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Identification of Herpes Simplex Virus RNAs That Interact Specifically with Regulatory Protein ICP27 in Vivo

2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês

10.1074/jbc.m302063200

ISSN

1083-351X

Autores

Marcus Sokolowski, J. E. Scott, Robert P. Heaney, Arvind H. Patel, J. Barklie Clements,

Tópico(s)

RNA regulation and disease

Resumo

Herpes simplex virus type 1 (HSV-1) protein ICP27 has an essential regulatory role during viral replication, in part by post-transcriptional control of gene expression, and has a counterpart in all herpes viruses sequenced so far. Although much is known about the functions of this signature herpesvirus protein, little is known about its RNA binding capabilities; ICP27 interacts with specificity for a subset of intronless HSV-1 RNAs and poly(G), through its RGG box. We performed an in vivo yeast three-hybrid screen of an HSV-1 genomic library, searching for ICP27 interacting RNAs. Comparable with a yeast genomic screen, 24 of 55 single inserts mapped to antisense strands of HSV-1 transcribed regions or non-transcribed regions. The 31 HSV-1 sense RNAs identified were 35 to 225 nucleotides in length and interacted with preferred specificity for ICP27 as compared with an unrelated RNA-binding protein. They map to 10 monocistronic and 10 polycistronic transcripts of all kinetic classes and represent 28 open reading frames encoding predominantly essential viral proteins with roles in viral DNA replication and virion maturation. Several studies show regulatory effects by ICP27 on the majority of these transcripts, consistent with its regulation of the early-late switch in the HSV-1 life cycle. Deletion of the ICP27 RGG box and the ICP27 M15 mutation, both lethal in virus, abolished or severely reduced the ICP27-RNA interactions, indicating their biological relevance. The study facilitates continued study of gene regulation by ICP27 by further defining its interactions with viral RNAs. Herpes simplex virus type 1 (HSV-1) protein ICP27 has an essential regulatory role during viral replication, in part by post-transcriptional control of gene expression, and has a counterpart in all herpes viruses sequenced so far. Although much is known about the functions of this signature herpesvirus protein, little is known about its RNA binding capabilities; ICP27 interacts with specificity for a subset of intronless HSV-1 RNAs and poly(G), through its RGG box. We performed an in vivo yeast three-hybrid screen of an HSV-1 genomic library, searching for ICP27 interacting RNAs. Comparable with a yeast genomic screen, 24 of 55 single inserts mapped to antisense strands of HSV-1 transcribed regions or non-transcribed regions. The 31 HSV-1 sense RNAs identified were 35 to 225 nucleotides in length and interacted with preferred specificity for ICP27 as compared with an unrelated RNA-binding protein. They map to 10 monocistronic and 10 polycistronic transcripts of all kinetic classes and represent 28 open reading frames encoding predominantly essential viral proteins with roles in viral DNA replication and virion maturation. Several studies show regulatory effects by ICP27 on the majority of these transcripts, consistent with its regulation of the early-late switch in the HSV-1 life cycle. Deletion of the ICP27 RGG box and the ICP27 M15 mutation, both lethal in virus, abolished or severely reduced the ICP27-RNA interactions, indicating their biological relevance. The study facilitates continued study of gene regulation by ICP27 by further defining its interactions with viral RNAs. In a lytic infection with herpes simplex virus type 1 (HSV-1), 1The abbreviations used are: HSV-1, herpes simplex virus type 1; aa, amino acids; β-gal, β-galactosidase; RGG, arginine- and glycine-rich; KH, K homology; S.D., synthetic dropout; AD, activation domain; IRE, iron responsive element; IRP1, iron regulatory protein 1; ORF, open reading frame; IE, immediate-early; wt, wild type. the viral genes are expressed in a cascade-like fashion and are subject to regulation (1Honess R.W. Roizman B. J. Virol. 