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Series GSE11432 Query DataSets for GSE11432
Status Public on Aug 31, 2008
Title Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein
Platform organism Human gammaherpesvirus 8
Sample organisms Chlorocebus aethiops; Human gammaherpesvirus 8
Experiment type Expression profiling by array
Summary Lytic reactivation from latency is critical for the pathogenesis of KSHV. We previously demonstrated that the 691 amino acid KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta’s direct targets during reactivation by generating a conditionally nuclear variant of Rta. WT Rta protein is constitutively localized to cell nuclei, and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS-2; aa 516-530) was required for nuclear localization of Rta, and relocalized eGFP exclusively to cell nuclei. Analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and stimulation of viral reactivation. Fusion of Rta_NLS-1,2 to the hormone binding domain of the murine estrogen receptor generated a variant of Rta whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled post-translationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Only eight KSHV genes were activated by Rta in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different magnitudes, and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/vIL-6, ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that induction of most of the KSHV lytic cycle genes requires additional protein expression post-Rta.
Keywords: Comparative transcriptome analysis by oligonucleotide microarray
 
Overall design KSHV infected cells were transfected with empty expression vector or with vector expressing RtadeltaNLS1,2-ER (RtadeltaNLS1,2 fused to murine hormone binding domain of estrogen receptor (ER)). Cells were treated with 4-hydroxytamoxifen (4-OHT) to induce nuclear re-localization of Rta fusion protein. Cells were pre-treated with hygromycin to eliminate de novo cellular and viral protein expression. Direct transcriptional targets of Rta were identified by comparing Rta +/- 4-OHT to vector (negative control) +/- 4-OHT.
 
Contributor(s) Bu W, Palmeri D, Aris VM, Soteropoulos P, Lukac DM
Citation(s) 18715905
Submission date May 13, 2008
Last update date Mar 19, 2012
Contact name David M. Lukac
E-mail(s) lukacdm@njms.rutgers.edu
Phone 973 972-4868
Organization name Rutgers Univ./NJ Medical School
Department Microbiology, Biochemistry and Molecular Genetics
Street address 225 Warren St.; ICPH E350C; PO Box 1709
City Newark
State/province NJ
ZIP/Postal code 07101
Country USA
 
Platforms (1)
GPL6852 UMDNJ/Lukac Human herpesvirus 8/107+GAPDH
Samples (36)
GSM288239 VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample1-Repl-1
GSM288242 VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample1-Repl-2
GSM288244 VectorTransfected-Vero-rKSHV.219cells-Hygro-Plus/Minus4-OHT-Sample1-Repl-3
Relations
BioProject PRJNA106457

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