The ORF57 protein is an essential KSHV factor with multiple roles in viral gene expression (Boyne and Whitehouse, 2006
; Conrad, 2009
; Majerciak and Zheng, 2009
; Swaminathan, 2005
). While it likely plays a role in transcription, most of its proposed activities involve posttranscriptional regulation of RNA metabolism and its RNA-binding properties are important for these functions. However, the mechanisms ORF57 uses to recognize its ligand RNAs are unknown. Here, we delineate a core ORF57-responsive element in PAN RNA, a known ORF57 target in both cultured and infected cells (Han and Swaminathan, 2006
; Kirshner et al., 2000
; Majerciak et al., 2007
; Nekorchuk et al., 2007
; Sahin et al., 2010
). Conservatively, we define the core ORE to be a 30-nt sequence, SL2-T, because it fits all three criteria tested in the present work. That is, SL2-T 1) binds to ORF57 in vitro
, 2) enhances intronless β-globin RNA levels in the presence of ORF57, and 3) is necessary for ORF57-responsiveness of PAN RNA. Most likely, the core activity is contained in an even smaller region. Specifically, the nucleotides AUGGAUUUU in the unstructured loop at the top of the core ORE are essential for binding and for full ORF57-responsiveness in PAN RNA. Thus, these nine nucleotides may constitute an ORF57 RNA binding motif.
ORF57 post-transcriptionally enhances the expression of many viral genes as well as various reporter constructs, so it may seem that its binding to RNA is relatively nonspecific. Indeed, in our β-globin assays ( and ) there is a consistent background of ~4–5-fold enhancement by ORF57 in the absence of any ORE. This relatively non-specific effect may be important for the biology of ORF57. In mammalian cells, the stages of nuclear pre-mRNA processing are tightly coupled, and abrogation of one activity often affects other processing steps (Le Hir et al., 2003
; Pandit et al., 2008
; Reed, 2003
; Vinciguerra and Stutz, 2004
). Pre-mRNA splicing is central to this coupling, but most KSHV transcripts lack introns (Zheng, 2003
). As a result, KSHV genes are missing an important component of efficient expression. One ascribed function of ORF57 is to enhance the expression of intron-lacking viral mRNAs. As such, it seems likely that ORF57 would evolve a relatively promiscuous binding specificity to enable it to bind the large pool of intronless viral mRNAs expressed during lytic phase. Peculiarities of individual viral transcripts may require more robust binding by ORF57 to yield a higher activity. For example, the nuclear accumulation of PAN RNA appears to require a stable complex with ORF57 to protect it from nuclear decay enzymes. Perhaps in these cases, transcripts evolved cis
-acting OREs like the one described here.
While this work was in preparation, Zheng and colleagues published a different ORF57-responsive element derived from the viral interleukin-6 (vIL-6) mRNA (Kang et al., 2011
). The vIL-6 MRE (M
lement) overlapped with a miRNA binding site and the authors proposed that binding of ORF57 antagonized the down-regulatory effects of the miRNA. While this mechanism is unlikely to be responsible for ORF57-dependent stabilization of the nuclear PAN RNA, binding of ORF57 to the vIL-6 MRE or to the PAN ORE is essential for its regulatory function. Comparison of the core PAN ORE with the vIL-6 MRE reveals little overall sequence homology. However, there are two interesting similarities. Consistent with the idea that high-affinity ORF57 binding is driven by sequence rather than structure, both elements are predicted to fold into stem-loops, but neither shows particularly strong secondary structures. Even more compelling, the uppermost loops of both the vIL-6 MRE and the PAN ORE share the tetranucleotide GGAU. This element is necessary for ORE activity because mutation of this motif (UG→AA40–41
) abrogates binding in vitro
and ORF57-responsiveness in cells. Thus, these data suggest that the GGAU motif may provide sequence specificity for ORF57 binding that is utilized in multiple transcripts. Interestingly, the herpesvirus saimiri ORF57 protein has been reported to preferentially bind a purine-rich GGAGRG element (Colgan et al., 2009
), so there may be at least loose conservation of binding-site specificity with respect to the GGA trinucleotide. Identification and detailed examination of additional ORF57-responsive elements is necessary to test whether this sequence is a general feature of ORF57 regulated RNAs.