1974; 14: 8-19Crossref PubMed Google Scholar, 2Honess R.W. Roizman B. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 1276-1280Crossref PubMed Scopus (324) Google Scholar). The ∼80 genes have been grouped into one of three kinetic classes based on their temporal expression during productive infection; immediate-early (IE; α), early (β), and late (γ) genes, respectively (reviewed in Ref. 3Roizman B. Knipe D.M. Knipe D.M. Howley P. Fields Virology. 4th Ed. Vol. 2. Lippincott, Williams, & Wilkins, Philadelphia2001: 2399-2460Google Scholar). Four of the five IE gene products act to regulate expression of the early and late genes (3Roizman B. Knipe D.M. Knipe D.M. Howley P. Fields Virology. 4th Ed. Vol. 2. Lippincott, Williams, & Wilkins, Philadelphia2001: 2399-2460Google Scholar). The essential (4Sacks W.R. Greene C.C. Aschman D.P. Schaffer P.A. J. Virol. 1985; 55: 796-805Crossref PubMed Google Scholar) UL54 IE gene product ICP27 (IE63) has roles in the early-late switch by regulating viral gene expression at the post-transcriptional level (4Sacks W.R. Greene C.C. Aschman D.P. Schaffer P.A. J. Virol. 1985; 55: 796-805Crossref PubMed Google Scholar, 5McLauchlan J. Phelan A. Loney C. Sandri-Goldin R.M. Clements J.B. J. Virol. 1992; 66: 6939-6945Crossref PubMed Google Scholar, 6Phelan A. Clements J.B. Sem. Virol. 1998; 8: 309-318Crossref Scopus (33) Google Scholar, 7Sandri-Goldin R.M. Mendoza G.E. Genes Dev. 1992; 6: 848-863Crossref PubMed Scopus (172) Google Scholar, 8Smith I.L. Hardwicke M.A. Sandri-Goldin R.M. Virology. 1992; 186: 74-86Crossref PubMed Scopus (238) Google Scholar), for example by affecting nucleocytoplasmic export of HSV-1 mRNAs (9Soliman T.M. Sandri-Goldin R.M. Silverstein S.J. J. Virol. 1997; 71: 9188-9197Crossref PubMed Google Scholar, 10Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 7600-7609Crossref PubMed Scopus (32) Google Scholar, 11Phelan A. Dunlop J. Clements J.B. J. Virol. 1996; 70: 5255-5265Crossref PubMed Google Scholar, 12Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (217) Google Scholar, 13Koffa M. Clements J.B. Izaurralde E. Wadd S. Wilson S. Mattaj I.W. Kuersten S. EMBO J. 2001; 20: 5769-5778Crossref PubMed Scopus (139) Google Scholar, 14Lengyel J. Guy C. Leong V. Borge S. Rice S.A. J. Virol. 2002; 76: 11866-11879Crossref PubMed Scopus (47) Google Scholar, 15Chen I. Sciabica K. Sandri-Goldin R.M. J. Virol. 2002; 76: 12877-12889Crossref PubMed Scopus (133) Google Scholar) or pre-mRNA 3′-end processing (5McLauchlan J. Phelan A. Loney C. Sandri-Goldin R.M. Clements J.B. J. Virol. 1992; 66: 6939-6945Crossref PubMed Google Scholar, 16McGregor F. Phelan A. Dunlop J. Clements J.B. J. Virol. 1996; 70: 1931-1940Crossref PubMed Google Scholar). Many studies have highlighted the multifunctional nature of this protein, which also acts transcriptionally; ICP27 associates with cellular RNA polymerase II (17Zhou C. Knipe D.M. J. Virol. 2002; 76: 5893-5904Crossref PubMed Scopus (84) Google Scholar) and affects transcription of certain late genes (18Jean S. Le Van K.M. Song B. Levine M. Knipe D.M. Virology. 2001; 283: 273-284Crossref PubMed Scopus (69) Google Scholar). Other viral proteins may regulate HSV-1 gene expression post-transcriptionally, as suggested for VP13/14 (19Donnelly M. Elliott G. J. Virol. 2001; 75: 2566-2574Crossref PubMed Scopus (61) Google Scholar). The IE ICP27 (IE63) phosphoprotein (20Wilcox K.W. Kohn A. Sklyanskaya E. Roizman B. I. J. Virol. 1980; 71: 167-182Crossref Google Scholar) shuttles between the nucleus and cytoplasm (9Soliman T.M. Sandri-Goldin R.M. Silverstein S.J. J. Virol. 