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. 2011 Feb;85(4):1528-40.
doi: 10.1128/JVI.01709-10. Epub 2010 Nov 24.

Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Vladimir Majerciak  1 , Hiroaki Uranishi, Michael Kruhlak, Guy R Pilkington, Maria Julia Massimelli, Jenifer Bear, George N Pavlakis, Barbara K Felber, Zhi-Ming Zheng
Affiliations

Kaposi's sarcoma-associated herpesvirus ORF57 interacts with cellular RNA export cofactors RBM15 and OTT3 to promote expression of viral ORF59

Vladimir Majerciak et al. J Virol. 2011 Feb.

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes ORF57, which promotes the accumulation of specific KSHV mRNA targets, including ORF59 mRNA. We report that the cellular export NXF1 cofactors RBM15 and OTT3 participate in ORF57-enhanced expression of KSHV ORF59. We also found that ectopic expression of RBM15 or OTT3 augments ORF59 production in the absence of ORF57. While RBM15 promotes the accumulation of ORF59 RNA predominantly in the nucleus compared to the levels in the cytoplasm, we found that ORF57 shifted the nucleocytoplasmic balance by increasing ORF59 RNA accumulation in the cytoplasm more than in the nucleus. By promoting the accumulation of cytoplasmic ORF59 RNA, ORF57 offsets the nuclear RNA accumulation mediated by RBM15 by preventing nuclear ORF59 RNA from hyperpolyadenylation. ORF57 interacts directly with the RBM15 C-terminal portion containing the SPOC domain to reduce RBM15 binding to ORF59 RNA. Although ORF57 homologs Epstein-Barr virus (EBV) EB2, herpes simplex virus (HSV) ICP27, varicella-zoster virus (VZV) IE4/ORF4, and cytomegalovirus (CMV) UL69 also interact with RBM15 and OTT3, EBV EB2, which also promotes ORF59 expression, does not function like KSHV ORF57 to efficiently prevent RBM15-mediated nuclear accumulation of ORF59 RNA and RBM15's association with polyadenylated RNAs. Collectively, our data provide novel insight elucidating a molecular mechanism by which ORF57 promotes the expression of viral intronless genes.

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Figures

FIG. 1.
FIG. 1.
Ectopic RBM15 promotes and cooperates with ORF57 in ORF59 expression. HEK293 cells at 2.5 × 105 per ml were cotransfected with 20 ng of RBM15-FLAG expression vector in combination with 300 ng of ORF59-FLAG expression vector in the absence or presence of an increased amount of ORF57-FLAG expression vector as indicated. Protein and RNA samples were prepared 24 h after cotransfection and analyzed, respectively, by Western blot (WB) and Northern blot (NB) assays. Tubulin served as a protein loading control for Western blotting, and GAPDH RNA served as an RNA loading control for Northern blotting. (A) RBM15 regulation of ORF59 expression in the absence or presence of ORF57. Relative ORF59 protein levels (fold) were quantified based on each band's density after normalization to that of tubulin as a sample loading control, with the ORF59 protein level in lane 1 being set as 1. (B) Bar graphs show relative level of ORF59 RNA in each sample from Northern blot analysis in panel A after being normalized to the level of the corresponding GAPDH RNA for each sample loaded. Shown above the bar graph is the relative ratio of ORF59 RNA in the presence versus the absence of RBM15 for each pair of samples.
FIG. 2.
FIG. 2.
Distinct effects of ORF57 and RBM15 in promotion of ORF59 expression. HEK293 cells were cotransfected with an ORF59-FLAG expression vector (300 ng) together with the indicated amount of ORF57-FLAG or RBM15-FLAG vector. Total protein and RNA, as well as fractionated RNA, were prepared and analyzed, respectively, by Western blot (WB) and Northern blot (NB) assays. (A and C) Dose-dependent effect of ORF57 but not RBM15 on the expression of ORF59 protein and RNA. Bar graphs show relative level of ORF59 RNA in each sample from Northern blot analysis after being normalized to the level of GAPDH RNA for sample loading. (B and D) ORF57 promotes ORF59 RNA accumulation both in the cytoplasm and in the nucleus, but RBM15 preferentially promotes ORF59 RNA accumulation in the nucleus. Bar graphs below each Northern blot show the relative level of ORF59 RNA in each sample after normalization to the level of the corresponding GAPDH for sample loading. U6 results indicate the nuclear fractionation efficiency. The cytoplasmic-to-nuclear RNA ratio (C/N ratio) of ORF59 RNA in each pair of samples from the Northern blot assay was calculated after normalization to the level of the corresponding GAPDH loading control and is shown above the bar graph.
