Identification of genome-wide viral RNA targets of KSHV ORF57 by CLIP assays.
To search for genome-wide viral RNA targets of ORF57 in KSHV-infected cells, we established a very reliable and efficient CLIP assay modified from our previous publication where we reported that ORF57 protein associates with a viral ORF59 RNA and can be cross-linked to the associated ORF59 RNA in cells by UV irradiation (25
). In this study, the RNA-protein complexes pulled down with anti-ORF57 antibody in the modified CLIP assay were briefly digested with RNase T1
to remove the protein-free parts of the RNAs within the complexes and then thoroughly digested with proteinase K to remove ORF57 protein from the remaining RNA fragments. The resultant ORF57-bound RNA fragments protected from RNase digestion were extracted and ligated to a 32
P-labeled 3′ adaptor. Following gel purification, the ligated RNA fragments were reverse transcribed, amplified, and cloned for sequence analysis (Fig. ). After screening nearly 9,500 bacterial clones, we found a cDNA insert in 5,791 clones, and 199 of them came from 30 viral transcripts (Table ; see also Table S1 in the supplemental material). We chose viral transcripts with the same sequence cloned twice or more as putative viral targets of ORF57. With this cutoff, we were able to identify 11 viral transcripts as putative ORF57 targets. Among them, ORF56, ORF59, and PAN RNA were authentic ORF57 targets previously reported in other studies (18
FIG. 1. Isolation of RNAs in ORF57-RNA complexes obtained from butyrate-activated JSC-1 cells. (A) Key steps of the ORF57 CLIP assay. Chemically induced JSC-1 cells were UV irradiated. The protein-RNA complexes cross-linked in the cell lysates were pulled down (more ...)
Genome-wide viral RNA targets of ORF57 identified from CLIP assaysa
Identification of viral IL-6 RNA as a major target of ORF57.
Interestingly, we found that one of the major ORF57 targets was viral IL-6 (vIL-6 or K2) (Table ), the viral homolog of hIL-6. This result is intriguing because vIL-6 is a critical factor for maintaining tumor cell proliferation during KSHV infection (2
). The interaction between vIL-6 mRNA and ORF57 was verified using CLIP/RT-PCR (Fig. ), where vIL-6 RNA was specifically pulled down by using an anti-ORF57 antibody under UV-cross-linking conditions. In contrast, an abundant cellular RNA, coding for GAPDH, was not associated with ORF57. Similarly to results reported previously (36
), vIL-6 expression at both RNA and protein levels was increased in lymphoma-derived B cell lines JSC-1 and BCBL-1 (Fig. ) with lytic KSHV induction. Bac36 wt cells with stable transfection of a wt KSHV genome also responded to virus induction for vIL-6 expression, whereas Bac36 Δ57 cells stably transfected with an ORF57-null KSHV genome (23
) did not (Fig. ). These results were confirmed further by vIL-6 immunofluorescence assays (Fig. ), showing a remarkable number of cells being activated for vIL-6 expression in Bac36 wt cells but only a few in Bac36 Δ57 cells (Fig. , bar graph). We found that cells expressing vIL-6 in the cytoplasm always coexpressed ORF57 in the nucleus (Fig. ). These data indicate that vIL-6 expression in viral lytic infection is associated with ORF57.
FIG. 2. vIL-6 expression depends on viral ORF57. (A) ORF57 is associated with vIL-6 RNA during lytic KSHV infection. RT-PCR was performed by using a vIL-6- or GAPDH-specific primer pair on RNA isolated from input lysates or from the CLIP complexes obtained with (more ...)
