One of the current models for the mechanism of action of SM and its homologs in other herpesviruses, such as HSV-1 ICP27, HCMV UL69, and KSHV ORF57 protein/Mta, postulates that these proteins bind to intronless viral mRNAs and, by binding to one or more cellular nuclear export factors, facilitate the export of viral RNAs (for a review, see reference 38
). A central aspect of this model is that SM and its homologs compensate for the lack of export factor recruitment that normally occurs concomitant with splicing of intron-containing genes (22
). However, a basic question that is not addressed by such models is whether RNA-specific binding occurs and whether it is required for the functions of these proteins.
While much evidence exists for the ability of EBV SM, HSV-1 ICP27, HCMV UL69, and KSHV ORF57/Mta proteins to bind RNA, relatively little is known about the target specificity of these interactions, and the mechanism by which these proteins bind to specific viral RNAs or RNA motifs remains poorly characterized (4
). HSV ICP27 has been shown to associate with seven intronless HSV transcripts, but not with two intron-containing HSV transcripts, during lytic HSV infection (37
). ICP27 was also shown to associate with 31 HSV RNAs in a yeast three-hybrid assay (41
). In this study, the only identified characteristic of RNAs binding to ICP27 was the presence of multiple G nucleotide repeats. Aside from the suggestion that poly(G) sequences may increase ICP27 interaction, which is consistent with a previous study comparing ICP27 binding to poly(G) and poly(U) (25
), there was no additional characterization of the relative affinities of various RNAs for ICP27. More recently, systematic evolution of ligands by exponential enrichment has been used to define a potential RNA motif targeted by herpesvirus saimiri ORF57 protein (9
). Repetitive selection with oligonucleotide libraries identified GAAGRG as a potential ORF57 protein binding site. The motif was found in multiple herpesvirus saimiri ORFs, and deletion of the motif from a herpesvirus saimiri RNA led to loss of ORF57 protein binding.
In the case of EBV SM, very little specific binding to RNA targets has been demonstrable. An arginine-rich region of 33 amino acids has been identified as an RNA binding region in SM (17
). This region does not strongly resemble classic RNA binding protein motifs, such as RGG boxes, KH domains, or RRM motifs. However, it contains two RS dipeptides and three other arginine residues. In vitro, no target-specific binding could be demonstrated using a peptide containing this motif (17
). Limited comparisons of the affinity of SM for heterologous reporter gene RNAs suggested that SM bound equally well to luciferase and chloramphenicol acetyltransferase despite having markedly greater effects on chloramphenicol acetyltransferase RNA accumulation (33
). Attempts to demonstrate site-specific RNA binding by in vitro cross-linking of SM to RNA revealed some preferential binding, but an ability to bind all the RNAs tested to some degree was observed (46
). Gene-specific activation of expression despite apparently nonselective RNA binding by SM has been explained as possibly due to inherent differences in target transcripts (17
). For example, SM might bind nonspecifically to all RNAs but only affect the expression of those RNAs that are unstable or incapable of independent nuclear export. Alternatively, SM might act in a manner similar to that of cellular RNA binding proteins that have broad RNA binding properties yet achieve extremely specific effects on target RNAs. SR proteins that bind to short, degenerate sequences and hnRNPs, which have both nonspecific and specific RNA binding properties, are classic examples of such RNA binding proteins that exert gene-specific effects on alternative splicing. Such specific effects are achieved by combinatorial effects, context-dependent binding, and protein-protein interactions (40
It is in this context that we investigated whether SM exhibits any preferential association with EBV RNAs during the course of lytic replication. In the current study, we chose to analyze SM-RNA interactions without UV cross-linking, under relatively gentle isolation conditions, in an attempt to preserve physiologic interactions. Overall, it is clear that there are large differences in the affinities of various RNAs for SM despite the fact that under the conditions used in these experiments, SM associated with the majority of EBV mRNAs.
While the abundance of an RNA species would be expected to affect the percentage found complexed with SM if RNA binding activity were nonspecific, it is clear that the extent to which an RNA is associated with SM is not simply dependent on its abundance. It is likely, however, that the timing of the experiment, which was chosen to maximize the number of lytic RNAs that might be detected, affected the representation of RNAs that were detected in association with SM. These data therefore indicate that while SM does associate with a large number of RNAs in vivo, a hierarchy of affinities for SM exists among the various RNAs. Thus, it is unlikely that SM binds to or affects only RNAs that contain a critical response element, in the manner of human immunodeficiency virus Rev.
One caveat in our experiment is that the qRT-PCR primers were directed against individual ORFs. Hence, we do not know how ORF length, 3′ or 5′ UTR length, or the presence of multiple SM binding regions affect our quantitation. We therefore used ranking-based statistics (44
) to identify ORFs that are encoded by mRNAs of as yet unspecified size that were bound by SM.
The finding that two of the most highly SM-associated RNAs in both P3HR1 and B95-8 cells exhibit region-specific binding to SM in vitro and in vivo suggests that binding of at least some RNAs to SM may be highly sequence or structure specific. Significantly, SM binding to these regions was correlated with the ability to enhance expression, suggesting that the specificity of SM action is mediated at least in part by binding specificity. Preferential binding to the 5′ and 3′ UTRs, albeit of only two transcripts, is similar to the behavior of other RNA regulatory proteins, such as eIF4E, which bind to so-called USER codes in the 5′ and 3′ UTRs of target mRNAs (10
). Such USER codes consist of long, highly structured sequences, as well as relatively short (40-nt) sequences in the case of eIF4E. It is tempting to speculate that SM and its herpesvirus homologs may perform coordinate regulation of transcripts as an RNA regulon, as proposed by Keene and Tenenbaum (20
). In this model, multiple transcripts may be regulated as a posttranscriptional operon, allowing a protein that binds to common elements in the UTRs of target genes to perform coordinated functions, such as nuclear export, on all such target mRNAs. The utility of an RNA regulon in herpesvirus replication is apparent when one considers the multitude of lytic transcripts that must be stabilized, exported, and translated in an efficient manner in a limited time. Whether one or more specific SM response elements are present in EBV transcripts, and possibly cellular transcripts, remains to be experimentally confirmed. There is no obvious homology between the 5′ UTRs of BFRF3 and BDLF3, nor is there an obvious bias in the nucleotide compositions of these regions. Further mapping of SM binding by in vitro cross-linking experiments such as the ones described here may allow the definition of one or more minimal binding sites. However, by analogy to SR proteins, which bind to degenerate consensus sequences and exhibit both general and specific RNA binding, definition of SM target specificity by bioinformatics analysis alone is unlikely to be successful, and further biochemical characterization of SM RNA interactions and definition of the crystal structure of SM and its homologs may be required (16
). The identification of transcripts that are highly associated with SM in vivo should allow further characterization of specific elements involved in coordinate posttranscriptional regulation by SM.