EBV SM protein is known to be essential for production of infectious EBV. However, the role that it plays in regulating EBV gene expression to allow productive replication remains to be fully defined. Establishing the functions of SM in lytic replication is complicated by the fact that although SM is an RNA-binding protein with a posttranscriptional mechanism of action, relatively little is known of its RNA target specificity. Previous work has shown that SM, while acting on a number of reporter genes and EBV lytic cycle genes, does exhibit gene specificity (2
). For example, SM enhances accumulation of chloramphenicol acetyltransferase but not β-galactosidase mRNA (26
). However, no specific RNA sequence motif has been identified that is required for SM binding. Possible explanations for SM specificity include underlying differences in the stability or exportability of target mRNAs, with SM preferentially enhancing the accumulation of unstable or poorly exported intronless mRNAs. Alternatively, unidentified RNA secondary structures may exist which act as determinants of SM binding.
In contrast to previously published findings, we demonstrate that lytic DNA replication and production of linear EBV genomes do not occur in the absence of SM. Two early genes essential for EBV DNA replication, coding for EBV primase and polymerase, were shown to be SM dependent. In this respect, SM is similar to ICP27, as an ICP27 mutant of HSV (Δ27 HSV) also fails to replicate viral DNA (31
). The DNA replication defect in Δ27 HSV has been attributed to its failure to efficiently express six of the genes required for HSV lytic replication (23
). Interestingly, expression of one essential gene, coding for ICP8, does not seem to require ICP27. The situation therefore parallels that in EBV; although the SM-dependent replication complex genes are fewer (polymerase and primase), there seems to be a selective dependence of the essential DNA replication genes on SM. Why only a subset of the genes that act coordinately to carry out DNA replication is regulated by SM is not obvious, but it is clear that SM may act as a key element of the temporal regulation of DNA replication.
By transfection of the primase and polymerase genes, a partial but significant rescue of DNA replication in the SM-KO virus could be accomplished (Fig. ). The level of DNA replication was approximately 20 to 25% of that achieved with transfection of SM. Previous studies have demonstrated replication of EBV oriLyt using cosmids that expressed six EBV genes: BALF5 (DNA polymerase), BMRF1 (polymerase processivity factor), BALF2 (single-stranded DNA-binding protein), BSLF1 (primase), BBLF4 (helicase), and BBLF2/3 (helicase-primase-associated protein) (14
). Although initially it was thought that SM was directly involved in replication, it was subsequently demonstrated that the role of SM was in fact indirect, enhancing expression of the proteins involved in the actual DNA replication (13
). Our data suggest that the role of SM in vivo is similar to its role in replication assays in vitro. However, it is also clear that SM enhances DNA replication by mechanisms in addition to enhancing expression of these six minimally essential genes, since full rescue of DNA replication requires SM. It is likely that other genes induced by SM, such as those coding for thymidine kinase and the small unit of ribonucleotide reductase, which, while not absolutely essential, contribute to efficient replication in vivo.
Nevertheless, the level of DNA replication achieved by expression of primase and polymerase should have been sufficient to allow virus production, albeit at a reduced level, if the block to DNA replication were the primary reason for the lack of late gene expression observed in the absence of SM. However, transfection of polymerase and primase genes was unable to rescue infectious virus production, under conditions where even production of 1% of wild-type levels of virus should have been detectable (as GFP expression within infected Raji cells). Furthermore, noninfectious virus was also not produced, indicating that there are additional genes whose expression is SM dependent and not merely a consequence of the block to DNA replication in the absence of SM. Thus, it is clear that while DNA replication enhances late gene expression, possibly by effects on genome copy number as well as on transcription, SM is required for full productive late gene expression.
