The essential role of SM in EBV replication, like that of its homologs in other herpesviruses, is multifactorial. The multiple roles played by each of these proteins are uniquely evolved to facilitate the replication of the parent virus. The distinct regulatory niche of each protein is evinced by the fact that although they perform similarly in many functional assays that measure target gene activation or mRNA export, they nevertheless fail to complement each other in the ability to rescue virus replication (3
). In this study, we show that in addition to enhancing expression of the majority of EBV lytic genes, SM is involved in downregulation of IE and early gene expression. The particular effect on BHRF1 appears to be at least partly mediated by downregulation of R at the posttranscriptional level. However, as R autoactivates the R promoter (55
), any posttranscriptional inhibitory effects of SM on R are also likely to result in decreased R transcription. Control of R transcription is complex, and Z is an important and central activator of Rp, which acts preferentially on methylated Rp (2
). Cellular transcriptional activators important in Rp regulation include YY1, Sp1, and EGR-1 (40
). In the experiments reported here, it is unlikely that SM exerts effects on R levels or activity by downregulating Z or Na, since both Z and Na expression actually increased during lytic replication in the presence of SM. These experiments do not address the possibility of SM acting to modulate Z expression during the course of spontaneous lytic replication in vivo as Z was transfected to induce replication.
BHRF1 downregulation as lytic replication progresses may serve several functions. While BHRF1 is not required for reactivation from latency in vitro, it is likely that it prevents apoptosis of infected cells in vivo. There is a significant induction of BHRF1 with the onset of EBV lytic replication, which may maintain cell viability during early lytic replication. It is possible that reducing BHRF1 synthesis during later stages of lytic replication facilitates viral production by allowing cell death to occur. Interestingly, BHRF1 has been shown to be a potent epitope for CD4+
cytotoxic T lymphocytes directed against EBV-infected cells (37
), and downregulation of BHRF1 may thus be important in helping EBV evade destruction by the immune system during the later stages of virus production. It is also possible that BHRF1 transcription may facilitate DNA replication from oriLyt, similar to the enhancement of Kaposi's sarcoma-associated herpesvirus lytic origin usage by transcription in this region (72
). Downregulation of BHRF1 promoter activity in this region could then act to curtail DNA replication during the late phase of lytic replication.
Feedback regulation of IE and early lytic gene expression during progression of the lytic cascade in HSV is well described (30
). ICP4 may participate in autoregulation by inhibiting transcription from its own promoter (39
). ICP8, the single-stranded DNA binding protein, inhibits ICP4 transcription and that of other HSV early genes (17
). The mRNA degradation activity of vhs operates on HSV mRNAs and cellular mRNA, and this activity acts to modulate levels of lytic HSV mRNAs of all classes, as shown by the temporal profiles of lytic RNA expression in vhs mutant-infected cells (48
). Perhaps most relevant to the role of SM in modulating EBV lytic gene expression is the repressive function of HSV ICP27 on IE gene expression during HSV replication (44
). Many of ICP27's repressive effects may be indirect, mediated through activation of other HSV genes, such as ICP8, for example. As in the case of HSV, it is likely that negative regulatory effects of SM include indirect effects. Since mRNA levels of approximately 40% of EBV lytic genes are severely reduced and EBV DNA replication is limited in the absence of SM (25
), there are likely many cell and viral genes affected by SM which could act to inhibit IE and early gene expression.
The interactions between cellular transcriptional activators, Z, R, and Na generate a complex system of lytic gene regulation with multiple points of control. The finding that R levels decrease during lytic replication and that BHRF1 and SM promoters appear to be particularly susceptible to SM-mediated inhibition suggests that promoters that bind R directly may be differentially regulated. Indeed, the particular activity of Rta on the BHRF1 promoter was observed in early experiments characterizing Rta function (8
). It should be noted that unlike HSV lytic replication occurring during primary infection, reactivation of EBV from latency requires exogenous chemical induction or transfection of Z. Thus, Z levels may also be subject to negative autoregulation, which would not be detected in experiments in which exogenous Z is expressed by transfection. RAZ, a protein encoded by an alternatively spliced product of the R-Z bicistronic mRNA, may also exert a dominant-negative effect on Z function (14
). Such feedback control of IE and early genes is likely to be an integral component of an efficient and ordered lytic cascade of gene expression in herpesviruses. The data reported here suggest that feedback control of IE and early gene expression occurs during EBV replication and that SM may play a negative as well as positive role in lytic gene expression.