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Rta, an Epstein-Barr virus (EBV)-encoded immediate-early protein, governs the reactivation of the viral lytic program by transactivating a cascade of lytic gene expression. Cellular transcription factors such as Sp1, ATF2, E2F, and Akt have been demonstrated to mediate Rta transactivation of lytic genes. We report herein that Rta associates with another potent transcription factor, tumor susceptibility gene 101 (TSG101), to promote the activation of EBV late genes. Results from an EBV cDNA array reveal that depletion of TSG101 by siRNA potently inhibits the transcription of five Rta-responsive EBV late genes, BcLF1, BDLF3, BILF2, BLLF1, and BLRF2. Depletion of TSG101 impairs the Rta transactivation of these late promoters severely. Moreover, a concordant augmentation of Rta transactivating activity is observed when TSG101 is overexpressed following ectopic transfection. Mechanistically, Rta interaction with TSG101 causes the latter to accumulate principally in the nuclei, wherein the proteins colocalize and are recruited to the viral promoters. Of note, TSG101 is crucial for the efficient binding of Rta to these late promoters. As a result, cells with defective TSG101 fail to express late viral proteins, leading to a decrease in the yield of virus particles. Thus, the contribution of TSG101 to Rta-mediated late gene activation is of great importance for completion of the EBV productive lytic cycle. These observations consolidate a role for TSG101 in the replication of EBV, a DNA virus, that differs from what is observed for RNA viruses, where TSG101 aids mainly in the endosomal sorting of enveloped late viral proteins for assembly at the plasma membrane.

The Zta protein is a key transactivator involved in initiating the Epstein-Barr virus (EBV) lytic cascade. In addition to transactivating many viral genes, Zta has the capacity to influence host cellular signals by binding to promoter regions or by interacting with several important cellular factors. Based on the observation that tyrosine kinases play central roles in determining the fate of cells, a kinase display assay was used to investigate whether cells expressing Zta have an altered pattern of kinase expression. The assay revealed that TRK-related tyrosine kinase (TKT) is expressed at significant levels in Zta transfectants but not in control cells. Additional evidence was obtained from Northern and Western blotting. Importantly, the upregulation of phosphorylated TKT and TKT downstream effector matrix metalloproteinase 1 in Zta transfectants hinted that TKT might initiate a signaling cascade in Zta-expressing cells. In addition, deletion analysis of the Zta protein revealed that the transactivation and dimerization domains were both essential for the upregulation of TKT transcription. Moreover, correlation of expression levels of Zta and TKT transcripts in nasopharyngeal carcinoma biopsy specimens was clearly demonstrated by quantitative PCR (Q-PCR), which provides the first evidence for an effect of Zta on cellular gene expression in vivo. These findings offer insight into the virus-cell interactions and may help us elucidate the role of EBV in tumorigenesis.