1997; 71: 9188-9197Crossref PubMed Google Scholar, 21Mears W.E. Rice S.A. Virology. 1998; 242: 128-137Crossref PubMed Scopus (98) Google Scholar, 22Phelan A. Clements J.B. J. Gen. Virol. 1997; 78: 3327-3331Crossref PubMed Scopus (79) Google Scholar) and is capable of interaction with RNA (12Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (217) Google Scholar, 23Brown C. Nakamura M. Mosca J. Hayward G. Straus S. Perera L. J. Virol. 1995; 69: 7187-7195Crossref PubMed Google Scholar, 24Ingram A. Phelan A. Dunlop J. Clements J.B. J. Gen. Virol. 1996; 77: 1847-1851Crossref PubMed Scopus (40) Google Scholar, 25Mears W. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar) and proteins (15Chen I. Sciabica K. Sandri-Goldin R.M. J. Virol. 2002; 76: 12877-12889Crossref PubMed Scopus (133) Google Scholar, 26Panagiotidis C.A. Lium E.K. Silverstein S.J. J. Virol. 1997; 71: 1547-1557Crossref PubMed Google Scholar, 27Bryant H.E. Matthews D.A. Wadd S. Scott J.E. Kean J. Graham S. Russell W.C. Clements J.B. J. Virol. 2000; 74: 11322-11328Crossref PubMed Scopus (49) Google Scholar, 28Wadd S. Bryant H.E. Filhol O. Scott J.E. Hsieh T.-Y. Everett R.D. Clements J.B. J. Biol. Chem. 1999; 274: 28991-28998Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). ICP27 functions include an involvement in host cell shutoff by inhibiting pre-mRNA splicing (29Hardy W.R. Sandri-Goldin R.M. J. Virol. 1994; 68: 7790-7799Crossref PubMed Google Scholar, 30Bryant H.E. Wadd S. Lamond A.I. Silverstein S.J. Clements J.B. J. Virol. 2001; 75: 4376-4385Crossref PubMed Scopus (85) Google Scholar, 31Lindberg A. Krievi J.P. Virology. 2002; 294: 189-198Crossref PubMed Scopus (54) Google Scholar), redistribution of splicing factors (32Phelan A. Carmo-Fonseca M. McLauchlan J. Lamond A.I. Clements J.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9056-9060Crossref PubMed Scopus (126) Google Scholar, 33Sandri-Goldin R.M. Hibbard M.K. Hardwicke M.A. J. Virol. 1995; 69: 6063-6076Crossref PubMed Google Scholar), and its requirement for efficient viral DNA replication (34McCarthy A.M. McMahan L. Schaffer P.A. J. Virol. 1989; 63: 18-27Crossref PubMed Google Scholar). The versatile functions observed are mirrored in the different protein domain structures reported in ICP27. A nuclear export signal (12Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (217) Google Scholar), an export control sequence (10Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 7600-7609Crossref PubMed Scopus (32) Google Scholar), and nuclear and nucleolar localization signals (35Mears W.E. Lam V. Rice S.A. J. Virol. 1995; 69: 935-947Crossref PubMed Google Scholar) may control ICP27 subcellular localization, an N-terminal acidic region performs essential functions in lytic replication (36Rice S.A. Lam V. Knipe D.M. J. Virol. 1993; 67: 1778-1787Crossref PubMed Google Scholar), and C-terminal regions are involved in transactivation and transrepression (36Rice S.A. Lam V. Knipe D.M. J. Virol. 1993; 67: 1778-1787Crossref PubMed Google Scholar, 37Rice S.A. Knipe D.M. J. Virol. 1990; 64: 1704-1715Crossref PubMed Google Scholar, 38Rice S.A. Lam V. J. Virol. 1994; 68: 823-833Crossref PubMed Google Scholar). The C-terminal region also contains a putative Sm homology domain (39Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 2814-2825Crossref PubMed Scopus (56) Google Scholar), a structure that mediates protein-protein interactions in certain spliceosomal proteins (40Hermann H. Fabrizio P. Raker V.A. Foulaki K. Hornig H. Brahms H. Luhrmann R. EMBO J. 1985; 14: 2076-2088Crossref Scopus (215) Google Scholar). Apparently, protein-protein interactions play key roles in its multiple functions as ICP27 interacts with several cellular proteins; heterogeneous nuclear ribonucleoprotein K (28Wadd S. Bryant H.E. Filhol O. Scott J.E. Hsieh T.