PRIMA-1MET induces nucleolar translocation of Epstein-Barr virus-encoded EBNA-5 protein

The low molecular weight compound, PRIMA-1MET restores the transcriptional transactivation function of certain p53 mutants in tumor cells. We have previously shown that PRIMA-1MET induces nucleolar translocation of p53, PML, CBP and Hsp70. The Epstein-Barr virus encoded, latency associated antigen EBNA-5 (also known as EBNA-LP) is required for the efficient transformation of human B lymphocytes by EBV. EBNA-5 associates with p53-hMDM2-p14ARF complexes. EBNA-5 is a nuclear protein that translocates to the nucleolus upon heat shock or inhibition of proteasomes along with p53, hMDM2, Hsp70, PML and proteasome subunits. Here we show that PRIMA-1MET induces the nucleolar translocation of EBNA-5 in EBV transformed B lymphoblasts and in transfected tumor cells. The PRIMA-1MET induced translocation of EBNA-5 is not dependent on the presence of mutant p53. It also occurs in p53 null cells or in cells that express wild type p53. Both the native and the EGFP or DSRed conjugated EBNA-5 respond to PRIMA-1MET treatment in the same way. Image analysis of DSRed-EBNA-5 expressing cells, using confocal fluorescence time-lapse microscopy showed that the nucleolar translocation requires several hours to complete. FRAP (fluorescence recovery after photobleaching) and FLIP (fluorescence loss in photobleaching) measurements on live cells showed that the nucleolar translocation was accompanied by the formation of EBNA-5 aggregates. The process is reversible since the aggregates are dissolved upon removal of PRIMA-1MET. Our results suggest that mutant p53 is not the sole target of PRIMA-1MET. We propose that PRIMA-1MET may reversibly inhibit cellular chaperons that prevent the aggregation of misfolded proteins, and that EBNA-5 may serve as a surrogate drug target for elucidating the precise molecular action of PRIMA-1MET

Associations between SNPs in candidate immune-relevant genes and rubella antibody levels: a multigenic assessment

<p>Abstract</p> <p>Background</p> <p>The mechanisms of immune response are structured within a highly complex regulatory system. Genetic associations with variation in the immune response to rubella vaccine have typically been assessed one locus at a time. We simultaneously assessed the associations between 726 SNPs tagging 84 candidate immune response genes and rubella-specific antibody levels. Blood samples were obtained from 714 school-aged children who had received two doses of MMR vaccine. Associations between rubella-specific antibody levels and 726 candidate tagSNPs were assessed both one SNP at a time and in a variety of multigenic analyses.</p> <p>Results</p> <p>Single-SNP assessments identified 4 SNPs that appeared to be univariately associated with rubella antibody levels: rs2844482 (p = 0.0002) and rs2857708 (p = 0.001) in the 5'UTR of the LTA gene, rs7801617 in the 5'UTR of the IL6 gene (p = 0.0005), and rs4787947 in the 5'UTR of the IL4R gene (p = 0.002). While there was not significant evidence in favor of epistatic genetic associations among the candidate SNPs, multigenic analyses identified 29 SNPs significantly associated with rubella antibody levels when selected as a group (p = 0.017). This collection of SNPs included not only those that were significant univariately, but others that would not have been identified if only considered in isolation from the other SNPs.</p> <p>Conclusions</p> <p>For the first time, multigenic assessment of associations between candidate SNPs and rubella antibody levels identified a broad number of genetic associations that would not have been deemed important univariately. It is important to consider approaches like those applied here in order to better understand the full genetic complexity of response to vaccination.</p