalence is expected to double by 2025 PPARG) KCNJ11) TCF2) WFS1) PPARGKCNJ11KCNQ1CDKAL1CDKN2A-2BCDC123-CAMK1DMTNR1BTCF7L2TCF2 (HNF1B), HHEX-KIF11-IDEJAZF1IGF2BP2SLC30A8THADAADAMTS9WFS1FTONOTCH2TSPAN8KCNQ1; individually, these variants confer a modest risk (odds ratio [OR] = 1.1–1.25) of developing T2D. KCNQ1 was identified as a T2D susceptibility gene in three GWA scans in Japanese individuals, highlighting the need to extend large-scale association efforts to different populations, such as Asian populations CDKAL1, CDKN2A-2B, IGF2BP2, TCF7L2, SLC30A8, HHEX, and KCNJ11) with T2D were also replicated in the Japanese population Much effort has been devoted to finding common T2D genes, including genome-wide linkage, candidate-gene, and genome-wide association studies (GWAS). Whole-genome linkage scans have identified chromosomal regions linked to T2D; however, with the exception of regions 1q KCNQ1 and CDKAL1, CDKN2A-2B, MTNR1B, TCF7L2, HNF1β, and KCNJ11To date, a GWA scan for T2D has not been conducted in the Han Chinese population, although the association of some known loci have been confirmed, including P = 0.111, Fst statistics between populations <0.001)  . Multidi <0.001) . Further <0.001) .P values of <10−5 at stage 1 with any of the genotype, allele, trend, dominant, and recessive models for subsequent cross-platform validation using Sequenom , we searched for novel susceptibility candidates for T2D as implicated by (1) gene function identified by a bioinformatics approach and (2) an animal model showing defects in glucose homeostasis caused by genes within the same subfamily. Therefore, we selected SNP rs17584499 (P = 2.4×10−5 under best model) for further investigation. rs17584499 lies within protein tyrosine phosphatase receptor type D (PTPRD). We hypothesized that PTPRD might play a role in the regulation of insulin signaling, because its subfamily members leukocyte common antigen-related (LAR) and protein tyrosine phosphatase sigma (PTPRS) exhibit defects in glucose homeostasis and insulin sensitivity in knockout and/or transgenic mice We selected eight SNPs in seven regions: rs9985652 and rs2044844 on 4p13, rs7192960 on 16q23.1, rs7361808 on 20p13, rs1751960 on 10q11.23, rs4845624 on 1q21.3, rs391300 on 17p13.3, and rs648538 on 13q12.3. These SNPs had association Sequenom . For SNPKCNQ1, which was previously reported to be a diabetes susceptibility gene in a Japanese population, as well as in populations of Korean, Chinese, and European ancestry p<10−5, rs17584499, and rs231361—were cross-platform validated and yielded consistent results using both Illumina and Sequenom. The concordance rate for stage 1 samples typed on the Illumina and Sequenom platforms was 99.1%±0.84%  within 1%±0.84% .PTPRD at 9p24.1-p23, rs231359 in KCNQ1 at 11p15.5, and rs391300 in serine racemase (SRR) at 17p13.3  block forward to replicate in 3,803 additional samples . The average call rate for each sample was 96.13%±4.66%. After applying stringent quality control criteria, high-quality genotypes for 35 SNPs (89.7%) were obtained, with an average call rate of 98.96%±0.24% . Of the  17p13.3 . We wereanalysis . Joint aanalysis . These a of 0.05 .PTPRD  . The nearby SNP rs4523957 also demonstrated a significant association . SNPs rs391300 and rs4523957 were in tight LD with one another (r2 = 0.942 in HapMap HCB), and were located within the serine racemase gene (SRR).Two previously unknown loci were detected in our joint analysis of GWAS data. The strongest new association signal was found for rs17584499 in intron 10 of 36–1.82) . The secKCNQ1, had a less significant association with T2D, and was selected in stage 1   (r2 = 1 in HapMap HCB), and were located approximately 164 kb upstream of SNP rs2237897, which was previously reported to be significantly associated with T2D in a Japanese population P = 3.43×10−8, OR = 1.33, 95% CI = 1.2–1.48; rs231361: P = 2.89×10−7, OR = 1.3, 95% CI = 1.18–1.44).SNP rs231361, located in intron 11 of 17–1.64) . We furt14–1.61) . rs23136KCNQ1   . Conditi19–1.40) .KCNQ1 gene in 50 individuals with T2D, and identified 42 polymorphic variations, including one nonsynonymous P448R polymorphism and two novel SNPs with minor allele frequency >0.03. We then genotyped the two novel SNPs and one nonsynonymous