-Y. Everett R.D. Clements J.B. J. Biol. Chem. 1999; 274: 28991-28998Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), casein kinase 2 (28Wadd S. Bryant H.E. Filhol O. Scott J.E. Hsieh T.-Y. Everett R.D. Clements J.B. J. Biol. Chem. 1999; 274: 28991-28998Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), cellular protein p32 (27Bryant H.E. Matthews D.A. Wadd S. Scott J.E. Kean J. Graham S. Russell W.C. Clements J.B. J. Virol. 2000; 74: 11322-11328Crossref PubMed Scopus (49) Google Scholar), spliceosome-associated protein 145 (30Bryant H.E. Wadd S. Lamond A.I. Silverstein S.J. Clements J.B. J. Virol. 2001; 75: 4376-4385Crossref PubMed Scopus (85) Google Scholar), and RNA export factor Aly/REF (13Koffa M. Clements J.B. Izaurralde E. Wadd S. Wilson S. Mattaj I.W. Kuersten S. EMBO J. 2001; 20: 5769-5778Crossref PubMed Scopus (139) Google Scholar, 15Chen I. Sciabica K. Sandri-Goldin R.M. J. Virol. 2002; 76: 12877-12889Crossref PubMed Scopus (133) Google Scholar). Consistent with its functions as a post-transcriptional regulator, ICP27 interacts with various RNAs in vitro (23Brown C. Nakamura M. Mosca J. Hayward G. Straus S. Perera L. J. Virol. 1995; 69: 7187-7195Crossref PubMed Google Scholar, 24Ingram A. Phelan A. Dunlop J. Clements J.B. J. Gen. Virol. 1996; 77: 1847-1851Crossref PubMed Scopus (40) Google Scholar, 25Mears W. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar) and forms UV light-induced cross-links to poly(A)+ RNA and seven HSV-1 intronless mRNAs from all kinetic classes during infection (12Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (217) Google Scholar, 39Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 2814-2825Crossref PubMed Scopus (56) Google Scholar). Structural domains of ICP27 implicated in RNA binding are an arginine- and glycine-rich region (RGG box) (25Mears W. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar) and three putative K homology (KH)-like domains (39Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 2814-2825Crossref PubMed Scopus (56) Google Scholar), initially identified as RNA-binding domains in heterogeneous nuclear ribonucleoproteins (41Kiledjian M. Dreyfuss G. EMBO J. 1992; 11: 2655-2664Crossref PubMed Scopus (516) Google Scholar, 42Siomi H. Matunis M.J. Michael W.M. Dreyfuss G. Nucleic Acids Res. 1993; 21: 1193-1198Crossref PubMed Scopus (459) Google Scholar). The RGG box acted as a ICP27 protein domain necessary for interaction with RNA (12Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (217) Google Scholar, 25Mears W. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar) and was a determinant of in vivo methylation (25Mears W. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar). A viral substitution mutation that mapped to KH-like motif 3 displayed a reduced efficiency of in vivo UV cross-linking to poly(A)+ RNA (39Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 2814-2825Crossref PubMed Scopus (56) Google Scholar), but the KH-like motifs in ICP27 have not been demonstrated to be directly involved in interaction with HSV-1 RNAs. A specific ICP27-interacting RNA binding site has not been identified to date, and it is unknown whether other HSV-1 mRNAs may interact with ICP27. In the present study, we address these points by using a yeast three-hybrid system (reviewed in Ref. 43Bernstein D. Buter N. Stumpf C. Wickens M. Methods. 2002; 26: 123-141Crossref PubMed Scopus (113) Google Scholar). This approach is the first to identify in vivo ICP27 interactions against a library of HSV-1 RNAs of various lengths that represent the entire viral genome. Plasmids—The following plasmids have been described elsewhere: pACTII-CAN, pIIIA/MS2–2 (44Zhang B. Kraemer B. Sengupta D. Fields S. Wickens M. Methods Enzymol. 2000; 318: 399-419Crossref PubMed Google Scholar), pM15 (38Rice S.A. Lam V. J. Virol. 1994; 68: 823-833Crossref PubMed Google Scholar), pMd4–5 (35Mears W.E. Lam V. Rice S.A. J. Virol. 