FIG. 3.
FIG. 3.
OTT3 functions like RBM15 in promotion of ORF59 expression. (A) ORF59 expression in HEK293 cells 24 h after cotransfection with 300 ng of ORF59 and 100 ng of ORF57, RBM15, or OTT3. Bar graphs show relative level of ORF59 RNA in each sample from Northern blot analysis after being normalized to the level of GAPDH RNA for sample loading. (B) OTT3, which functions like RBM15, preferentially promotes ORF59 RNA accumulation in the nucleus. Bar graphs below each Northern blot show relative level of ORF59 RNA in each sample after normalization to the level of the corresponding GAPDH for sample loading. U6 results indicate nuclear fractionation efficiency. The C/N ratio of ORF59 RNA in each sample set calculated from the Northern blot is shown above the bar graph.
FIG. 4.
FIG. 4.
Endogenous RBM15 and full-length RBM15 or OTT3 are essential to promote ORF59 expression. (A) Endogenous RBM15 is required for ORF59 expression. HeLa cells, with or without knockdown of RBM15 expression by RNAi, were transfected with 500 ng of ORF59 expression vectors for 24 h and then examined for ORF59 and RBM15 expression by Western blot (WB) or Northern blot (NB) assays. The numbers below the Northern blots show the relative level of ORF59 RNA in each sample after normalization to the level of the corresponding GAPDH for sample loading. NS, nonspecific. (B and C) Full-length RBM15 and OTT3 are essential for ORF59 expression. HEK293 cells were cotransfected with 300 ng of ORF59-FLAG and 100 ng of full-length, N-terminal, or C-terminal RBM15-FLAG (B) or OTT3-HA (C). Protein samples were prepared 24 h after transfection and blotted with anti-FLAG or anti-HA antibodies. *, nonspecific. Tubulin served as sample loading control.
FIG. 5.
FIG. 5.
ORF57 offsets the nuclear RNA accumulation activity of RBM15 in promoting ORF59 expression. (A) ORF57 coordinates with RBM15 to additively promote ORF59 expression. (B) ORF57 promotes ORF59 RNA accumulation in the cytoplasm by offsetting the nuclear RNA accumulation mediated by ectopic RBM15. HEK293 cells were cotransfected with 300 ng of ORF59-FLAG in combination with or without 20 ng of RBM15-FLAG in the presence or absence of the indicated ORF57-GFP expression vector (100 ng). mtNLS2 + 3 (mt2 + 3), an inactive ORF57 mutant with point mutations in its nuclear localization signals 2 and 3, served as a control. A GFP-only plasmid was used as a vector control. Protein samples, total RNA, and fractionated RNA were prepared 24 h after cotransfection. Protein samples were examined by Western blot (WB) assay with anti-FLAG and anti-GFP antibodies (A, upper panel). Tubulin served as sample loading control. All RNA samples were analyzed by Northern blot (NB) assay (A, lower panel; B, upper panel). Bar graphs below each Northern blot in panels A and B show the relative level of ORF59 RNA in each sample after normalization to the level of the corresponding GAPDH for sample loading. U6 results show nuclear fractionation efficiency. Relative C/N ratio of ORF59 RNA in each sample set calculated from the Northern blot in panel B is shown above the bar graph.
FIG. 6.
FIG. 6.
Determination of the RBM15-mediated hyperpolyadenylation of nuclear ORF59 RNA. (A) Strategy to determine RNA poly(A) tail length by RNase H digestion assay. Oligo(dT16) was annealed to purified RNAs, and poly(A) tails of RNAs were removed by incubation with RNase H, specifically digesting the RNA within the double-stranded DNA/RNA region. The digested RNA was separated in agarose gel and analyzed by Northern blotting. Undigested RNA served as a control. Bold lines represent mRNA transcripts with an m7G RNA cap (filled circle) on the 5′ end and a poly(A) tail (AAAAAAA) on the 3′ end. (B) Northern blot analyses of RNase H-digested total and fractionated nuclear RNA (5 μg/lane) extracted from HEK293 cells 24 h after cotransfection with 300 ng of ORF59-FLAG vector and 100 ng of ORF57, RBM15, or an empty vector. ORF59 transcripts were detected with a 32P-labeled oligonucleotide probe. Total GAPDH RNA served as a control RNA in the digestion. An, transcript with poly(A) tail; A0, transcript without poly(A) tail.
FIG. 7.
FIG. 7.