polymorphism; however, none of these SNPs showed an association with T2D , none of the P values for any of the SNPs within or near the genes reached 10−5 using allele, genotype, trend, dominant, or recessive models , e models . Three Sown loci . No signPTPRD. The overall Fst among 11 HapMap groups for rs17584499 was estimated to be 0.068 P<0.0001, chi-square test )  subfamily of protein tyrosine phosphatases (PTPs). The R2A PTP subfamily comprises LAR, PTPRS, and PTPRD. The R2A family has been implicated in neural development, cancer, and diabetes LAR- and PTPRS-deficient mice were demonstrated to have altered glucose homeostasis and insulin sensitivity LAR in skeletal muscle show whole-body insulin resistance PTPRD could play a role in T2D pathogenesis and should be further characterized.The strongest new signal was observed for rs17584499 in e test ) . PTPRD iSRR. SRR encodes a serine racemase that synthesizes D-serine from L-serine P = 2.49×10−6); this SNP resides 3.8 kb upstream from SRR, within Smg-6 homolog, nonsense mediated mRNA decay factor (C. elegans) (SMG6). rs216193 was in tight LD with rs391300 (r2 = 0.942 in HapMap HCB). Based on their biological functions and the association results, neither SMG6 nor any of the nearby genes TSR1, SGSM2, MNT, and METT10D were compelling candidates for association withT2D. However, SRR was significantly associated with T2D; thus, we suggest that dysregulation of D-serine could alter glutamate signaling and affect insulin or glucagon secretion in T2D pathogenesis.The second new association locus was found for rs391300 and rs4523957 in the biologically plausible candidate gene P = 1.32×10−5; OR = 1.21, 95% CI = 1.11–1.33). This SNP which lies approximately 211 kb downstream of v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) (MAF) and 170 kb downstream of WW domain containing oxidoreductase (WWOX). WWOX is a tumor suppressor gene that spans the second most common human fragile site FRA16D MAF encodes the transcription factor c-Maf, a member of the Maf family of basic-Zip (bZip) transcription factors. c-Maf is involved in development and differentiation of the lens rs7192960 also had a suggestive association with T2D , which is involved in repolarization of the action potential in cardiac muscle KCNQ1 cause long QT syndrome KCNQ1 is widely expressed, including in the heart, brain, kidney, liver, intestine, and pancreas KCNQ1 knockout mice have cardiac dysfunctions KCNQ1, rather than in the coding region. We did not find an association between either CDKAL1 or IGF2BP2 and T2D, in contrast with the results described in a previous study Our GWAS revealed that KCNQ1 with T2D. The novel T2D risk loci may involve genes that are implicated in insulin sensitivity and control of glucagon and insulin secretion: PTPRD may participate in the regulation of insulin action on its target cells, while SRR variants may alter glutamate signaling in the pancreas, thus regulating insulin and/or glucagon secretion. Our study suggests that in different patient populations, different genes may confer risks for diabetes, which may lead to a better understanding of the molecular pathogenesis of T2D.In conclusion, we identified two previously unknown loci that are associated with T2D in a Han Chinese population, and confirmed the reported association of The study was approved by the institutional review board and the ethics committee of each institution. Written informed consent was obtained from each participant in accordance with institutional requirements and the Declaration of Helsinki Principles.1C ranging from 3.4 to 6, and (3) BMI<32. The two control groups were comparable with respect to BMI, gender, age at study, and level of HbA1C. All of the participating T2D cases and controls were of Han Chinese origin, which is the origin of 98% of the Taiwan population. Details of demographic data are shown in A total of 2,798 unrelated individuals with T2D, age >20 years, were recruited from China Medical University Hospital (CMUH), Taichung, Taiwan; Chia-Yi Christian Hospital (CYCH), Chia-Yi, Taiwan; and National Taiwan University Hospital (NTU), Taipei, Taiwan. All of the T2D cases were diagnosed according to medical records and fasting plasma glucose levels using American Diabetic Association Criteria. Subjects