Next, we demonstrated that ORF57 promotes vIL-6 expression. The ORF57-mediated enhancement of vIL-6 expression is independent of other viral factors as indicated by cotransfection of several human epithelial cell lines. From our CLIP assays, we found that ORF57 interacts with the coding region of vIL-6; therefore, when designing expression vectors for vIL-6, we included only the coding region of vIL-6 mRNA. When cotransfecting these expression vectors with ORF57, we found that ORF57 by itself was capable of efficiently promoting vIL-6 production in HEK293 (Fig. ) and HeLa and HCT116 (Fig. ) cells but not the production of the GFP control (Fig. ). We also found, in HEK293 cells, that the increase in vIL-6 production was dependent on the amount of ORF57 present (Fig. ). The direct effects of ORF57 protein on vIL-6 expression in vivo were also examined by transfection of recombinant ORF57 protein into BCBL-1 and JSC-1 cells. Similarly, direct transfection of ORF57 protein into these cells stabilized vIL-6 RNA and increased vIL-6 protein expression (see Fig. S1A and B in the supplemental material), whereas the control (transfection of β-galactosidase protein) had no effect on vIL-6 expression. Although the transfected ORF57 protein appeared toxic to the cells as indicated by tubulin level (Fig. S1A), we observed a 2-fold increase in both vIL-6 RNA and protein levels due to the presence of ORF57 protein compared to the β-galactosidase controls. Together, these data indicate that the enhanced expression of vIL-6 depends on viral ORF57.
vIL-6 RNA has an ORF57-responsive element that contains two ORF57 binding sites, one of them overlapping an miR-1293 seed match.
We then set out to determine the vIL-6 sequence that ORF57 specifically interacts with. Our ORF57 CLIP assay allowed us to identify an RNA sequence protected by bound ORF57 protein from partial RNase digestion, and therefore, we could determine the specific vIL-6 region that interacts with ORF57 (Fig. ). We named this sequence of the vIL-6 RNA an MTA-responsive element (MRE). Subsequent RNA folding analysis indicated that the vIL-6 MRE is composed of two separate stem-loop structures, MRE-A and MRE-B (Fig. ). Therefore, we designed a series of RNA oligomers (oNP41 to oNP44) covering the majority of the MRE and examined each RNA oligomer for its binding affinity to ORF57 protein. When ORF57 was expressed in either HEK293 or JSC-1 cells, it was observed to interact with oNP42, oNP43, and oNP44 but not with oNP41 (Fig. ). As oNP43 covers a partial sequence of oNP44 and showed only a weak affinity for ORF57, we designated the vIL-6 RNA sequences covered by oNP42 and oNP44 active MRE-A (indicated in blue) and MRE-B (indicated in red) motifs, respectively. Interestingly, the MRE-B motif was also identified as an miR-1293 binding site that regulates vIL-6 expression and shows no similarity to any other cellular miRNA seed matches (J.-G. Kang and Z.-M. Zheng, unpublished observation). Together, these data suggest that vIL-6 RNA has at least two distinct sites for ORF57 binding and that the MRE-B motif is a dual binding site for both miR-1293 and ORF57.
FIG. 3. Sequence motif of vIL-6 RNA in association with ORF57 protein. (A) Sequences of 18 vIL-6 cDNA clones from CLIP assays. Numbers above the sequences are the nucleotide positions in the KSHV genome (39). Numbers in parentheses indicate the same sequences (more ...) The vIL-6 MRE is crucial for RNA stability and translation.
To further analyze the function of the newly discovered vIL-6 MRE motifs, various deletion mutants of vIL-6 (Fig. ) were compared with a wt vIL-6-expressing vector in response to the presence of ORF57 in HEK293 cells. As shown in Fig. , deletion of the entire MRE (ΔB) exhibited vIL-6 RNA instability and reduced vIL-6 expression in the presence of ORF57. This instability of vIL-6 RNA in the ΔB mutant and unresponsiveness to ORF57 were gradually reversed when the deletion was narrowed down to only MRE-B (Fig. ). The data indicate that the MRE functionally mediates ORF57-enhanced vIL-6 expression, with MRE-A functioning specifically to stabilize vIL-6 RNA. Unexpectedly, we noticed that deletions 5′ to 3′ of the MRE increased vIL-6 protein expression, regardless of whether ORF57 was present in cells. This was most noticeable in regard to the ΔD mutant, which carries a deletion of the MRE-B sequence. Even in the absence of ORF57, the ΔD mutant exhibited a remarkable amount of vIL-6 protein that was almost comparable to the level observed in the presence of ORF57 (Fig. ). This result indicates that MRE-B is mainly involved in translational repression of vIL-6, in contrast to the role of MRE-A in vIL-6 RNA instability.