1995; 69: 935-947Crossref PubMed Google Scholar) (kind gifts from S. Rice), pIII/MS2-IRE, pADIRP1 (45Sengupta D. Zhang B. Kramer B. Pochart P. Fields S. Wickens M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8496-8501Crossref PubMed Scopus (437) Google Scholar), and pSG130 (46Sekulovich R. Leary K. Sandri-Goldin R.M. J. Virol. 1988; 62: 4510-4522Crossref PubMed Google Scholar). pAD27 was constructed by first PCR amplifying a 10-amino acid (aa) N-terminal truncated form of the ICP27 open reading frame (ORF) from plasmid pSG130 using oligonucleotides ICP27Δ9(S) (5′-GCAGATCTACCTCGGCCTGGACCTCTCCGACAGC-3′) and ICP27(A) (5′-CGAGATCTCTAAAACAGGGAGTTGCAATAAAAATAT-3′) that introduced BglII restriction enzyme recognition sites (underlined) at both ends of the ORF. The PCR product was digested with BglII and ligated to pACTII-CAN digested with BglII and treated with calf intestinal alkaline phosphatase, resulting in pAD27. pAD27 encodes a fusion protein containing the GAL4 activation domain fused to ICP27 aa 10 to 512. To generate pADM15, ICP27 aa 10 to 512 containing the M15 mutation (38Rice S.A. Lam V. J. Virol. 1994; 68: 823-833Crossref PubMed Google Scholar) was PCR-amplified from plasmid pM15 and cloned into pACTII-CAN as outlined above. To generate pADΔRGG, pADΔRGG/KH2/3, and pADΔRGG/KH1/2/3, various 10-aa N-terminal truncated ICP27 fragments, also having the RGG box (aa 138–152) internally deleted, were PCR amplified from plasmid pMd4–5 (35Mears W.E. Lam V. Rice S.A. J. Virol. 1995; 69: 935-947Crossref PubMed Google Scholar) using ICP27Δ9(S) and ICP27(A), ICP27Δ9(S), and ICP27ΔKH2(A) (5′-GAGATCTCTAGATGCACATCTTGCACCACG-3′) or ICP27Δ9(S) and ICP27 ΔKH1(A) (5′-GAGATCTCTAGGCGGGAAACGGCTGCCCCC-3′), respectively. The PCR products were digested with BglII and ligated to pACTII-CAN, digested with BglII, and treated with calf intestinal alkaline phosphatase, resulting in pADΔRGG, pADΔRGG/KH2/3, and pADΔRGG/KH1/2/3. Plasmid structures were confirmed by digestion with restriction enzymes and DNA sequencing. Construction of HSV-1 Genomic DNA Plasmid Library—The source of HSV-1 strain 17syn+ genomic DNA was a set of five previously described cosmids (47Cunningham C. Davison A.J. Virology. 1993; 197: 116-124Crossref PubMed Scopus (176) Google Scholar), whose inserts overlap and represent the entire viral genome (cosmids 6, 14, 28, 48, and 56; a kind gift from Dr. A. J. Davison). Cosmids were digested with various combinations of 11 different restriction enzymes, partially or to completion, and the ends were made blunt by treatment with T4 DNA polymerase and DNA polymerase I (Klenow fragment). DNA fragments ranging from 20 to 400 bp were purified from an agarose gel, followed by ligation to pIIIA/MS2–2, digested with SmaI, and treated with calf intestinal alkaline phosphatase. This resulted in insertion of various HSV-1 genomic DNA fragments, in sense or antisense orientation, upstream of the sequences encoding the MS2 RNA. Different ligations were pooled and used to transform electrocompetent Escherichia coli DH5α cells. The library was amplified by growing transformants on solid LB media overnight, followed by growth of the pooled transformants for 4 h in liquid LB media. Yeast Three-hybrid RNA Screen—The yeast three-hybrid RNA screen was performed essentially as described previously (44Zhang B. Kraemer B. Sengupta D. Fields S. Wickens M. Methods Enzymol. 