ORF57 colocalizes with RBM15 and OTT3 in vivo. (A and B) The confocal images were taken from HeLa cells 24 h after cotransfection with an equal amount (500 ng each) of wt ORF57-GFP (A) or its inactive mutant mtNLS2 + 3 (mt2 + 3) (B) and FLAG-tagged RBM15 or OTT3 constructs. Cells were stained with anti-FLAG antibody in immunofluorescent staining to visualize FLAG-tagged proteins. Cell nuclei were counterstained by DAPI. Scale bar, 10 μM.
FIG. 8.
FIG. 8.
ORF57 interacts with RBM15 and OTT3. (A) ORF57 interacts with endogenous RBM15 in vivo. ORF57-FLAG expressed in HEK293 cells at 24 h after transfection was immunoprecipitated (IP) with anti-FLAG antibody. The proteins in the IP pulldowns were blotted with a polyclonal anti-RBM15 antibody or anti-ORF57. (B) RBM15 and OTT3 interact with the N-terminal nuclear localization signals (NLS) 2 and 3 of ORF57. HEK293 cell lysates were prepared 2 days after transfection and immunoprecipitated using anti-FLAG antibody. The IP complexes were blotted with anti-GFP for ORF57 or anti-FLAG antibody for RBM15 and OTT3. Shown at the top are diagrams of ORF57 proteins without (wt) or with (mtNLS2 + 3) mutation of NLS2 and NLS3. Lower panel shows interaction of wt ORF57 but not the inactive mutant mtNLS2 + 3 (mt2 + 3) with RBM15 and OTT3 in cotransfected HEK293 cells by IP-Western blotting. (C) Mapping of ORF57 interaction domains of RBM15 and OTT3. Shown at the top are the structures of wt RBM15 and OTT3 or their deletion mutant proteins. Both RBM15 and OTT3 contain an N-terminal portion with three RRMs (RNA recognition motifs) and a C-terminal SPOC domain. Shown for each protein, at a, b, and c for RBM15 and d, e, and f for OTT3, are diagrams of full-length (FL; a, d), N-terminal (b, e), and C-terminal (c, f) mutants that were cloned into a 3×FLAG vector. Numbers represent amino acid positions. Lower panel shows that RBM15 and OTT3 interact with ORF57. HEK293 cells were cotransfected with an ORF57-GFP expression vector in combination with an RBM15- or OTT3-FLAG expression vector. See more details for IP-Western blot assay in the description for panel B. (D) In vitro direct binding of ORF57 to the SPOC domain of RBM15 and OTT3. ORF57 and control proteins (UAP56 and firefly luciferase) that were expressed and metabolically labeled in reticulocyte extracts were tested for binding to E. coli-produced, purified GST-RBM15 (aa 530 to 977), GST-OTT3 (aa 488 to 890), or GST only. The 35S-radiolabeled proteins in the bound (GST pull-down) fractions and 1% aliquots of the respective input (load) and unbound fractions were analyzed by SDS-PAGE and autoradiography.
FIG. 9.
FIG. 9.
ORF57 affects RBM15-RNA interactions in vivo. (A and B) ORF57-RBM15 SPOC domain interaction interferes with RBM15's association with polyadenylated RNA. HEK293 cells (5 × 105/well in a 6-well plate) were cotransfected with the following expression vectors (500 ng each): FLAG-tagged ORF59, FLAG-tagged full-length RBM15 (A) or SPOC domain deletion mutant (aa 1 to 530) (B) and GFP-tagged wild-type ORF57 (wt) or NLS2 + 3 mutant (mt). Empty vector pEGFP-N1 served as an ORF57 negative control. At 24 h after transfection, the cells were UV irradiated, and the polyadenylated [poly(A)] RNA transcripts were isolated. After RNase digestion, the proteins associated with poly(A) RNAs were detected by Western blotting by using anti-FLAG antibody for RBM15. Total cell lysates prior to poly(A) RNA selection served as input for each sample in Western blotting. (C to F) ORF57 interaction with RBM15 affects the binding of RBM15 to ORF59 RNA. HEK293 cells 24 h after cotransfection with RBM15 and ORF59 in the absence or presence of wt or mutant ORF57 were examined by poly(A) selection (C) or CLIP assays (D to F) followed by Western blot (C) or RT-PCR (D to F) analysis. (C) RBM15 protein but not ORF59 associates with poly(A) RNAs, which is preventable by wt ORF57. Lanes 3 and 4 contain cell lysate input prior to poly(A) mRNA selection, showing ectopic expression of FLAG-tagged RBM15 and ORF59. (D) RBM15 protein binds ORF59 RNA in the absence of ORF57. The RBM15-ORF59 RNA complex in the CLIP assay, after proteinase K digestion, was analyzed by RT-PCR analysis for ORF59 RNA or GAPDH RNA as a nonspecific RNA control. Preimmune rabbit serum IgG served as an IP control. RT, reverse transcriptase; M, 100-bp DNA ladder; DNA, ORF59 plasmid DNA for size control. (E and F) Coexpression of wt ORF57 with RBM15 reduces RBM15 binding to ORF59 RNA. See other details in the panel D legend. (E, left) Results show the same amount of ORF59 RNA from the input cell extracts as used for CLIP. GAPDH RNA in the extracts served as a loading control. (E, right) Results show that wt ORF57 but not ORF57 mtNLS2 + 3 prevents RBM15 association with ORF59 RNA in CLIP assays. (F) Bar graphs with means ± standard deviations show the ORF59 RNA level from each anti-FLAG CLIP, quantified by qRT-PCR and analyzed by two-tailed Student's t test. n = 2, each in triplicate. Ab, antibody.