FIG. 4. The MRE in vIL-6 RNA is responsible for vIL-6 RNA stability and translation regulation. (A) Diagrams of wt vIL-6 and its mutants with the nucleotide positions in the KSHV genome (39) of the deletions indicated. MRE-A (striped box) and MRE-B (black box) (more ...) An miR-1293 seed match in the vIL-6 MRE-B sequence is required for efficient vIL-6 expression in response to ORF57.
To determine the importance of the vIL-6 MRE-B interaction with ORF57 in modulating vIL-6 translation, we transiently cotransfected HEK293 cells with ORF57 and either vIL-6 or miR-re vIL-6 expression vectors. The miR-re vIL-6 expression vector used in this assay contains a disrupted miR-1293 seed match (Fig. ). As shown in Fig. , disruption of the miR-1293 seed match in vIL-6 RNA remarkably increased, in the absence of ORF57, the miR-re vIL-6 protein expression compared to that of wt vIL-6 when the same levels of mRNAs were assayed. Despite the fact that ORF57 was capable of promoting the expression of both wt vIL-6 and miR-re vIL-6 RNAs, it appeared more potent at increasing the production of wt vIL-6 protein. Our results indicate the importance of miR-1293 in modulating the translational repression of vIL-6. Given that ORF57 stimulates IL-6 expression and interacts directly with the vIL-6 MRE-B containing an miR-1293 binding site, we find that ORF57 functions to disrupt the miR-1293-mediated repression of vIL-6 translation.
FIG. 5. An miR-1293 seed match in vIL-6 MRE-B is responsible for repression of vIL-6 expression and association with Ago2 and for ORF57 to function. (A) The sequences of the miR-1293 seed match in MRE-B of wt vIL-6 mRNA and its point mutations in the miR-re vIL-6. (more ...)
Additional experiments further supported our findings: first, we found that ORF57 enhanced vIL-6 expression in wt RKO cells but not in RKOdicer−
cells (see Fig. S2A in the supplemental material); second, ORF57 disrupts miR-1293-mediated repression of vIL-6 translation in the cytoplasm (Fig. S2B to D). By using a cytoplasmic version of the N-terminal half (aa 1 to 251) of ORF57, containing point mutations in all three of its nuclear localization signals (25
), we observed that this mutant ORF57 stabilized vIL-6 RNA less efficiently than did the N-terminal half of wt ORF57. However, the mutant ORF57 functioned similarly to wt N-terminal ORF57 in terms of vIL-6 protein expression in relief of miR-1293-mediated repression of vIL-6 translation when the protein/RNA ratio was taken into account (Fig. S2B and C). In addition, the mutant ORF57 also increased vIL-6 translation in a dose-dependent manner (Fig. S2D).
ORF57 prevents Ago2/miR-1293 binding to vIL-6 RNA in vivo and in vitro.
To understand the molecular mechanism by which ORF57 disrupts miR-1293-mediated translational repression of vIL-6, we performed a CLIP assay in cotransfected HEK293 cells expressing HA-Ago2. In the assay an anti-HA antibody was used to measure the vIL-6 RNA in miR-1293-mediated association with Ago2, a major component of RISC. The assay was performed either in the presence or in the absence of ORF57. miR-re vIL-6, which lacks an miR-1293 binding site, served as a negative control. As expected, cotransfection with ORF57 promoted the accumulation of both vIL-6 and miR-re IL-6 RNA, but cotransfection with a luciferase vector did not (Fig. ). Strikingly, we found that vIL-6 mRNA was associated with Ago2 efficiently in the presence of luciferase but much less efficiently in the presence of ORF57 (Fig. ; also compare with the corresponding level of total vIL-6 RNA in Fig. ). In contrast, miR-re vIL-6 lacking an miR-1293 binding site has no association with Ago2 either in the presence of luciferase or in the presence of ORF57. As miRNAs are tightly associated with RISC in vivo
), our data indicate that Ago2 association with vIL-6 is mediated specifically by cellular miR-1293 in HEK293 cells and that this association is disrupted in the presence of ORF57.
