Role of the epstein-barr virus RTA protein in activation of distinct classes of viral lytic cycle genes

Initiation of the Epstein-Barr virus (EBV) lytic cycle is controlled by two immediate-early genes, BZLF1 and BRLF1. In certain epithelial and B-cell lines, their protein products, ZEBRA and Rta, stimulate their own expression, reciprocally stimulate each other's expression, and activate downstream viral targets. It has been difficult to examine the individual roles of these two transactivators in EBV-infected lymphocytes, as they are expressed simultaneously upon induction of the lytic cycle. Here we show that the Burkitt lymphoma cell line Raji represents an experimental system that allows the study of Rta's role in the lytic cycle of EBV in the absence and presence of ZEBRA. When expressed in Raji cells, exogenous Rta does not activate endogenous BZLF1 expression, yet Rta remains competent to transactivate certain downstream viral targets. Some genes, such as BaRF1, BMLF1, and a late gene, BLRF2, are maximally activated by Rta itself in the absence of detectable ZEBRA. The use of the Z(S186A) mutant form of ZEBRA, whose transactivation function is manifest only by coexpression of Rta, allows identification of a second class of lytic cycle genes, such as BMRF1 and BHRF1, that are activated in synergy by Rta and ZEBRA. It has already been documented that of the two activators, only ZEBRA stimulates the BRLF1 gene in Raji cells. Thus, there is a third class of viral genes activated by ZEBRA but not Rta. Moreover, ZEBRA exhibits an inhibitory effect on Rta's capacity to stimulate the late gene, BLRF2. Consequently ZEBRA may function to repress Rta's potential to activate some late genes. Raji cells thus allow delineation of the combinatorial roles of Rta and ZEBRA in control of several distinct classes of lytic cycle genes.     

Interplay between PKCδ and Sp1 on histone deacetylase inhibitor-mediated Epstein-Barr virus reactivation

Epstein-Barr virus (EBV) undergoes latent and lytic replication cycles, and its reactivation from latency to lytic replication is initiated by expression of the two viral immediate-early transactivators, Zta and Rta. In vitro, reactivation of EBV can be induced by anti-immunoglobulin, tetradecanoyl phorbol acetate, and histone deacetylase inhibitor (HDACi). We have discovered that protein kinase C delta (PKCδ) is required specifically for EBV reactivation by HDACi. Overexpression of PKCδ is sufficient to induce the activity of the Zta promoter (Zp) but not of the Rta promoter (Rp). Deletion analysis revealed that the ZID element of Zp is important for PKCδ activation. Moreover, the Sp1 putative sequence on ZID is essential for PKCδ-induced Zp activity, and the physiological binding of Sp1 on ZID has been confirmed. After HDACi treatment, activated PKCδ can phosphorylate Sp1 at serine residues and might result in dissociation of the HDAC2 repressor from ZID. HDACi-mediated HDAC2-Sp1 dissociation can be inhibited by the PKCδ inhibitor, Rotterlin. Furthermore, overexpression of HDAC2 can suppress the HDACi-induced Zp activity. Consequently, we hypothesize that HDACi induces PKCδ activation, causing phosphorylation of Sp1, and that the interplay between PKCδ and Sp1 results in the release of HDAC2 repressor from Zp and initiation of Zta expression.     

DNA-binding-defective mutants of the Epstein-Barr virus lytic switch activator Zta transactivate with altered specificities.

The Epstein-Barr virus BRLF1 and BZLF1 genes are the first viral genes transcribed upon induction of the viral lytic cycle. The protein products of both genes (referred to here as Rta and Zta, respectively) activate expression of other viral genes, thereby initiating the lytic cascade. Among the viral antigens expressed upon induction of the lytic cycle, however, Zta is unique in its ability to disrupt viral latency; expression of the BZLF1 gene is both necessary and sufficient for triggering the viral lytic cascade. We have previously shown that Zta can activate its own promoter (Zp), through binding to two Zta recognition sequences (ZIIIA and ZIIIB). Here we describe mutant Zta proteins that do not bind DNA (referred to as Zta DNA-binding mutants [Zdbm]) but retain the ability to transactivate Zp. Consistent with the inability of these mutants to bind DNA, transactivation of Zp by Zdbm is not dependent on the Zta recognition sequences. Instead, transactivation by Zdbm is dependent upon promoter elements that bind cellular factors. An examination of other viral and cellular promoters identified promoters that are weakly responsive or unresponsive to Zdbm. An analysis of a panel of artificial promoters containing one copy of various promoter elements demonstrated a specificity for Zdbm activation that is distinct from that of Zta. These results suggest that non-DNA-binding forms of some transactivators retain the ability to transactivate specific target promoters without direct binding to DNA.