Transcription Activation of Polyadenylated Nuclear RNA by Rta in Human Herpesvirus 8/Kaposi's Sarcoma-Associated Herpesvirus

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is a member of the gammaherpesvirus subfamily, which also includes herpesvirus saimiri, murine gammaherpesvirus 68 (MHV-68) and Epstein-Barr virus (EBV) (5, 29, 35). Gammaherpesviruses have been shown to be associated with tumor formation in infected hosts. HHV-8 is commonly found in all types of KS, including AIDS-associated KS, classic KS, endemic forms of KS, and renal transplant-related KS (1, 2, 14, 16, 25, 28, 32, 40, 43). HHV-8 is also associated with primary effusion lymphoma (PEL) (4) and multicentric Castleman's disease (38). While most tumor cells are latently infected with HHV-8, in a small number of cells in tumor lesions, the virus undergoes lytic replication (8, 33, 39, 40, 43, 51). Recent data have demonstrated the expression of viral macrophage inflammatory proteins and viral interleukin-6 in a subset of tumor cells (3, 40, 43). In these cells, while undergoing lytic replication, the virus expresses viral cytokines, which may play an important role in the pathogenesis of HHV-8 (9, 26), for example, by creating a favorable environment for supporting growth of latently infected neighboring cells. Studies of HHV-8 gene expression have revealed a novel transcript, polyadenylated nuclear (PAN) RNA (also referred as T1.1 or nut-1) (41, 51). PAN RNA was initially identified in KS tumor tissue (51), as well as in PEL cells latently infected with HHV-8 (41). The expression of PAN RNA in PEL cells was induced by chemicals. More than 80% of cDNA clones from induced BC-1 cells represented a single transcript, PAN RNA, indicating the abundance of PAN RNA during lytic replication. PAN RNA is the most abundant viral transcript of HHV-8. Its copy number per cell has been estimated by a Northern analysis to be up to 2.5 × 105 to 5.0 × 105 copies in PEL cells (41) and 10,000 to 25,000 copies in KS tumor cells (41, 51). PAN RNA is expressed with early kinetics, and high levels of expression are maintained until late stages of lytic replication (43). PAN RNA is unique in that it contains features of both mRNAs and small nuclear RNAs (snRNAs). In common with mRNA, PAN RNA has a canonical poly(A) signal and is polyadenylated. PAN RNA is also transcribed by RNA polymerase II, based on its sensitivity to α-amanitin (5 μg/ml), an inhibitor of RNA polymerase II (41). However, similar to snRNAs, PAN RNA appears to be a noncoding transcript, as deduced from the following (41). First, 57 stop codons are distributed randomly in all three reading frames throughout the transcript, making the longest possible open reading frame (ORF) of 61 amino acids. Second, this ORF is located in a region where codon usage is not typical of that most commonly found in eukaryotes. In addition, PAN RNA was not associated with ribosomes, according to sucrose gradient sedimentation. Instead, PAN RNA was found by fluorescence in situ hybridization to be in the nuclear speckles (41) and is known to form a ribonucleoprotein complex in the nucleoplasm (41, 50). Although it has been speculated that PAN RNA is involved in RNA processing in the nucleus, its function has yet to be defined. However, elucidating the mechanism controlling the expression of PAN RNA, the most abundant transcript of HHV-8, may help us understand how the virus maximizes its expression during lytic replication. Upon reactivation from latency, viral lytic genes of HHV-8 (immediate-early, early, and late genes) are expressed in a highly regulated manner. rta, an immediate-early gene, is conserved among all gammaherpesviruses, including EBV (23), herpesvirus saimiri (45), bovine herpesvirus 4 (44), and MHV-68 (20, 48), and plays a central role in the switch of the viral life cycle from latency to lytic replication. Rta acts as a transcriptional activator, and its expression has been demonstrated to be sufficient for viral reactivation, since ectopic expression of Rta has been found to reactivate the latent HHV-8 genome to lytic replication in PEL cell lines (22, 42). In addition, a dominant-negative form of Rta was shown to suppress viral reactivation, indicating that Rta is necessary for the switch of the viral life cycle (21). It has been recently reported that in MHV-68, a gamma-2 herpesvirus closely related to HHV-8, Rta alone is sufficient to disrupt latency and activate viral lytic replication, indicating a conserved role of Rta in the reactivation of gamma-2 herpesviruses (48). Two immediate-early genes of EBV (a gamma-1 herpesvirus), ZEBRA (also referred to as BZLF1, Zta, or Z) and rta, play essential roles in disruption of viral latency, activating downstream genes in a cooperative fashion (6, 7, 31, 49). However, there is no evidence that K-bZIP (known as K8), the ZEBRA homologue of HHV-8, can function as a switch gene for reactivation of HHV-8 (12, 19, 42, 52). The mechanism by which Rta controls lytic gene expression of gammaherpesviruses is currently being investigated. Consistent with the facts that PAN RNA is an early lytic gene transcript of HHV-8 and that Rta is a potent transactivator of early genes, Rta has been shown to induce the expression of PAN RNA from the endogenous viral genome (42) and activate the PAN promoter linked to a heterologous gene (22). Promoters of other early genes of HHV-8 are also responsive to Rta but are expressed at lower levels than PAN RNA. Therefore, it is intriguing how HHV-8 produces PAN RNA at such high levels. Here, we address this mechanism by measuring the abundance of PAN RNA, mapping the Rta-responsive element (RRE) in the PAN promoter and demonstrating the direct binding of Rta to this RRE.