To verify biochemically that the association of Ago2 with vIL-6 RNA is miRNA specific and that ORF57 functionally modulates this association, the influences of the MRE-A (Fig. ) and MRE-B (Fig. ) RNA sequences of vIL-6 on Ago2 binding were compared in RNA-protein pulldown assays by using HEK293 cell lysates with ectopically expressed miR-NC or miR-1293. ORF57 protein was added to the cell lysates before the RNA pulldown. The vIL-6 MRE-A motif, which does not contain an miRNA binding site, exhibited a minimal background level of binding to Ago2, independently of a specific miRNA and ORF57 (Fig. ). In contrast, the MRE-B motif, which has an miR-1293 binding site, exhibited strong binding of Ago2 only in the presence of miR-1293. Not surprisingly, ORF57, which interacts with the MRE-B RNA, could block this binding in a dose-dependent manner (Fig. ).
We further examined the specific interaction of miR-1293 with vIL-6 seed match region by a RISC assembly and RNase protection assay using in vitro-transcribed vIL-6 RNAs. The protein-RNA complexes mediated by miR-1293 in HEK293 cell lysates containing HA-Ago2 were UV cross-linked, immunoprecipitated with an anti-HA antibody, digested by RNase T1, and finally incubated with proteinase K. We found, by gel electrophoresis, that the vIL-6 RNA with the MRE-B region was partially protected by association with HA-Ago2 only in the presence of miR-1293 (Fig. , compare RNA ΔD to RNA ΔE), indicating that miR-1293 mediated specific association of Ago2 with MRE-B. However, this specific association could be inhibited by ORF57 (Fig. ). Collectively, these data provide evidence that ORF57 prevents association of miR-1293-specific RISC with vIL-6 RNA and, therefore, disrupts miRNA-mediated translational repression of the target and leads to increased expression of vIL-6.
FIG. 6. miR-1293-mediated specific association of Ago2 with MRE-B in the context of vIL-6 mRNA is preventable in the presence of ORF57. (A) The 3′ halves of vIL-6 RNAs with deletion of MRE-B (ΔD) or MRE-A (ΔE). (B) miR-1293-mediated Ago2 (more ...) Ago2/miR-1293 competes with ORF57 for the same binding site in MRE-B of vIL-6 RNA.
We next used a reciprocal approach to confirm how ORF57 disrupts the miR-1293-mediated translational repression of vIL-6 by competition with miR-1293/Ago2 for the same binding site. We observed that the endogenous miR-1293-mediated Ago2 association with MRE-B of vIL-6 RNA prevents ORF57 from binding to the MRE-B region. This was accomplished by using an anti-Ago2 antibody to deplete Ago2 from HEK293 cell lysates, and then the binding of ORF57 to the MRE-B RNA was compared to that with non-Ago2-depleted lysates in RNA pulldown assays. By Western blotting (Fig. ), we observed that Ago2 binding to the MRE-B RNA was reduced by 50% in the presence of ORF57. Conversely, depletion of Ago2 (RISC) from the lysates increased ORF57 binding to the MRE-B RNA by 70%, indicating a competition between ORF57 and Ago2 (RISC) for MRE-B RNA binding. The binding of miR-1293/Ago2 to the MRE-B RNA of vIL-6 was also examined in RNA-protein pulldown assays using HEK293 cell lysates with ectopically expressed miR-1293 in the presence of BSA or ORF57. The MRE-B RNA was found to be strongly bound by miR-1293-associated endogenous Ago2, and this binding, as expected (Fig. ), was reduced by 30% in the presence of ORF57 (Fig. ). Consistent with this result, miR-1293 binding to the MRE-B RNA was also decreased by 30% in the presence of ORF57 compared to the control BSA (Fig. ). Altogether, these data provide further evidence that ORF57 competes with the miR-1293-specific RISC for binding to vIL-6 RNA.
