Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012-2013. Here, we describe an antibody, F045-092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045-092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045-092 extends its recognition to divergent subtypes, including H1, H2, and H13, using the enhanced avidity of its IgG to overcome lower affinity Fab binding, as observed with other receptor-binding site antibodies. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small molecule therapeutics.

Influenza A viruses are classified into 16 subtypes according to the serotypes of hemagglutinin (HA). It is generally thought that neutralizing antibodies (Abs) are not broadly cross-reactive among HA subtypes. We examined the repertoire of neutralizing Abs against influenza viruses in humans. B lymphocytes were collected from donors by apheresis, and Ab libraries were constructed by using phage-display technology. Anti-HA clones were isolated by screening with H3N2 viruses. Their binding activity was examined, and four kinds of Abs showing broad strain specificity were identified from one donor. Two of the Abs, F045-092 and F026-427, were extensively analyzed. They neutralized not only H3N2 but also H1N1, H2N2, and H5N1 viruses, although the activities were largely varied. Flow cytometry suggested that they have the ability to bind to HA and HA1 artificially expressed on the cell surface. They show hemagglutination inhibition activity and do not compete with C179, an Ab thought to bind to the stalk region. F045-092 competes with Abs that recognize sites A and B for binding to HA. Furthermore, the serine at residue 136 in site A could be a part of the epitope. Thus, it is likely that F045-092 and F026-427 bind to a conserved epitope in the head region formed by HA1. Interestingly, while the V H 1-69 gene can encode MAbs against the HA stem that are group 1 specific, F045-092 and its relatives that recognize the head region also use V H 1-69. The possible epitope recognized by these clones is discussed.

, it has been reported that DREF can bind to a sequence in the hsp70 scs chromatin boundary element that is also recognized by boundary element-associated factor, and thus DREF may participate in regulating insulator activity. To examine DREF function in vivo, we established transgenic flies in which ectopic expression of DREF was targeted to the eye imaginal discs. Adult flies expressing DREF exhibited a severe rough eye phenotype. Expression of DREF induced ectopic DNA synthesis in the cells behind the morphogenetic furrow, which are normally postmitotic, and abolished photoreceptor specifications of R1, R6, and R7. Furthermore, DREF expression caused apoptosis in the imaginal disc cells in the region where commitment to R1/R6 cells takes place, suggesting that failure of differentiation of R1/R6 photoreceptor cells might cause apoptosis. The DREF-induced rough eye phenotype was suppressed by a half-dose reduction of the E2F gene, one of the genes regulated by DREF, indicating that the DREF overexpression phenotype is useful to screen for modifiers of DREF activity. Among Polycomb/trithorax group genes, we found that a half-dose reduction of some of the trithorax group genes involved in determining chromatin structure or chromatin remodeling (brahma, moira, and osa) significantly suppressed and that reduction of Distal-less enhanced the DREF-induced rough eye phenotype. The results suggest a possibility that DREF activity might be regulated by protein complexes that play a role in modulating chromatin structure. Genetic crosses of transgenic flies expressing DREF to a collection of Drosophila deficiency stocks allowed us to identify several genomic regions, deletions of which caused enhancement or suppression of the DREF-induced rough eye phenotype. These deletions should be useful to identify novel targets of DREF and its positive or negative regulators.The promoters of Drosophila genes related to DNA replication, such as those for the 180-kDa catalytic subunit and 73-kDa subunit polypeptide of DNA polymerase ␣ and proliferating-cell nuclear antigen (PCNA), contain a common 8-bp palindromic sequence (5Ј-TATCGATA), named the DNA replication-related element (DRE) (21) in addition to E2F recognition sites (13, 54). The DRE requirement for promoter activation has been confirmed in both cultured cells and transgenic flies (22, 51, 52). Studies using the latter have shown that DRE is required for the function of the PCNA promoter throughout development except in adult females. We found a specific DRE-binding factor (DREF) consisting of an 80-kDa polypeptide homodimer, and molecular cloning of its cDNA has led to confirmation that DREF is a trans activator for DRE-containing genes (22).Recently, we found that the N-terminal fragment of the DREF polypeptide containing a region responsible for DRE binding and dimer formation acts as dominant negative effector against DREF (23). Expression of a dominant-negative form of DREF in cells of salivary glands and eye imaginal discs using the GAL4-upstream activation site (UAS...

A human homologue (hDREF/KIAA0785) of Drosophila DREF, a transcriptional regulatory factor required for expression of genes involved in DNA replication and cell proliferation, was identified by BLAST search. Amino acid sequences corresponding to three regions highly conserved between two Drosophila species also proved to be very similar in the hDREF/KIAA0785 polypeptide. A consensus binding sequence (5-TGTCG(C/T)GA(C/ T)A) for hDREF/KIAA0785, determined by the CASTing method, overlapped with that for the Drosophila DREF (5-TGTCGATA). We found hDREF/KIAA0785 binding sequences in the promoter regions of human genes related to cell proliferation. Analyses using a specific antibody revealed that an hDREF/KIAA0785 binds to the promoter region of the histone H1 gene. Co-transfection experiments with an hDREF/KIAA0785-expressing plasmid and a histone H1 promoter-directed luciferase reporter plasmid in HeLa cells revealed possible activation of the histone H1 promoter. Immunohistochemical analysis demonstrated that hDREF/KIAA0785 is localized in the nuclei. Although the expression level of the factor was found to be low in serum-deprived human normal fibroblasts, the amount was increased by adding serum to cultures and reached a maximum during S phase. RNA interference experiments targeting hDREF/KIAA0785 resulted in inhibition of S phase entry and reduction of histone H1 mRNA in HeLa cells. These results suggest that expression of hDREF/KIAA0785 may have a role in regulation of human genes related to cell proliferation.Promoters of Drosophila genes related to DNA replication, such as those for the 180-kDa catalytic subunit of DNA polymerase ␣ and proliferating cell nuclear antigen (PCNA), 1 contain a common 8-bp palindromic sequence (5Ј-TATCGATA), named the DRE (DNA replication-related element) (1), in addition to E2F recognition sites (2-5). Our previous studies (6, 7) performed in vitro and in vivo suggested that the DRE sequence and the E2F binding sites function synergistically in activation of PCNA, DNA polymerase ␣, and dE2F genes. We found a specific DREF (DRE-binding factor) consisting of an 80-kDa polypeptide homodimer, and molecular cloning of its cDNA has allowed confirmation that DREF is a trans-activator for DREcontaining genes (8). An important role of the DRE/DREF regulatory system has been indicated by the finding that DRE/ DREF is a target of some differentiation signals. The zen (zerknullt) gene encoding a homeodomain-containing protein, Zen, which is expressed in the dorsal region of the early embryo at the cellular blastoderm stage, is involved in differentiation of the amnioserosa and the optic lobe (9). Zen expression in cultured cells results in repression of DRE-containing genes by reducing the DREF activity (10). Thus, the DRE/DREF system may occupy a cross-road position in growth and differentiation signaling pathways.Recently, Hart et al. (11,12) proposed a novel function of DREF as an antagonist of the boundary element-associated factor (BEAF), which is involved in the boundary activity of t...