FIG. 10.
FIG. 10.
The interaction with RBM15 and OTT3 proteins is conserved among ORF57 homologs. (A to C) Myc-tagged EBV EB2 (A), GFP-tagged HSV-1 ICP27 (B), and V5-tagged VZV IE4 (C) were expressed in HEK293 cells alone or together with FLAG-tagged RBM15 or OTT3, and the complexes were coimmunoprecipitated with anti-FLAG antibody under stringent salt conditions (400 mM). The IP complexes were blotted with anti-myc for EB2 (A), with anti-GFP for ICP27 (B), with anti-V5 to detect IE4 (C), and with anti-FLAG for RBM15 and OTT3 (A, B, and C, lower panels). (D) To determine the interaction of CMV UL69 with RBM15 and OTT3 in vivo, HEK293 cells were cotransfected with FLAG-tagged UL69 and HA-tagged RBM15 or HA-tagged OTT3. The protein complexes were coimmunoprecipitated with anti-FLAG antibody (UL69) under stringent salt conditions (400 mM) and blotted with anti-HA to detect RBM15 and OTT3 and with anti-FLAG to detect UL69.
FIG. 11.
FIG. 11.
EBV EB2 does not prevent RBM15-mediated nuclear accumulation of ORF59 RNA and does not block RBM15-RNA interactions. (A) Colocalization of EB2-myc and RBM15-FLAG in HeLa cells by cotransfection. Scale bar, 10 μm. (B and C) EB2 promotes ORF59 expression but does not prevent RBM15-mediated nuclear accumulation of ORF59 RNA. HEK293 cells (2.5 × 105 cells/ml) were transfected with 300 ng of ORF59-FLAG with or without 20 ng RBM15-FLAG in addition to 100 ng empty vector, ORF57-FLAG, or EB2-myc. Twenty-four hours after transfection, the cells were collected for total protein and RNA analyses by Western blot (WB) and Northern blot (NB) assays, respectively (B). Fractionated cytoplasmic and nuclear RNA was also analyzed in Northern blot assays by using a 32P-labeled oligonucleotide probe specific for ORF59 RNA (C). (D) EB2 does not prevent RBM15's association with polyadenylated RNAs. HEK293 cells (5 × 105) were transfected with 500 ng each of ORF59-FLAG and RBM15-FLAG vectors together with 500 ng of an empty FLAG vector, ORF57-FLAG, or EB2-myc. Twenty-four hours after transfection, the cells were exposed to UV light and polyadenylated RNAs were isolated. RBM15 proteins bound onto polyadenylated RNAs were analyzed by Western blotting. SE, short exposure; LE, long exposure.
FIG. 12.
FIG. 12.
Proposed model for the effects of RBM15 and ORF57 on ORF59 expression. (A) RBM15 at physiological level promotes the expression of ORF59 at the early stage of KSHV lytic replication when ORF57 is at low concentration. Although ORF57 promotes ORF59 expression, ORF57 expression is also dependent on RBM15 (18). At a later stage of infection when ORF57 is high, its interaction with RBM15 prevents RBM15's association with ORF59 RNA and, consequently, reduces the function of RBM15 to promote ORF59 RNA export. ORF57 accumulates ORF59 RNA by increasing ORF59 RNA stability by an unknown mechanism. (B) Overexpression of RBM15 preferentially increases the nuclear over the cytoplasmic level of ORF59 RNA and stimulates hyperpolyadenylation of nuclear ORF59 RNA. In the presence of ORF57, ORF57 interacts with RBM15 and prevents the RBM15-mediated nuclear accumulation of ORF59 RNA.
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