with type 1 diabetes, gestational diabetes, and maturity-onset diabetes of the young (MODY) were excluded from this study. For the two-stage GWAS, we genotyped 995 T2D cases and 894 controls in the first exploratory genome-wide scan (stage 1). In the replication stage (stage 2), we genotyped selected SNPs in additional samples from 1,803 T2D cases and 1,473 controls. The controls were randomly selected from the Taiwan Han Chinese Cell and Genome Bank P<10−7). Genotyping validation was performed using the Sequenom iPLEX assay . In the replication stage (stage 2), SNPs showing significant or suggestive associations with T2D and their neighboring SNPs within the same LD block were genotyped using the Sequenom iPLEX assay. The neighboring SNPs in the same LD were selected from the HapMap Asian (CHB + JPT) group data for fine mapping the significant signal.Genomic DNA was extracted from peripheral blood using the Puregene DNA isolation kit . In stage 1, whole genome genotyping using the Illumina HumanHap550-Duo BeadChip was performed by deCODE Genetics . Genotype calling was performed using the standard procedure implemented in BeadStudio , with the default parameters suggested by the platform manufacturer. Quality control of genotype data was performed by examining several summary statistics. First, the ratio of loci with heterozygous calls on the X chromosome was calculated to double-check the subject's gender. Total successful call rate and the minor allele frequency of cases and controls were also calculated for each SNP. SNPs were excluded if they: (1) were nonpolymorphic in both cases and controls, (2) had a total call rate <95% in the cases and controls combined, (3) had a minor allele frequency <5% and a total call rate <99% in the cases and controls combined, and (4) had significant distortion from Hardy–Weinberg equilibrium in the controls  the case and control labels were shuffled and redistributed to subjects, and (2) the test statistics of the corresponding association test was calculated based on the shuffled labels. The empirical P value was defined as the number of permutations that were at least as extreme as the original divided by the total number of permutations. Detection of possible population stratification that might influence association analysis was carried out using principle component analysis, multidimensional scaling analysis, and genomic control , dominant, and recessive models. The most significant test statistic obtained from the five models was chosen. SNPs with  control . Quantilibutions .Figure S1P = 0.111, and Fst statistics between populations <0.001).Principle component analysis (PCA) plot. The PCA plot shows the first two principal components, estimated by EIGENSTRAT , based on genotype data from 76,673 SNPs with equal spacing across the human genome. No population stratification between the 995 T2D cases (green x) and 894 controls (red +) was detected ((1.18 MB TIF)Click here for additional data file.Figure S2P = 0.192598 for T1: case/control less similar).Multidimensional scaling analysis (MDS) plot. The MDS plot shows the first two principal components, estimated by PLINK , based on genotype data from 516,212 SNPs. No population stratification between the 995 T2D cases (red) and 894 controls (blue) was detected Click here for additional data file.Figure S3LD block between rs231361 and rs223787.(0.17 MB TIF)Click here for additional data file.Figure S4NOTCH2, (B) THADA, (C) PPARG, (D) IGF2BP2, (E) ADAMTS9, (F) WFS1, (G) CDKAL1, (H) JAF1, (I) SLC30A8, (J) CDKN2AB, (K) HHEX, (L) CDC123/CAMK1D, (M) TCF7L2, (N) KCNJ11, (O) MTNR1B, (P) TSPAN8/LGR5, (Q) FTO, and (R) TCF (HNF1B) regions, the −log10P values from the primary scan are plotted as a function of genomic position (NCBI Build 36). The reported SNPs in previous GWAS are denoted by blue diamonds. Estimated recombination rates (right y-axis) based on the Chinese HapMap population are plotted to reflect the local LD structure around the significant SNPs. Gene annotations and numbers of transcripts were taken from NCBI.Comparisons to susceptible regions reported by previous GWAS. For each of the (A) (4.17 MB TIF)Click here for additional data file.Figure S5Quantile-quantile (QQ) plots. QQ plots are shown for the four association tests, (A) allelic, (B) genotype, (C) dominant, and (D) recessive, based on the 516,212 quality SNPs of the initial analysis of 995 cases and 894 controls. The upper and lower boundaries of the 95% confidence bands are represented by the red lines.