2000; 318: 399-419Crossref PubMed Google Scholar, 48Sengupta D. Wickens M. Fields S. RNA. 1999; 5: 596-601Crossref PubMed Scopus (60) Google Scholar), with certain procedural modifications. The HSV-1 genomic plasmid library was transformed into yeast strain L40coat (L40), pre-transformed with plasmid pAD27. Transformants were plated onto synthetic dropout (S.D.) medium lacking leucine and histidine (leu–/his–) and supplemented with 0.2 to 1.0 mm 3-aminotriazole. White colonies were picked after 6 days and patched onto new S.D. leu–/his– plates containing 0.5 mm 3-aminotriazole, for regrowth to confirm expression of the HIS3, ADE2, and LEU2 genes, followed by analysis of β-galactosidase (β-gal) activity by using an X-gal filter assay. LacZ+/HIS+ colonies were made to loose the pAD27 plasmid, retaining only the library plasmid, by the procedure described previously (44Zhang B. Kraemer B. Sengupta D. Fields S. Wickens M. Methods Enzymol. 2000; 318: 399-419Crossref PubMed Google Scholar). Transformants that lost the pAD27 plasmid were patched to S.D. ura– plates and analyzed for loss of β-gal activity. LacZ– transformants in the absence of pAD27 were then mated with yeast strain R40coat, pre-transformed with plasmids pAD27 or pADIRP1. Diploids that restored β-gal activity in the presence of pAD27, but not in the presence of pADIRP1, expressed HSV-1 RNAs that likely interacted specifically with ICP27 protein. Library plasmid DNAs were isolated from yeast and sequenced. Library plasmids that encoded RNAs corresponding to single inserts of transcribed HSV-1 genes were retransformed back into L40, followed by mating with R40 pre-transformed with plasmids pAD27 or pADIRP1. The specificity of these RNAs for ICP27 was again tested by measuring β-gal activity of resulting diploids using a liquid assay and confirmed by analysis of β-gal activity in the presence of ICP27 mutants. Assays of β-Gal Activity—Using the X-gal filter assay, lawns of yeast transformants were grown on appropriate S.D. plates. Yeast transformants were lifted onto Whatman (grade 5) filter paper, freeze-thawed once in liquid nitrogen, and placed onto Whatman 3MM paper presoaked in Buffer Z (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4, 50 mm β-mercaptoethanol, pH 7.0) containing 300 mg/ml X-gal. Filters were examined for development of blue color after incubation for 3 h at 30 °C. To assay β-gal activity quantitatively, the liquid assay was used with chlorophenol red β-d-galactopyranoside as substrate (49Miller J.H. Schmeissner V. J. Mol. Biol. 1979; 131: 223-248Crossref PubMed Scopus (57) Google Scholar, 50Iwabuchi K. Li B. Bartel P. Fields S. Oncogene. 1993; 8: 1693-1696PubMed Google Scholar). Briefly, cultures were grown in appropriate S.D. medium overnight, followed by reinocculation of fresh cultures and regrowth to A 0.8. Cells were pelleted, washed in appropriate enzyme buffer once, and freeze-thawed three times in between liquid nitrogen and 37 °C. Enzyme buffer containing CPRG substate was added, and following color development, the reactions were stopped to yield optical density values in the linear range of the assay. Measurements below one unit (in reactions stopped after 2 h of color development) were not in the linear range of the assay and were referred to as 3 × 104 E. coli transformants was generated, and sequence analysis of more than twenty clones revealed that inserts varied in length from 20 to 400 nucleotides and corresponded to different locations on the HSV-1 genome. This confirmed the construction of a small-insert library, similar to that used to screen the Saccharomyces cerevisiae genome (48Sengupta D. Wickens M. Fields S. RNA. 1999; 5: 596-601Crossref PubMed Scopus (60) Google Scholar), and having a >15-fold theoretical coverage of the entire HSV-1 genome. To generate the second protein hybrid component, the ICP27 ORF was inserted inframe with the GAL4 activation domain (AD) in plasmid pACTII-CAN, generating plasmid pAD27 (resembling plasmid p502CAD used in yeast two-hybrid screens; see Refs. 28Wadd S. Bryant H.E. Filhol O. Scott J.E. Hsieh T.-Y. Everett R.D. Clements J.B. J. Biol. Chem. 1999; 274: 28991-28998Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar and 30Bryant H.E. Wadd S. Lamond A.I. Silverstein S.J. Clements J.B. J. Virol. 