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Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a transforming protein that affects multiple cell signaling pathways and contributes to EBV-associated oncogenesis. LMP1 can be expressed in some states of EBV latency, and significant induction of full-length LMP1 is also observed frequently during virus reactivation into the lytic cycle. It is still unknown how LMP1 expression is regulated during the lytic stage and whether any EBV lytic protein is involved in the induction of LMP1. In this study, we first identified that LMP1 expression is associated with the spontaneous virus reactivation in EBV-infected 293 cells and that its expression is a downstream event of the lytic cycle. We further found that LMP1 can be induced by ectopic expression of Rta, an EBV immediate-early lytic protein. The Rta-mediated LMP1 induction is independent of another immediate-early protein, Zta. Northern blotting and reverse transcription-PCR analysis revealed that Rta upregulates LMP1 at the RNA level. Reporter gene assays further demonstrated that Rta activates both the proximal and distal promoters of the LMP1 gene in EBV-negative cells. Both the amino and carboxyl termini of the Rta protein are required for the induction of LMP1. In addition, Rta transactivates LMP1 not only in epithelial cells but also in B-lymphoid cells. This study reveals a new mechanism to upregulate LMP1 expression, expanding the knowledge of LMP1 regulation in the EBV life cycle. Considering an equivalent case of Kaposi's sarcoma-associated herpesvirus, induction of a transforming membrane protein by a key lytic transactivator during virus reactivation is likely to be a conserved event for gammaherpesviruses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]