(3.10 MB TIF)Click here for additional data file.Table S1Quality control of the subject participants in stage 1.(0.03 MB DOC)Click here for additional data file.Table S2Quality control of the genotyping results.(0.03 MB DOC)Click here for additional data file.Table S3Association results in stage 1.(0.05 MB DOC)Click here for additional data file.Table S4Concordance rates for the 10 SNPs with significant associations in stage 1.(0.05 MB DOC)Click here for additional data file.Table S5Power Calculation using CaTS.(0.05 MB DOC)Click here for additional data file.Table S6Association of additional SNPs within KCNQ1 in all T2D cases and controls in the joint analysis.(0.06 MB DOC)Click here for additional data file.Table S7Conditional analysis on rs2237895.(0.03 MB DOC)Click here for additional data file.Table S8Previously reported loci and SNPs associated with T2D.(0.13 MB DOC)Click here for additional data file.Table S9Genotype frequency and allele frequency of rs17584499 (founders only) from HapMap3.(0.05 MB DOC)Click here for additional data file.Table S10Clinical characteristics of the subjects.(0.04 MB DOC)Click here for additional data file.Text S1Supplementary methods.(0.03 MB DOC)Click here for additional data file.


We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus. The natural hosts of influenza A viruses are aquatic birds. On rare occasions these viruses may be transmitted to humans and then give rise to an influenza pandemic. Human influenza is therefore a typical re-emerging infection. Evidence is increasing that the viral polymerase, an enzyme that has to enter into the nucleus of the infected cell in order to promote replication and transcription of the viral genome, is a major determinant of host range. Thus, in a comparative study of an avian influenza strain and its mouse adapted variant we have previously shown that adaptation to mice depended exclusively on mutations in the polymerase proteins. These findings supported the concept that adaptation of the polymerase to host factors is an important mechanism underlying interspecies transmission. In the present study, we have identified importin α1, a component of the nuclear pore complex, as such a host factor. We show that adaptive mutations in polymerase subunits improve binding to importin α1 in mammalian, but not in avian cells. As a result, nuclear transport of these proteins and efficiency of replication are enhanced in mammalian cells. These observations demonstrate that the interaction of the viral polymerase with the nuclear import machinery is an important determinant of host range. Orthomyxoviridae family influenza A viruses have a segmented RNA genome of negative polarity. The eight segments are present in enveloped virus particles as ribonucleoprotein (RNP) complexes with the nucleocapsid protein (NP) and the three subunits of the RNA-dependent RNA-polymerase . In the infected cell, the polymerase is responsible for transcription and replication of the viral genome [The natural host of influenza A viruses is waterfowl where these agents occur in large variety defined by 16 hemagglutinin and 9 neuraminidase subtypes. Avian influenza viruses are the source of devastating outbreaks in poultry. Moreover, because of their potential to cross species barriers, to adapt to new hosts, and to cause on rare occasions pandemics, they are also a constant threat to human health [e PB1, PB, PA. In l genome .Evidence is increasing that the viral polymerase plays a major role in host adaptation. Thus, in a comparative study of the avian strain SC35 H7N7) and its mouse-adapted variant SC35M we showed that adaptation to mice was the result of seven mutations in the polymerase proteins, six of which  were responsible for enhanced polymerase activity in mammalian cells. In avian cells, replication and transcription of SC35M were reduced, whereas both activities were increased with SC35. Furthermore, the differences in polymerase activity were paralleled by differences in pathogenicity: SC35 was highly pathogenic for chickens and had