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The Cellular RNA-Binding Protein EAP Recognizes a Conserved Stem:Loop in the Epstein-Barr Virus Small RNA EBER 1 DAVID P

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The Cellular RNA-Binding Protein EAP Recognizes a Conserved Stem:Loop in the Epstein-Barr Virus Small RNA EBER 1 DAVID P MOLECULAR AND CELLULAR BIOLOGY, Jan. 1993, p. 703-710 Vol. 13, No. 1 0270-7306/93/010703-08$02.00/0 Copyright © 1993, American Society for Microbiology The Cellular RNA-Binding Protein EAP Recognizes a Conserved Stem:Loop in the Epstein-Barr Virus Small RNA EBER 1 DAVID P. TOCZYSKI* AND JOAN A. STEITZ Department ofMolecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University, 295 Congress Avenue, New Haven, Connecticut 06536-0812 Received 17 August 1992/Returned for modification 1 October 1992/Accepted 23 October 1992 EAP (EBER-associated protein) is an abundant, 15-kDa cellular RNA-binding protein which associates with certain herpesvirus small RNAs. We have raised polyclonal anti-EAP antibodies against a glutathione S-transferase-EAP fusion protein. Analysis of the RNA precipitated by these antibodies from Epstein-Barr virus (EBV)- or herpesvirus papio (HVP)-infected cells shows that >95% of EBER 1 (EBV-encoded RNA 1) and the majority of HVP 1 (an HVP small RNA homologous to EBER 1) are associated with EAP. RNase protection experiments performed on native EBER 1 particles with affinity-purified anti-EAP antibodies demonstrate that EAP binds a stem-loop structure (stem-loop 3) of EBER 1. Since bacterially expressed glutathione S-transferase-EAP fusion protein binds EBER 1, we conclude that EAP binding is independent of any other cellular or viral protein. Detailed mutational analyses of stem-loop 3 suggest that EAP recognizes the majority of the nucleotides in this hairpin, interacting with both single-stranded and double-stranded regions in a sequence-specific manner. Binding studies utilizing EBER 1 deletion mutants suggest that there may also be a second, weaker EAP-binding site on stem-loop 4 of EBER 1. These data and the fact that stem-loop 3 represents the most highly conserved region between EBER 1 and HVP 1 suggest that EAP binding is a critical aspect of EBER 1 and HVP 1 function. Epstein-Barr virus (EBV) is a B-cell lymphotrophic her- EBERs. Whereas previous experiments had suggested that pesvirus which is able to transform primary cells in vitro (for EAP bound both EBER 1 and EBER 2 (32), the binding a review, see references 18 and 25). During latency, most studies presented here indicate that EAP's association with EBV genes are transcriptionally silent (for a review, see EBER 1 is by far the more significant. In contrast to that of reference 19). A striking exception, however, is the genes for many other RNA-binding proteins, EAP's ability to bind the two small RNAs called EBER 1 and EBER 2 (for Epstein- third stem-loop of EBER 1 is reduced by many mutations in Barr-encoded RNA). These RNAs are each present at ap- double-stranded regions, even when secondary structure is proximately 5 x 10' copies per cell in latently infected B-cell maintained. Characterization of this binding site should aid lines (22). Herpesvirus papio (HVP), a related virus which in identifying the cellular RNAs associated with EAP. infects baboons, encodes a homologous pair of small RNAs. These RNAs, HVP 1 and HVP 2, are 83 and 65% identical to the EBERs and are present at approximately 5 x 106 and 5 MATERIALS AND METHODS x 105 to 10 x 105 copies per cell, respectively (15). The EBER genes are unusual in that they are transcribed Glutathione transferase fusion protein and antibody pro- by RNA polymerase III and yet they utilize promoter duction. A glutathione S-transferase (GST)-EAP fusion vec- elements normally associated with protein-coding (class II) tor was prepared by inserting an EAP-encoding DNA frag- genes (16). As with most RNA polymerase III transcripts, ment containing a 5'-terminal BamHI site into an expression the 3' ends of the EBERs (and those of the HVPs) are vector (pGEX) (2) encoding 27 kDa of the GST protein with composed of a short stretch of uridine residues. The La a BamHI site at its 3' end. The EAP-encoding insert con- protein, a 50-kDa phosphoprotein first identified as an au- tained an open reading frame with a BamHI site followed by toantigen, binds this poly(U) tail on >90% of the EBER and a factor X cleavage site and the entire EAP sequence, HVP RNAs (15). We previously identified a second protein, excluding the initiating methionine. A stop codon immedi- EAP (EBER-associated protein), which also binds to these ately after the EAP C terminus was followed by a short viral small RNAs (32). EAP is an abundant, 15-kDa cellular polylinker including a BamHI and a Sall site. This insert was protein which appears to be the mammalian homolog of a generated by the polymerase chain reaction from an EAP previously