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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Gene Expression I Abstract 65 KSHV ORF57 AND RNA EXPORT FACTORS: ROLES OF UAP56, URH49, RBM15, AND OTT3 IN ORF57 EXPRESSION AND FUNCTION Merlyn Deng 1 , Vladimir Majerciak 1 , Barbara K. Felber 2 , and Zhi-Ming Zheng 1 1HIV and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD. USA. 2 Human Retrovirus Pathogenesis Section, Vaccine Branch, National Cancer Institute, Frederick, MD, USA Abstract KSHV ORF57 promotes the expression of a subset of viral lytic genes at posttranscriptional level and is essential for virus production. To investigate how ORF57 might function to promote RNA export of its targets, we examined the cellular export factors UAP56, URH49, RBM15 and OTT3 for their possible roles in ORF57-enhanced expression of KSHV ORF59. We found that reducing the expression of each of these proteins with gene-specific siRNAs significantly reduced the expression of ORF59 in cotransfection experiments. Surprisingly, we also found that all four proteins affected ORF57 expression, indicating that the reduced ORF59 expression was likely due to the reduction of ORF57. The effect became most severe when two genes were knocked down simultaneously. To investigate whether the downregulation of ORF57 expression was due to a reduction of ORF57 mRNA export, we fractionated the nuclear RNA from the cytoplasmic RNA of cells transfected with an ORF57 expression vector and UAP56 and/URH49 siRNAs. A substantial reduction of cytoplasmic ORF57 RNA was observed along with substantial accumulation of nuclear ORF57 RNA. A greater reduction of cytoplasmic ORF57 RNA was noticed in double-knockdown cells. Although overexpression of UAP56 had no effect on ORF57 or ORF59, the presence of RBM15 or OTT3 greatly increased expression of ORF59, but suppressed ORF57 expression. Collectively, our data suggest that ORF57 expression is controlled by the four RNA export factors analyzed. The role of ORF57 is likely to substitute cellular RBM15 and OTT3 in promotion of ORF59 expression by sequestering them from RNA export machinery. Presenting author Email: zhengt@exchange.nih.gov 94
The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Gene Expression II Abstract 66 DYNAMICS OF K-RTA RECRUITMENT ON THE KSHV GENOME REVEAL NOVEL REGULATION BY NF-KB Thomas J. Ellison 1 , Chie Izumiya 1 , Paul A. Luciw , Hsing-Jien Kung 1 , Yoshihiro Izumiya 1 1 nd 2 UC Davis Cancer Center, 4645 2 Ave. Sacramento, California 95817, USA. Department of Pathology, UC Davis, 1 Sheilds Ave. Davis, Davis, California 95616, USA. Abstract KSHV is regulated epigenetically and transcriptionally, subject to cellular factors including NF-kB. K-Rta and K-bZIP are two key factors that control reactivation and lytic replication. In this work, we performed genome-wide chromatin immunoprecipitation anaylsis with a viral promoter-chip (ChIP-on-Vchip) containing all 83 putative KSHV promoter regions. The recruitment of K-Rta and K-bZIP were examined in BCBL-1, as well as association with acetylated histone 3 as a marker for chromatin state. K12 and Ori-RNA promoters were major sites for recruitment of K-Rta, and a number of K-bZIP binding sites were also identified. To examine the dynamics of recruitment, ten viral promoters were selected for a time-course. K-Rta recruitment was most evident at intermediate-strength target promoters, whereas the primary sites of K-bZIP binding were its repression targets, to which it was co-recruited with K-Rta by 4-12 hours post induction. Because NF-kB has been implicated in K-Rta transactivation, it was also examined using the viral promoter library. Interestingly, the only two viral promoters not responsive to NF-kB mediated inhibition were the K-Rta major binding promoters. Overexpression of NF-kB strongly inhibited recruitment of K-Rta to the ORF57 and KbZIP promoters but not the K12 promoter during viral reactivation. These results were further tested by in vitro DNA binding assay using RBPjk, RelA, NF-kB1, and K-Rta. NFkB sequestered RBP-jk from the ORF57 promoter, and was found to form a complex with RBP-jk through their Rel homology domains. These studies set the stage for further analysis of regulation of KSHV reactivation. Presenting author Email: yizumiya@ucdavis.edu 95
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