The KSHV RTA protein is responsible for the switch from latency to lytic replication, and this RTA activity requires its interaction with the RBP-Jκ transcription factor (18
). Here, we demonstrate that RTA strongly induced CD21 and CD23a gene expression through the RBP-Jκ binding sites at the first intron region of CD21 and CD23a core promoter region, respectively. Furthermore, RTA-induced expression of CD21 surface glycoprotein effectively facilitated EBV infection, and RTA-induced expression of sCD23 glycoprotein activated primary human B and T lymphocytes. These results demonstrate that KSHV RTA regulates RBP-Jκ-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.
EBV EBNA2 contributes to B-cell immortalization, most likely by its ability to act as a transcriptional modulator of cellular and viral gene expression. It activates the transcription of B-cell activation markers CD21, CD23, and c-myc
) and tyrosine kinase Fgr (43
) and downregulates the expression of Igμ (37
). EBNA2 does not bind to DNA directly but is recruited to EBNA2-responsive elements by interaction with RBP-Jκ transcription factor, indicating that EBNA2 is a functional homolog of the activated Notch protein (47
). Indeed, cellular NIC has been shown to functionally substitute for EBNA2 in the context of EBV for primary B-cell transformation (7
). However, the cellular targets of cellular NIC do not completely overlap with those of EBNA2: NIC and EBNA2 both activate CD21 gene expression and repress Igμ expression, whereas EBNA2, but not NIC, activates CD23a gene expression (37
). We demonstrate that, like EBNA2, RTA activates CD21 and CD23a transcription. Interestingly, the first intronic sequence of CD21 has been shown to control appropriate B-cell-specific expression and also contain binding sites for RBP-Jκ and other transcription factors (25
). In concordance with these findings, RTA-induced activation of CD21 promoter activity required the first intronic sequence. This was further confirmed by the requirement of the first intronic sequence of CD21 for NIC-mediated activation (unpublished results). Besides the previously characterized RBP-Jκ binding site of the CD23a core promoter region that EBV EBNA2 primarily targets (22
), four additional sequences have also been shown to bind to RBP-Jκ (15
). Our serial deletion mutational analysis showed that, as seen with EBNA2, the first RBP-Jκ binding sequence of the CD23 core promoter region was primarily responsible for RTA-induced activation. Despite this similarity between KSHV RTA and EBV EBNA2 for the activation of CD21 and CD23a gene expression, RTA does not affect Igμ and c-myc
expression, which are strongly regulated by EBNA2. These results collectively indicate that KSHV RTA targets Notch signal transduction in a manner that is similar to but distinct from that of EBV EBNA2.
CD21 (also known as CR2, C3d, and EBV receptor) is a member of the regulators of complement activation gene family. CD21 is a B-cell receptor for CD23 and possibly for gamma interferon and also serves as the receptor for EBV gp350/220 (28
). We demonstrate that the upregulation of CD21 surface expression induced by RTA facilitates EBV infection. Since most PEL cells are coinfected with KSHV and EBV, our results suggest the possibility of hierarchically ordered infection: KSHV infection, followed by EBV infection. This hypothesis is under active investigation.
CD23 (also known as low-affinity IgE receptor and Fc
RII) is expressed as a type II extracellular protein on a variety of cells, such as B cells, monocytes, and macrophages, and is cleaved from the cell surface to generate several distinct fragments. This cleavage is mediated by a metalloprotease. This sCD23 then acts as a B-cell growth factor and is associated with EBV infection (15
). In fact, since the level of sCD23 in serum is higher in patients with autoimmune diseases and B-CLL, it has been used as an indicator of the progression of disease (15
). This suggests the intriguing possibility that KSHV-infected PEL patients may also have high levels of sCD23 in serum. Unfortunately, the limited availability of sera from PEL patients did not allow us to determine the levels of sCD23 in sera from PEL patients. Nevertheless, it will be interesting to pursue whether KSHV infection is associated with a high level of sCD23 in serum, which may provide a marker for KSHV infection and progression.
A recent study has demonstrated that sCD23 and soluble ICAM1, which are induced from macrophages by HIV Nef, contribute to the induction of the expression of CD80 and CD86 on the surfaces of B cells and the HIV permissivity of resting T lymphocytes (39
). We also showed that the expression of CD80 and CD86 costimulating molecules on the surfaces of primary B lymphocytes and the CD38 activation marker on primary T lymphocytes increased after treatment with the supernatants of TRExBJAB-RTA cells, whereas it was not affected after treatment with the supernatants of TRExBJAB-cDNA5 cells. These results suggest that sCD23 produced by RTA expression induces the stimulation of primary B and T cells through the potential paracrine and autocrine pathways, which may ultimately facilitate homotypic or heterotypic interactions of T and B cells. This also suggests that KSHV RTA potentially influences the activation state of infected B cells, thereby enhancing the ability of neighboring T cells to support the replication of HIV type 1. This activity may require the upregulation of CD80 and CD86 B-cell costimulating receptors involved in the alternative pathway of T-lymphocyte stimulation. Thus, although the full significance of RTA-mediated effects on the other coexisting viruses in KS and PEL patients needs to be studied further, our study may provide insight into the comprehensive mechanism by which KSHV RTA not only activates lytic reactivation by targeting KSHV gene expression but also creates a favorable milieu for viral infection and replication by activating cellular gene expression.