identified sea urchin protein. A cDNA encoding cDNA template. The terminal restriction sites and factor X the sea urchin protein, which was called 217, was identified cleavage site were included in the deoxyoligonucleotides: 5', in a screen for genes expressed in a developmentally specific CGGATCCTGATCGAGGGCAGGGCCCCCGTGAAGAA manner (8, 12). EAP and the 217 protein are 77% identical. GCTGGTGGTGAAGGGCGGAAAGAAGAAGAAGCA, Unfortunately, the functions of both remain a mystery. and 3', GGGATCCAGCTGTCGACTJAATCCTCGTCTT We have now raised anti-EAP antibodies and have used CCTCCTCTTCTTCGTCCTGG. The polymerase chain re- them to characterize the interaction between EAP and the action was carried out as described by Toczyski and Steitz (32). The EAP insert was cloned into pGEX-3X after diges- tion of both the insert and the vector with BamHI. The entire EAP insert was sequenced. T7 transcription, molecular * Corresponding author. cloning, bacterial techniques, DNA sequencing, and tissue 703 704 TOCZYSKI AND STEITZ MOL. CELL. BIOL. culture were carried out as described by Toczyski and Steitz varying amounts of antibody and determining, by Northern (32). (RNA) blot analysis, how much antibody was required to The GST-EAP fusion protein was isolated by inoculating deplete EBER 1. Two microliters of affinity-purified serum 12 1-liter flasks of Luria broth containing 200 ,g of carben- or 10 pl of whole serum was required for such a depletion. At icillin with 2 ml from a fresh overnight culture ofEscherichia least a twofold excess of serum was used unless otherwise coli (DH 1) transformed with the fusion protein-bearing or noted. wild-type GST plasmid. The cells were grown to an optical Immunoprecipitation, Northern blotting, RNA affinity se- density at 590 nm of 0.75 to 0.90. IPTG (isopropyl-O-D- lection, and RNase protection assays. Twelve-hour 32PO4 in thiogalactopyranoside) was then added to 0.05 mM, and the vivo labelling and immunoprecipitation were performed as cells were grown for another 2 to 3 h. Cells were harvested, described in reference 22. Urea polyacrylamide gels were sonicated in Tris-buffered saline (150 mM NaCl, 50 mM Tris Northern blotted onto Zeta-probe nylon membranes and [pH 7.5]) containing 1% Triton X-100 six times for 30 s on a probed with full-length antisense RNAs as specified by the Branson Sonifier at setting 7, and centrifuged for 15 min at 15 supplier (Bio-Rad). krpm in a Sorvall SA600 rotor. At this point, the cell sonicate 3'-end labelling of RNA for affinity selection was carried could be frozen after the addition of glycerol to 10%. The out by using 32pCp and RNA ligase (10). Fifty microliters of extract was then run over a 3- to 4-ml glutathione agarose (S bacterial sonicate (see "Affinity purification and Western linkage, Sigma) column and washed with 300 ml of Tris- blotting [immunoblotting]") was incubated with 20 mg of buffered saline containing 1% Triton X-100. After being glutathione agarose in 1 ml of TBS (50 mM Tris, 150 mM washed with 50 ml of 50 mM Tris, pH 8.0, the column was NaCl [pH 7.5]). The resin was then washed 10 times with 15 eluted with the same buffer containing 10 mM glutathione. A ml of TBS-1% Triton X-100 and incubated with pCp-labelled portion of this material was then boiled in 0.1% sodium RNA from 105 Raji cells. After a 15-min incubation at 25°C, dodecyl sulfate (SDS) for 5 min and used to raise antibodies the mixture was washed seven times with NET-2 (50 mM in rabbits; antibodies were produced by the Berkeley Anti- Tris, 150 mM NaCl, 0.05% Nonidet P-40 [pH 7.5]) at 4°C. body Company. RNA was then extracted with phenol-chloroform-isoamyl Affinity purification and Western blotting (immunoblot- alcohol (25:25:1), ethanol precipitated, and electrophoresed ting). The polymerase chain reaction EAP-encoding frag- on a urea polyacrylamide gel. ment mentioned above was cut with BamHI and SalI and For RNase protection experiments, sonicate from 106 cells cloned into pUR-292, an expression vector containing the was incubated with 50 ,ul of whole anti-EAP serum bound to entire 3-galactosidase gene with a polylinker at the carboxy 5 pLg of protein A-Sepharose. After 90 min at 4°C, samples terminus (29). This protein was overexpressed and purified were washed five times with 1 ml of NET-2 and once with according to the method described in reference 13. Approx- NET (NET-2 without Nonidet P-40). Samples were adjusted imately 50 mg of the 3-galactosidase-EAP fusion was dia- to 250 ,ul with NET containing 1,250 U of RNase T1 lyzed against 0.1 M borate-0.75 M NaCl-3 mM P-mercap- (Calbiochem). After being incubated at 30°C, samples were toethanol (pH 8.3) and incubated with 7 ml of CNBr- washed four times with cold NET-2 and incubated with activated Sepharose (Pharmacia; prewashed according to NET-2 containing 0.5% SDS-0.5 mg of proteinase K per ml the manufacturer's specifications) for 12 h at 4°C.
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