Rescue of DNA replication by transfected polymerase and primase allowed us to examine whether late genes were activated solely by DNA replication. These experiments clearly demonstrated that expression of a number of late lytic genes is stimulated by DNA replication alone in the absence of SM. In essence, these can be considered true γ2 genes of EBV in that their expression was enhanced by DNA replication per se. While some genes could be clearly shown to increase with DNA replication (seen in the middle left portion of the cluster map in Fig. ), they were further stimulated by SM expression. Conversely, there was also a group of late genes whose expression was not stimulated by DNA replication but was enhanced by SM (shown in the bottom third of the map in Fig. and listed in Fig. ). Such a stimulation of γ gene expression can also be distinguished from effects on viral DNA replication in the case of HSV ICP27 (19
). A previous study had shown that SM-KO EBV did not synthesize gp350 or VCA, both of which would be essential for infectious virus production (2
). In addition to the genes coding for these proteins, the current study has identified several other structural genes which are SM dependent, demonstrating that SM is directly required for efficient expression of multiple late genes.
It should be noted that there are some inherent limitations to the experiments described here. First, the temporal regulation of Z and SM likely does not represent that occurring during EBV reactivation in vivo (modeled in vitro here), in that both genes were expressed by transfection. In addition, these studies were performed in 293 cells, which may not accurately represent EBV replication in B cells or naturally infected epithelial cells. In addition, while this analysis has permitted the identification of essential roles of SM in DNA replication, virus production, and late gene expression, there are likely to be more subtle aspects of SM regulation of the replicative cycle as demonstrated by the kinetics of BMRF1 expression, where SM led to an earlier accumulation of BMRF1 mRNA.
The negative regulation of BHRF1 by SM was unexpected and intriguing. It is tempting to speculate that an antiapoptotic effect of BHRF1 expression is useful during an initial period of EBV DNA synthesis and protein production but is counterproductive at later times prior to cell lysis. A parallel again exists in HSV, where expression of certain early genes continues to increase unchecked in Δ27 mutants, suggesting that ICP27 also plays a viral gene-suppressive role. Certain early herpesvirus genes may be repressed by genome replication (31
). Whether effects on DNA replication or direct effects on BHRF1 transcription or processing are involved in the regulation of BHRF1 by SM remains to be determined.
The large number of transcripts whose accumulation is enhanced by SM raises the question of how SM specificity might be determined. There are clearly many transcripts that are strongly activated by Z and do not require SM for high-level expression. One possible explanation is that SM binding is nonspecific but only has effects on transcripts that are inherently poorly expressed. According to such a model, individual EBV transcripts vary in their exportability or stability, with SM enhancing the accumulation of poorly expressed transcripts. Conversely, SM would have relatively little effect on the levels of transcripts that are constitutively well expressed. Such differences in the ability of intronless mRNAs to serve as export substrates clearly exist and may be attributable to the presence of constitutive transport elements in some transcripts that are bound by cellular RNA-binding proteins. Alternative explanations that are not mutually exclusive also may apply. Although SM has been shown to exert posttranscriptional effects, transcriptional effects have not been ruled out. Nuclear run-on analyses have only been performed with transfected cell nuclei, where the effect of SM on a limited number of promoters has been examined (35
). Thus, it remains possible that SM, like its homologs in HSV and CMV, may have transcriptional effects on specific EBV promoters. The genes identified here provide a starting point to compare SM-responsive and SM-independent transcripts on the basis of these parameters.
In summary, the data presented above demonstrate that there is a clear demarcation of gene expression in the absence of SM. Lytic DNA replication is essentially curtailed without SM due to its effect on some early genes. While DNA replication does stimulate some late gene expression, SM is also required for late gene expression independent of its effects on DNA replication. Therefore, due to indirect (by permitting DNA replication) and direct effects on late transcripts, few late EBV lytic cycle genes are expressed in the absence of SM. SM also has kinetic effects on gene expression, leading to earlier expression of some genes and repressing the expression of one gene with antiapoptotic function. These multiple effects on the pattern of lytic cycle EBV gene expression explain the complete absence of infectious virus production in the absence of SM.