Comparison of the Rta/Orf50 transactivator proteins of gamma-2-herpesviruses - PubMed (original) (raw)

Comparative Study

Comparison of the Rta/Orf50 transactivator proteins of gamma-2-herpesviruses

Blossom Damania et al. J Virol. 2004 May.

Abstract

The viral immediate-early transactivator Rta/Orf50 is necessary and sufficient to initiate Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) reactivation from latently infected cells. Since Rta/Orf50 is conserved among all known gamma-2-herpesviruses, we investigated whether the murine gamma-68-herpesvirus (MHV-68) and rhesus monkey rhadinovirus (RRV) homologs can functionally substitute for KSHV Rta/Orf50. (i) Our comparison of 12 KSHV promoters showed that most responded to all three Rta/Orf50proteins, but three promoters (vGPCR, K8, and gB) responded only to the KSHV Rta/Orf50 transactivator. Overall, the activation of KSHV promoters was higher with KSHV Rta than with the RRV and MHV-68 Rta. (ii) Only the primate Rta/Orf50 homologs were able to interfere with human p53-depedent transcriptional activation. (iii) Transcriptional profiling showed that the KSHV Rta/Orf50 was more efficient than it's homologs in inducing KSHV lytic transcription from the latent state. These results suggest that the core functionality of Rta/Orf50 is conserved and independent of its host, but the human protein has evolved additional, human-specific capabilities.

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Figures

FIG. 1.

FIG. 1.

Three Rta/Orf50 proteins of the gamma-2-herpesvirus family. (A) ClustalW alignment of the three Rta/Orf50 proteins of KSHV, RRV, and MHV-68. Conserved amino acids are indicated by stars, and similarly charged residues are indicated by a semicolon. (B) Western blot of Rta/Orf50 protein expression after transient transfection into 293 cells. KSHV Rta/Orf50 was detected with an antibody against Rta/Orf50, and RRV and MHV-68 Rta/Orf50 proteins were epitope tagged and detected with antibodies against either an AU1 or Flag tag, respectively. (C) Western blot of the three Rta/Orf50 proteins in the four different cell lines. Lanes: 1 and 5, BJAB B cells; 2 and 6, BCBL-1 B cells; 3 and 7, SLK endothelial cells; 4 and 8, 293 epithelial cells. One hundred-fifty micrograms of total cell extract from the BJAB, BCBL, and SLK cells and 80 μg of cell extract from the 293 cells were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots were performed with either an AU1 antibody to detect RRV Orf50, Flag antibody to detect MHV-68 Orf50, or Rta antibody (K. Ueda) to detect KSHV Orf50.

FIG. 2.

FIG. 2.

Transactivation functions of KSHV, RRV, and MHV-68 Rta/Orf50. Panels A, B, and C depict the results of the transfection data from Table 1 for BJAB, 293, and SLK cells, respectively. (D) Inhibition of human p53 transactivation of a p53-responsive promoter/luciferase construct in p53-negative (10)1 cells. Promoter activity was measured as relative light units normalized to a cotransfected β-galactosidase promoter. The ability of all three Rta/Orf50 proteins to inhibit p53 activity is quantified as a percentage of inhibition.

FIG.3.

FIG.3.

Ability of the three Rta/Orf50 proteins to reactivate KSHV from latently infected BCBL-1 cells. (A) “Heatmap” representation of KSHV mRNA after transfection with KSHV, RRV, or MHV-68 Rta/Orf50 as determined by real-time quantitative RT-PCR (16). The three leftmost panels represent mock-treated cells and two replicate experiments of sorted, Rta/Orf50-transfected cells after 48 h. (B) KSHV mRNA levels in the total culture 72 h after transfection with KSHV, RRV, or MHV-68 Rta/Orf50. Darker shades of gray correspond to higher mRNA levels on a log 2 scale. (C) Plots of three individual relative mRNA (Δ CT) levels selected from panel A for KSHV v-cyclin (CYC), Mta/Orf57, and vGPCR on the vertical axis for each of the experiments on the horizontal axis. (D) mRNA levels (Δ CT) after electroporation with KSHV, RRV, or MHV68 Rta/Orf50 on the horizontal axis relative to the mRNA levels after electroporation with KSHV Rta/Orf50.

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