reduced pathogenicity in mice, whereas the opposite was the case with SC35M. Thus, the efficiency of the viral polymerase is a determinant of both host specificity and pathogenicity [N7 and itThe influenza virus polymerase is active in the nucleus of the infected cell where cytoplasmicly expressed PB1 and PA appear to be imported as a subcomplex which then assembles with separately imported PB2 . There iTo test this hypothesis we have compared now in avian and mammalian cells the interaction of the polymerase complex of SC35 and SC35M with importin α1, which not only binds to NP, but is also the most abundant importin in a variety of human cells  and may To test the hypothesis that the polymerase proteins of SC35 and SC35M differ in their interaction with the nuclear import machinery of mammalian and avian cells we have analyzed the binding of PB2 and NP to importin α1 in co-immunoprecipitation experiments. Cultures of human 293T cells and quail embryo cells (CEC-32) were transfected with plasmids encoding PB2 or NP. Importin α1 complexed with PB2 or NP was precipitated from lysates of transfected and mock transfected cells using specific antibodies against PB2 or NP, which were covalently coupled to an amine-reactive gel. Bound proteins were eluted three times from the columns using acidic elution buffer to allow quantitative estimation of interaction partners. All three eluates were used to detect importin α1, PB2 and NP, respectively, by Western blot analysis. In 293T cells transfected with PB2 of SC35M or PB2 D701N, 4 to 7 times more importin α1 was bound than in cells transfected with PB2 of SC35 A and 1C.It was now of interest to find out if increased binding of NP and PB2 to importin α1 affected nuclear transport in mammalian cells. First, we have carried out Western blot experiments to determine importin α1 in nuclear and cytoplasmic fractions prepared from infected cells early and late in the replication cycle. Since replication kinetics were different in avian and mammalian cells, time points with comparable virus titres were selected  . Early aSC35M and SC35-NPSC35M containing the PB2 or the NP gene of SC35M showed enhanced importin α1 and importin ß1 levels in the nuclear fractions, although the increase was not as distinct as with the parental SC35M virus  viruses containing one of the polymerase genes of SC35M in a SC35 background. Cells infected with SGR viruses were separated into nuclear and cytoplasmic fractions, and importin α1 and importin ß1 localization was determined . Only SG5M virus . With th5M virus . Since Sed cells . These fSC35M and SPM virus SC35-PB2701N, PB2 was also concentrated in the nucleus where it co-localized with importin α1 (714R (data not shown). We have then analyzed the nuclear import of NP by immunofluorescence , most of the cells infected with SC35 showed a nuclear and cytoplasmic distribution of PB2 A. This oortin α1 A. Nuclearescence B. NP of ey cells . Taken tIntracellular localization of NP, PB2 and importin α1 has also been analyzed in CEC-32 cells. With both viruses, PB2 was clearly present in the nucleus, but it was also detected in the cytoplasm. Importin α1 co-localized with PB2 in the cytoplasm as well as the nucleus A. NP disSC35M grew as well as SC35M, whereas titres of SGR virus SC35-PB2 SC35M were slightly reduced. In avian cells, parental and SGR viruses grew to equally high titres . This observation supports the concept that the virus when crossing the species barrier, goes through a phase that allows gradual acquisition of adaptive mutations without loosing fitness for the old host [NLSs have also been identified in PB1  and PA , but the293T (human embryonic kidney cells) and A549 (human lung carcinoma) cells were grown in DMEM  supplemented with 10% FCS . CEC-32 quail embryo fibroblasts  were groTM 2000 (Invitrogen) according to the manufacturers protocols. The medium was replaced with fresh growth medium at 6 h posttransfection and incubated for further 48 h for immunoprecipitation assays.Monolayers of 293T, A549 and CEC-32 cells were infected with recombinant virus and incubated for 30 min for absorption at 37°C. Cells were washed twice with phosphate-buffered saline (PBS) and incubated for 6 h, 12 h and 18 h at 37°C in appropriate medium containing 0.2% bovine serum