Reactivation of KSHV from latency requires the cellular protein RBP-Jk to specify transcriptional targets for the KSHV Rta lytic switch protein (45
). The KSHV genome includes 177 predicted binding sites for RBP-Jk. RBP-Jk also specifies transcriptional targets for EBV EBNA-2 and the activated form of the cellular Notch receptor NICD1. Despite the apparent mechanistic similarity of all three RBP-Jk-dependent transactivators, neither EBNA-2 nor NICD productively reactivates KSHV from latency (11
). These observations support the inference that these proteins are not always phenotypically interchangeable, and a noncanonical mechanism for regulating RBP-Jk promoter specification may operate in KSHV-infected cells. Since KSHV and EBV both maintain latency in B cells and coinfect 70% of the established PEL cell lines, we wanted to determine whether the KSHV lytic switch protein, Rta, could function in an EBNA-2-like fashion and cross talk to EBV in coinfected cells.
In the present study, we demonstrate that Rta transactivates the EBV LMP-1 and latency C promoters (Cp) in uninfected cells and in dually infected PEL cells (Fig. ). RBP-Jk was required for Rta to transactivate both EBV latency promoters (Fig. ), and Rta formed a ternary complex with RBP-Jk and both the Cp and the LMP-1 promoter DNAs (Fig. ). We showed that dominant-negative Rta (RtaΔSTAD) potentiated transactivation of Cp and LMP-1 by a constitutively active RBP-Jk allele (Fig. ). We have previously shown that all three mechanisms are used by Rta to transactivate the promoter of the essential delayed-early (DE) KSHV gene ORF57/Mta and correspond with the stimulation of RBP-Jk DNA binding by Rta (11
). Therefore, Rta regulates the EBV latency promoters as if they were KSHV DE promoters. Although RBP-Jk specifies these EBV promoters as Rta targets, it is likely that Rta stimulates RBP-Jk binding to them, a mechanism distinct from that of EBNA-2 (11
Since Rta and EBNA-2 shared similar promoter specificities, we tested their biological significance in EBV-infected cells that are conditionally immortalized by EBNA-2 (EREB2-5 cells). We showed that Rta maintains short-term growth of the cells when EBNA-2 is inactivated (Fig. ) and that this effect required DNA binding of Rta and RBP-Jk (Fig. ). Moreover, our data reveal that Rta's ability to maintain short-term viability of the cells in the absence of EBNA-2 is attributable to a paracrine effect (Fig. ). Therefore, the minor population of Rta-expressing cells supported the growth of the remaining cells that were not expressing Rta. Rta's complementation of the EBNA-2 deficiency also required LMP-1 signaling (Fig. ). Since LMP-1 expression induced paracrine signaling in other studies (19
), we hypothesize that LMP-1 is playing the same role in EREB2-5 cells transiently expressing Rta.
Although our short-term cell growth assays demonstrated an EBNA-2-like function for Rta, our array data suggest that short-term rescue of LCL growth by Rta is associated with a pattern of cellular proteome modulation distinct from that of EBNA-2. Although all of the EBNA-2 targets were shared with Rta, Rta uniquely activated the antiapoptotic proteins survivin, livin, IGF-1 receptor, and the cellular inhibitor of apoptosis protein (cIAP2) (Fig. ). Although we have not determined whether Rta and EBNA-2 differentially regulate the LCL transcriptome, we hypothesize that Rta's broader set of induced targets is attributable to its unique ability to stimulate RBP-Jk DNA binding (11
One question raised by our data is whether the three treatments rescue short-term LCL growth by unique effects on the cell proteome or whether the rescue is attributable to proteomic changes common to the three treatments. The data suggest that paracrine effects of expressing EBNA-2 and Rta are sufficient to rescue short-term LCL growth, without significantly repressing cellular protein expression within the subset of the proteome that we screened. It has long been recognized that LCL growth is dependent on paracrine/autocrine mechanisms (19
). The obvious cellular candidates for rescuing short-term growth are those secreted proteins induced by all three treatments. In particular, Rta and β-estradiol both repressed TNF-α, which suppresses LCL growth (21
) (Fig. ) (unfortunately, EBNA-2's effect on TNF-α is unknown since it was invalidated in our quality control procedure for the assay). If instead, the short-term growth of the LCLs is rescued by inducing unique proteomes, then another important question is whether the effect of Rta is sufficient for long-term outgrowth of LCLs. Partial rescue of EBNA-2 deficiency, and initialization, but not complete LCL transformation, have been previously reported for NICD1 (25
) and truncated LMP-1 (36
), respectively. In this scenario, the unique proteomic changes associated with re-addition of β-estradiol to EREB2-5 cells in our study would provide the most likely candidates for supporting long-term outgrowth of LCLs. Finally, it is currently unclear how induction of proapoptotic proteins in our experiments is consistent with maintaining short-term growth of the EREB2-5 cells (Fig. ).
