Standard deviations are shown as error bars

Standard deviations are shown as error bars. (+)-α-Tocopherol In order to analyze the capacity of all EBNA-2 mutants to induce endogenous transcripts we selected two viral, LMP1 and LMP2A, and two cellular target genes, CCL3 and CD23, for quantitative RT-PCR analyses in Eli-BL (Fig 5). of the EBNA-2 N-terminal dimerization (END) domain by heteronuclear NMR-spectroscopy. The END domain monomer comprises a small fold of four -strands and an -helix which form a parallel dimer by interaction of two -strands from each protomer. A structure-guided mutational analysis showed that hydrophobic residues in the dimer interface are required for self-association and purified with or without Z-tag under native conditions. The oligomerization status of the recombinant proteins was analyzed by analytical size exclusion chromatography (SEC) and static light scattering (SLS) (Table 1).The EBNA-2 N-terminal fragment lacking a Z-tag forms a single molecular species with a molecular mass of 13.1 kDa as expected for a dimer (2×6.7 kDa). Similarly, the EBNA-2 Z-tag fusion protein eluted as a single peak with a molecular mass of 46.3 kDa close to the theoretical molecular mass of a dimer (2×23.4 kDa). Table 1 Dimerization analysis of wild-type and mutant END domains by SEC/SLS and NMR. would also impair self-association of the full-length EBNA-2 protein [23, 24]. We expressed wild-type, deletion, surface and interface mutants as full-length EBNA-2 HA-tagged proteins and performed co-immunoprecipitation experiments in EBV negative DG75 cells [31] (Fig 3BC3E). For comparison we included HA-tagged mutants of EBNA-2 lacking amino acids 3C30 or 3C52 in our analyses (3C30 and 3C52, respectively). All EBNA-2 mutants were expressed well and could be co-expressed with a FLAG-tagged EBNA-2 fragment encompassing amino acid 1C199 (F199). Co-immunoprecipitation studies using HA-specific antibodies indicated that all EBNA-2 mutants efficiently bound to endogenous CBF1. Both EBNA-2N-terminal deletion mutants were significantly impaired for self-association as has been reported previously (Fig 3D) [24]. The residual binding of 3C30 and 3C52 to F199 might be supported by the second self-association domain, comprising residues 96C210, which is still present in (+)-α-Tocopherol the F199 protein [23]. The self-association domain of a non-conserved region [27] is not present in F199 and thus cannot account for residual dimerization. Next, (+)-α-Tocopherol we tested whether the interface mutants L16A, L16D, I50A and I50D can still mediate self-association with the EBNA-2 F199 fragment, which also harbors the END domain (Fig 3C, middle and right panel). While substitution of the Leu16 or Ile50 by alanine did not significantly affect F199 association, introduction of a negative charge by aspartic acid prevented self-association. These results confirmed the structural data indicating that hydrophobic residues facing each other across the dimer interface of the END domain are essential for EBNA-2 self-association. Surprisingly, 3C30 and 3C52 appeared to be less impaired than L16D and I50D. In order to further validate the structural integrity of the END domain in the context of the complete EBNA-2 protein we tested the surface mutants H15A, 1 and (+)-α-Tocopherol F51A for association with F199 (Fig 3E). Consistent with the Rat monoclonal to CD4.The 4AM15 monoclonal reacts with the mouse CD4 molecule, a 55 kDa cell surface receptor. It is a member of the lg superfamily,primarily expressed on most thymocytes, a subset of T cells, and weakly on macrophages and dendritic cells. It acts as a coreceptor with the TCR during T cell activation and thymic differentiation by binding MHC classII and associating with the protein tyrosine kinase, lck structural and biophysical data all surface mutants retained the capacity to self-associate, confirming that these residues are not essential for the dimerization of EBNA-2. Nuclear localization and formation of nuclear speckles is a typical feature of EBNA-2 [32]. In order to analyze whether the END domain mutants had retained these features all EBNA-2 mutants were expressed in HeLa cells and the subcellular distribution of the EBNA-2 proteins was analyzed by confocal microscopy (Fig E in S1 Text). All mutants still showed strict nuclear localization, (+)-α-Tocopherol which typically excludes the nucleoli. Moreover, all mutants formed granular speckles, which are characteristic of wild-type EBNA-2 protein. The surface mutations H15A and 1 affect the function of the EBNA-2 protein Based on previous work, EBNA-2 mutants impaired for dimerization were also severely impaired for activation of the viral target gene LMP1 [24]. In order to analyze the capacity of the EBNA-2 surface and interface mutants to activate the viral LMP genes we expressed EBNA-2 mutants in the EBV positive Burkitt’s lymphoma cell line Eli-BL [33]. This B cell line exhibits a specific viral gene expression program where.