FIG. 7. ORF57 competes with Ago2 and miR-1293 for binding to vIL-6 MRE-B RNA. (A) Depletion of endogenous Ago2 from cell lysates increases ORF57 binding to the miR-1293 binding site. Cell lysates of HEK293 cells were prepared in 1× RIPA buffer containing (more ...) ORF57 relieves translational repression of vIL-6 by miR-1293 in an in vitro translation assay.
To better separate ORF57-mediated translational enhancement from ORF57-mediated RNA accumulation, we examined whether ORF57 directly interferes with the miR-1293-mediated repression of vIL-6 translation, using an in vitro translation assay in the presence of purified human HA-Ago2. A firefly luciferase RNA that has no miR-1293 binding site served as an internal control in the study. We found that the translation of vIL-6 RNA in vitro, but not that of firefly luciferase RNA, could be inhibited by 32% in the presence of miR-1293 (Fig. ). When supplemented in the assays, ORF57 was found to relieve, in a dose-dependent manner, the miR-1293-mediated translational repression of a fixed amount of in vitro-transcribed vIL-6 mRNA (Fig. ). Together, these data confirm that ORF57 does exercise a major function in promoting vIL-6 translation by disrupting miR-1293-mediated translational repression, which is separate from its function in promoting vIL-6 RNA accumulation.
FIG. 8. ORF57 prevents the miRNA-mediated translational repression of vIL-6 in vitro. (A) A representative of multiple gels in in vitro translation assays. The mRNA and miRNA duplex were denatured at 70°C for 3 min and quenched on ice. hAgo2 (25 nM) with (more ...) ORF57 enhances hIL-6 expression via a mechanism similar to that for vIL-6 expression.
Lytic KSHV infection also induces hIL-6 expression in B cells (1
) and in TREx BCBL-1 RTA cells (Fig. ). The interaction between hIL-6 RNA and ORF57 in JSC-1 cells with lytic KSHV induction was detected by CLIP/RT-PCR (data not shown). Consistent with this, ORF57 itself promoted hIL-6 expression in cotransfection of HEK293 cells (Fig. ). ORF57 protein transfection into JSC-1 cells could also enhance hIL-6 expression (Fig. ). These data suggest that, during KSHV infection, enhanced hIL-6 expression is dependent on viral ORF57.
FIG. 9. ORF57 regulates hIL-6 expression by a mechanism similar to that for vIL-6. (A and B) KSHV lytic infection increases the expression of both vIL-6 and hIL-6. TREx BCBL-1 RTA cells carrying an episomal KSHV genome and a tetracycline-inducible RTA (ORF50) (more ...)
The hIL-6 ORF contains an miR-608 seed match in the corresponding region of vIL-6 MRE-B, and its expression could be greatly increased in RKOdicer− cells (Kang and Zheng, unpublished); therefore, it seems plausible that hIL-6, like vIL-6, could be an additional target of ORF57. To determine whether this seemed reasonable, a biotinylated RNA oligomer (oJGK50) was synthesized based on the hIL-6 MRE region containing an miR-608 seed match and was used for RNA pulldown assays. The cell lysates for the pulldown assays, either with or without ORF57, were prepared from HEK293 cells transfected with miR-NC (a negative control) or miR-608, and the proteins in the pulldowns were examined by Western blotting. As shown in Fig. , ORF57 binds to the MRE RNA (oJGK50) of hIL-6 in a dose-dependent manner, regardless of whether an HEK293 cell lysate with ectopically expressed miR-NC or miR-608 was used in the RNA pulldown assays. However, a high-affinity binding of Ago2 to the MRE RNA of hIL-6 could be conferred only when HEK293 cell lysates with ectopically expressed miR-608 were used in the assays. The binding could be gradually diminished by increased amounts of ORF57. In contrast, when HEK293 cell lysates containing miR-NC were used, we observed a very-low-affinity interaction (background) of Ago2 with the hIL-6 MRE. Overall, these results indicate that ORF57 promotes hIL-6 expression by modulating the interaction of miR-608-specific RISC with the hIL-6 MRE.