RBP-Jκ is a downstream transcription factor of the Notch pathway that is important in development and cell fate determination (44
). The intracellular domain of activated Notch receptor is released from the plasma membrane through proteolytic cleavage and is translocated to the nucleus, where it is directed to target promoters through RBP-Jκ interaction (42
). A previous report (37
) has shown that the KSHV RTA protein mimics cellular Notch signal transduction by interacting with RBP-Jκ and by activating RBP-Jκ-dependent activation of viral lytic gene expression. Here, we also demonstrate that the constitutively active form of the human Notch signaling molecule, hNIC, is partially exchangeable with RTA in viral lytic gene expression. A microarray showed that hNIC robustly induced expression of a number of viral genes, including vIL-6, K3, and K5, but was not capable of evoking the full repertoire of lytic viral gene expression. Further detailed analysis showed that hNIC targeted the specific RBP-Jκ binding sites of vIL-6 and K5 promoter regions to induce their gene expression. These results indicate that cellular Notch signal transduction not only is partially exchangeable with RTA in regard to activation of viral lytic gene expression but also provides a novel expression profile of KSHV growth and immune deregulatory genes.
RTA transcription factor that controls the switch from latency to lytic replication interacts with RBP-Jκ to activate viral gene expression. The fact that many KSHV lytic genes, including RTA itself, contain RBP-Jκ binding sites has raised the possibility that RTA/RBP-Jκ-mediated gene expression may be central to the switch from latency to lytic replication (7
). EBV EBNA2 has also been shown to be recruited to its responsive elements through interaction with RBP-Jκ (29
), indicating that EBNA2 and RTA may thus be regarded as functional homologs or mimickers of the activated Notch protein. Indeed, hNIC has been shown to be capable of functionally replacing EBNA2 in the context of EBV for primary B-cell transformation (21
). We also showed that hNIC was capable of partially replacing the RTA function in KSHV gene expression. Interestingly, while hNIC induced the expression of approximately 24 KSHV genes and RTA contained the functional RBP-Jκ binding site in its promoter region, RTA was not one of the genes induced by hNIC. A recent study has demonstrated that RBP-Jκ binding sites within the RTA promoter are critical for the repression rather than the activation of its expression (36
). Specifically, LANA physically associates with RBP-Jκ, and this interaction represses RTA expression by targeting this complex to the RBP-Jκ binding sites within its promoter, which ultimately leads to the maintenance of KSHV latency (36
). Thus, RBP-Jκ binding sites in viral promoters provide positive or negative effects on their gene expression, which is likely dependent on the interaction with target transcriptional factors. Nevertheless, these results indicate that hNIC-mediated activation of KSHV gene expression is specific and RTA independent.
Our previous report (7
) demonstrated that KSHV RTA induces both CD21 and CD23a surface expression on BJAB cells, whereas cellular NIC induces only CD21 surface expression on BJAB cells. However, NIC expression was not capable of inducing CD21 surface expression on KSHV-infected BCBL1 cells. This is likely because the origin of primary effusion lymphoma (PEL) cells is different from that of BJAB cells. BAJB cells that are derived from mature B cells show the surface expression of CD19, CD20, and CD21 mature B-cell markers (56
). In contrast, PEL cells that carry plasma cell types do not express any of mature B-cell markers, including CD19, CD20, and CD21 (6
). Since NIC expression was not capable of inducing CD21 surface expression on PEL cells, this indicates that cellular transcription factors other than Notch-mediated RBP-Jκ activity are necessary for CD21 gene expression.