2001; 75: 4376-4385Crossref PubMed Scopus (85) Google Scholar); because inclusion of ICP27 aa 1 to 9 fused to the GAL4 AD transactivates certain yeast promoters unspecifically in a yeast two-hybrid system (28Wadd S. Bryant H.E. Filhol O. Scott J.E. Hsieh T.-Y. Everett R.D. Clements J.B. J. Biol. Chem. 1999; 274: 28991-28998Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 30Bryant H.E. Wadd S. Lamond A.I. Silverstein S.J. Clements J.B. J. Virol. 2001; 75: 4376-4385Crossref PubMed Scopus (85) Google Scholar), an N-terminal truncated ICP27 ORF (aa 10 to 512) was used. The HSV-1 plasmid library was transformed into yeast strain L40, which had been pre-transformed with plasmid pAD27. Growth of transformants was performed on solid S.D. medium lacking leucine and histidine and containing 3-aminotriazole, selecting for plasmid pAD27 and activation of the HIS3 reporter gene, respectively. The transformation yielded >5 × 105 yeast transformants, of which >103 white colonies grew on the S.D. medium 6 days post-transformation (Fig. 1). By analysis of activation of the lacZ reporter gene of colonies picked at random, using a filter lift assay, we isolated 334 transformants that had β-gal activity (Fig. 1). Having isolated 334 colonies that activated expression of both reporter genes (LacZ+/His+), we investigated whether activation of the lacZ reporter gene was dependent on the GAL4 AD-ICP27 protein hybrid. Thus, transformants were grown in rich medium without selection, causing some cells to lose the pAD27 plasmid, followed by growth on S.D. medium lacking uracil to select for those cells that retained the library plasmid but had lost the pAD27 plasmid. These colonies were analyzed for loss of β-gal activity as compared with the initially isolated LacZ+/His+ transformants (which contained all three hybrid components). Of the 334 transformants analyzed, 113 showed β-gal activity in the absence of the GAL4 AD-ICP27 protein hybrid (Fig. 1), suggesting that they activated the lacZ reporter gene in a ICP27-independent manner. Similar RNA activators were reported in a screen of a yeast DNA library (48Sengupta D. Wickens M. Fields S. RNA. 1999; 5: 596-601Crossref PubMed Scopus (60) Google Scholar), in which 84% of the LacZ+/His+ transformants showed this property as compared with 34% observed here. The remaining 221 transformants, which showed loss of β-gal activity upon loss of the GAL4-ICP27 protein hybrid, were then mated with R40 (opposite mating type of L40), pre-transformed with plasmids pAD27, pADIRP1 (expressing a GAL4 AD-IRP1 protein hybrid), or pACTII-CAN (expressing the GAL4 AD alone). Analysis of β-gal activity by the filter lift assay showed that 68 single transformants had a restored activation of the lacZ gene upon reintroduction of the GAL4 AD-ICP27 protein hybrid, but not upon introduction of a GAL4 AD-IRP1 protein hybrid or the GAL4 AD alone (Fig. 1). The results suggested that 68 transformants expressed ICP27-interacting RNAs, but, mainly because of the qualitative nature of the filter lift assay, these RNAs were considered as only candidate ICP27-specific interactors at this stage. The library plasmids of the 68 transformants were isolated, and the DNA insert in each plasmid was sequenced. Computer searches of the HSV-1 strain 17+ genome and sequence analysis revealed that 31 of the isolated library plasmids encoded single-insert RNAs that were expressed in the same orientation as HSV-1 transcribed genes. Among these, two sets of RNAs corresponding to two different genomic regions were isolated more than once: clones 324/16.2 and 19/85/185 (see Fig. 2 and Table I). Furthermore, five sets of RNAs overlapped the same genomic region but were of different lengths: 251 and 19/85/185, 352 and 75, 17 and 247, 4 and 242, and 5 and 231 (see Fig. 2 and Table I). Comparable with the r

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