albumin . For transfection, we seeded the cells in 100-mm-dishes and used 10μg pHW2000-SC35-NP, 10 μg pHW2000-SC35M-NP, 30 μg pHW2000-SC35-PB2, or 30 μg pHW2000-SC35M-PB2 plasmid [−4 for multicycle replication. Virus inoculum was removed after 30 min of incubation at 37°C, and cells were washed two times with PBS pH5. Cells were then incubated in the appropriate medium containing 0.2% bovine serum albumin at 37°C. At time points 0, 3, 6, 9, 12, 15, 18, and 24 h for single cycle replication and at time points 0, 24, 48, 72, and 96 h for multicycle replication, we collected supernatants and determined plaque titers on MDCK cells. The growth curves shown are the average result of two independent experiments.293T, A549 and CEC-32 cells were inoculated with virus at a multiplicity of infection (MOI) of 2 for single cycle replication and at an MOI of 1060-mm-dishes of 293T and CEC-32 cells were infected with a MOI of 2. At the indicated time points, cells were washed twice with PBS and fractionated in 1ml PBS by using a Mixermill MM301 homogenizer (Retsch) at 20 Hz for 20 min. Lysates were mixed with 1% NP40 and separated into a nuclear (bottom) and a cytoplasmic (upper) fraction by centrifugation on a 20% sucrose cushion at 3000 rpm for 20 min. Nuclear fractions were sonicated for 15 min. Whole lysates and cell fractions were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blot analysis as described . Monoclon = 100) infected with recombinant viruses. The results shown represent three independent experiments.293T, A549 or CEC-32 cells were grown on glass cover slips and infected at a MOI of 2 with recombinant virus. At the indicated time points after infection, cells were fixed with PBS containing 2% paraformaldehyde and permeabilized with PBS containing 0.1% Triton-X-100. After blocking fixation with 3% BSA in PBS, we incubated the cells for 1h with monoclonal antibodies specific for importin α1 (BD Biosciences), PB2 (Santa Cruz) or NP . After incubation with primary antibodies we washed the cells three times with PBS and incubated for further 30 min with either FITC-coupled goat anti-rabbit , FITC-coupled goat anti-mouse , Rhodamine-coupled donkey anti-goat  or Rhodamine-coupled goat anti-mouse  secondary antibodies. Cells were washed three times with PBS and coverslips were mounted on glass plates. Cells were observed in an Axiovert 200M microscope equipped with an ApoTome device (Zeiss). Localization of importin α1, PB2, and NP in nucleus, nucleus/cytoplasm and cytoplasm was determined using the microscope by counting cells (TM Co-Immunoprecipitation Kit (PIERCE) according to the protocols provided by the manufacturer. Briefly, the antibodies specific for PB2 (Santa Cruz) or NP  [Transfected cells were washed twice with PBS and collected by centrifugation. Cell pellets were resuspended in 1 ml PBS and sonicated for 15 min. Complexes of importin α1 and viral proteins were determined by co-immunoprecipitation using the ProFound4 (H7N7) ) were co4 (H7N7) . For theFigure S1293T (A) and CEC-32 (B) cells were infected at MOI 2. Virus titers were determined in cell culture media by plaque assay at different time points.(383 KB EPS)Click here for additional data file.Figure S2293T cells were infected with SC35 and SC35M (MOI 2) and harvested 6 h p.i. (A) and 12 h p.i. (B). CEC-32 cells were infected with SC35 and SC35M (MOI 2) and harvested 12 h p.i. (C) and 18 h p.i. (D). NP, M1, importin α1, and importin β1 were detected in whole cell lysates by Western blotting.(1.4 MB EPS)Click here for additional data file.Figure S3SC35M, and SC35-PB2701N (MOI 2) and stained 6 h p.i. with antisera specific for PB2 (secondary antibody Rhodamine-coupled) and importin α1 (secondary antibody FITC-coupled) (A) or NP (secondary antibody FITC-coupled) and importin α1 (secondary antibody Rhodamine-coupled) (B).293T cells were infected with SC35, SC35M, SC35-PB2(2.1 MB EPS)Click here for additional data file.Figure S4(2.8 MB EPS)Click here for additional data file.Figure S5SC35M, SC35-PB2SC35M, SC35-PASC35M, and SC35-NPSC35M at an MOI 0.0001, and virus titers in the supernatant were determined by plaque assay at different time points.A549 (A) and CEC-32 (B) cells were infected with SC35, SC35M, SC35-PB1(424 KB EPS)Click here for additional data file.