In the same study that identified the vIL-6 MRE (Kang et al., 2011
), a nonbiased approach was taken to clone viral RNA fragments that interact with ORF57. Consistent with our results, they found that PAN RNA interacted with ORF57; 16 out of 91 PAN RNA clones (18%) overlapped the SL2-T core ORE sequence. Surprisingly, other clones spanned nearly every region of PAN RNA with close to 50% overlapping the ENE, a cis
-acting stability element in PAN RNA (Conrad et al., 2006
; Conrad et al., 2007
; Conrad and Steitz, 2005
). No validation of the cloned PAN RNA fragments for their ability to confer ORF57-responsiveness was performed in that study, but our data clearly show that ORF57 binding and response is conferred by sequences residing at the 5′ end of PAN RNA. In fact, deletion of the ENE actually increases ORF57-responsiveness, presumably due to the fact that ORF57’s stabilization effects are greater on an inherently less stable transcript (Sahin et al., 2010
). Moreover, our in vivo
and in vitro
UV cross-linking results do not support a direct interaction between ORF57 and the 3′ end of PAN RNA ( and ).
Our data are consistent, however, with the general model that the core ORE is not the only binding site in PAN RNA for ORF57. Sequences contained in nt 79–315 bind PAN RNA in vitro
and deletion or mutation of the core ORE is not sufficient to completely abrogate ORF57-responsiveness ( and ). In contrast, when the full-length ORE is deleted, virtually no ORF57 response is observed. We speculate that the core ORE efficiently recruits ORF57, which has been shown to be in a homomultimer complex of unknown number (Nekorchuk et al., 2007
). Once one ORF57 molecule is bound to the ORE, the RNA binding domain(s) on the unbound ORF57 molecule(s) are present in a high local concentration driving binding to weaker sites in adjacent sequences. Similar cooperative models have been proposed for other RNA-binding proteins like hnRNP A1 and the HIV REV protein (Daugherty et al., 2008
; Zhu et al., 2001
). The presence of high ORF57 levels and/or multiple weak ORF57-binding sites can drive this interaction in the absence of a high-affinity ORE leading to the observed ORF57 promiscuity.
The steady-state analyses of PAN RNA and β-globin used in this study monitored the accumulation of transcript as a measurement of ORF57 activity. These approaches are confounded by the presence of an uncharacterized enhancer of PAN RNA expression that overlaps the full-length ORE (Conrad and Steitz, 2005
; Sahin et al., 2010
). When driven by the PAN RNA promoter, deletion of the full-length ORE decreases PAN RNA levels by ~5-fold in the absence of ORF57. Use of the CMV promoter decreases the magnitude of the effect of this enhancer, but some up-regulation of RNA levels by this element is still observed. Close examination of the no ORF57 controls in this study suggest a subtle trend of increased RNA accumulation in the presence of the core ORE in the absence of ORF57 (–). To be sure, careful quantification of these data rarely yields a statistically significant effect (data not shown), so it is difficult to interpret the relevance of the trend. One formal possibility is that the core ORE does not drive ORF57-response directly, but rather it is an element that has a function in gene expression that lies upstream of ORF57 activity. However, this idea seems unlikely based on our in vitro
binding and β-globin reporter assays. We favor the idea that the element may bind to cellular factors that act in concert with ORF57 to promote gene expression. In the absence of ORF57, the activity of the cellular factor is minimized, but in its presence the effect is enhanced. Consistent with this idea, in vitro
binding of baculovirus-expressed ORF57 is enhanced by the presence of cellular extract (Majerciak et al., 2006
). Further experimentation is required to unravel the complex molecular mechanisms of these primary and secondary ORF57-responsive elements in PAN RNA.
ORF57 is a multifunctional regulator of gene expression that is essential for KSHV replication. In order to take a meaningful global approach to examine ORF57 targets, we must first employ reductionist techniques to define the requirements for ORF57-RNA interactions. Here we have identified a core element from a natural ORF57 target that is sufficient for ORF57 binding and response. These data provide a foundation to compare ORF57-responsive elements from novel viral and cellular targets as they are uncovered.