Despite the broad effect of hNIC on KSHV gene expression, hNIC was not capable of inducing the complete cycle of viral lytic replication, suggesting that a number of other cellular partners are required for RTA to evoke the full repertoire of lytic viral gene expression and thereby lytic replication. In fact, Liang and Ganem (39
) have also discussed as unpublished results that ectopic expression of the Notch intracellular domain or EBNA2 fails to induce KSHV lytic replication. The previous and current studies suggest that activation through the RBP-Jκ-mediated signal transduction is necessary for lytic induction but not sufficient for the completion of lytic replication. Interestingly, K5 and Orf11 genes showed the faster kinetics of expression upon hNIC expression compared to the rest of other viral genes, suggesting that they may be the initial targets for Notch signal transduction. Precise inspection reveals the potential six RBP-Jκ binding sites in the K5 promoter region. A series of mutational analysis indicated the complexity of transcriptional regulation of the K5 promoter. First, the R5 RBP-Jκ binding site within the K5 promoter likely played the major role in the hNIC-mediated activation. CHIP assay of K5 promoter and reporter assay with RBJ-Jκ dominant-negative mutant further supported this notion. However, the deletion mutations of the first four (R1, R2, R3, and R4) RBP-Jκ sites enhanced K5 promoter activity in both BJAB B cells and 293T epithelial cells, indicating that they might influence K5 promoter activity in a repressive way. These results were also supported by the study of RBP-Jκ-null murine fibroblasts. RBP-Jκ is a repressor in the ground state without upstream signal transduction, whereas upon stimulation its interaction with the Notch intracellular domain then relieves this repression and turns on target genes. This suggests that RBP-Jκ may initially target the first four R1, R2, R3, and R4 sites to repress K5 promoter activity and to keep the ground status in the absence of upstream Notch signaling. Upon stimulation, Notch intracellular domain is then recruited to the R5 site through RBP-Jκ interaction to evoke K5 promoter activation. This indicates that RBP-Jκ is an important transcription factor that regulates K5 gene expression in both positive and negative ways.
Tomescu et al. (62
) have described the surface downregulation of MHC-I, CD31, and CD54 immunoregulatory proteins by KSHV in newly infected endothelial cells. Analysis of viral mRNA expression both in vivo within Kaposi's sarcoma lesions and in vitro in infected PEL and endothelial cells indicates that KSHV gene expression is restricted to a small subset of latent genes (3
). While KSHV lytic protein K3 and K5 are capable of downregulating MHC-I and/or CD54, their contribution to immune evasion by KSHV during latency is less clear. We showed that Notch signal transduction induced K5 expression independent of RTA. This result may relate to the previous finding that K5 protein is detected in KSHV latently infected KS lesion (48
). It is possible that K5 may be expressed at a low level in latently infected cells, which is sufficient to induce surface molecule downregulation. In addition, under this condition, K5 expression is likely independent of the lytic transcription factor RTA but dependent on the Notch signal-mediated RBP-Jκ transcription factor. In fact, we found that KSHV-infected BCBL1 cells likely displayed the constitutive activation of ligand-mediated Notch signal transduction, evidenced by the expression of Jagged and the complete proteolytic process of Notch receptors (unpublished results). Furthermore, a recent report has shown that KSHV-infected cells have elevated not only the level of activated Notch and but also the activated level of Notch-mediated transcription activity (13
). Furthermore, inhibitors that block Notch activation resulted in apoptosis in primary and immortalized KS cells. The results suggest that targeting Notch signaling may be of therapeutic value in KS patients.
RBP-Jκ is a transcription factor whose function switches from a repressor in the ground state to an activator upon interaction with the Notch intracellular domain, which relieves its repression activity and then gains its activation activity to turn on target genes. Because of its important roles in a variety of cells, RBP-Jκ has been shown to be a common target for viruses, particularly gammaherpesviruses, which scrounge the Notch signaling pathway. Examples are KSHV RTA and LANA (36
), murine gammaherpesvirus 68 RTA (51
), and EBV EBNA2 and EBNA3 (21
). In all cases, these viral transcription factors interact with RBP-Jκ and this interaction activates target gene expression. Furthermore, the Notch pathway has also been reported to play a role in the pathogenesis of adenovirus (19
), simian virus 40 (1
), and human papillomavirus (28
). Here, we demonstrate that cellular Notch signal transduction not only is partially exchangeable with RTA in regard to activation of viral lytic gene expression but also provides a novel expression profile of KSHV growth and immune deregulatory genes. With all these activities of Notch signal transduction in mind, further study of Notch signal transduction should be informative for elucidation of the mechanisms of immune evasion and pathogenesis associated with KSHV latency.