While the EREB2-5 experiments were a key to investigating the similarities between Rta and EBNA-2, the biologically relevant experiments used PEL cells coinfected by KSHV and EBV. We showed that the dominant-negative Rta allele, RtaΔSTAD, but not a nonfunctional RtaΔSTADΔLR allele, inhibited growth of coinfected PEL cells (Fig. ). Importantly, the growth-inhibitory effect of RtaΔSTAD required cells to be infected by EBV and KSHV, since the dominant-negative had no effect on growth of uninfected BL-41 cells (Fig. ). Similar to EREB2-5 cells, we achieved only ca. 5% transfection efficiency in BC-1 PEL cells. Therefore, the growth-suppressing effect of RtaΔSTAD in BC-1 PELs supports two conclusions: (i) RtaΔSTAD is likely inhibiting a paracrine effect of Rta on cell growth of PELs, and (ii) a large proportion of the BC-1 cell population spontaneously expressed endogenous Rta at some point during the 7-day duration of the experiment. In the latter situation, spontaneous Rta expression must have been asynchronous and unsustained, since spontaneous Rta is typically only detected in ca. 5% of cells when assayed by immunofluorescence (48
). We recognize that immunofluorescence may underestimate the actual percentage of cells expressing Rta; however, regardless of the total number of Rta-expressing cells, the growth rescue of untransfected EREB2-5 cells by media conditioned by a small percentage of Rta-expressing cells (Fig. ) strongly supports a paracrine mechanism for Rta's effect. Further, we hypothesize that the growth-suppressing effect of the dominant-negative RtaΔSTAD in coinfected cells (Fig. ) might be due to a “feed-forward” mechanism in which RtaΔSTAD transfected cells inhibit spontaneous Rta expression in the cell population in a paracrine fashion, also. We note that we have previously shown that RtaΔSTAD completely inhibits spontaneous KSHV reactivation in singly infected cells (47
We propose a model for convergence of KSHV reactivation with EBV latency in coinfected B cells through their common cellular target, the Notch effector RBP-Jk (Fig. ). Rta activates the EBV type III latency promoters as if they were KSHV DE promoters (Fig. ), in a background of type I latency. We propose that the small amount of LMP-1 detected in PEL cell lines and primary tumors (6
) may be dependent on spontaneous Rta expression. The set of cellular proteins induced by EBNA-2 or Rta only partially overlap (Fig. ). Previous publications have revealed negative, reciprocal feedback between KSHV and EBV in dually infected cells (33
). Most notably, LMP-1 expression inhibits KSHV Rta expression and reactivation (Fig. ) (69
). We surmise that LMP-1 provides a negative-feedback loop to promote a cellular environment in which Rta indirectly contributes to PEL growth without productively reactivating KSHV and lysing the infected cell. Although we have not investigated the role of KSHV oncogenes in our studies, we point out that a similar negative-feedback relationship has been demonstrated for Rta and its transcriptional target, the KSHV oncogene ORF74/vG-protein-coupled receptor (vGPCR) (8
FIG. 12. Model of convergence of KSHV reactivation with EBV latency in KSHV+/EBV+ B cells. Rta transactivates EBV latency promoters as if they were KSHV delayed early promoters. Rta activates EBV latency type III promoters in a background of latency (more ...)