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A Role of RNA Helicase A in cis-Acting Transactivation Response Element-mediated Transcriptional Regulation of Human Immunodeficiency Virus Type 1

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.m006892200

ISSN

1083-351X

Autores

Ryouji Fujii, Mika Okamoto, Satoko Aratani, Takayuki Oishi, Takayuki Ohshima, Kazunari Taira, Masanori Baba, Akiyoshi Fukamizu, Toshihiro Nakajima,

Tópico(s)

RNA Interference and Gene Delivery

Resumo

RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHAK236E) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA. RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHAK236E) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA. RNA helicase A cAMP-responsive element-binding protein CREB-binding protein RNA polymerase II double-stranded RNA-binding domains human immunodeficiency virus, type 1 long terminal repeat cis-acting transactivation response element Rev response element human embryonic kidney hemagglutinin glutathione S-transferase positive transcription elongation factor b kilobase pair wild type TAR-binding protein polymerase chain reaction single-stranded RNA dsRNA-activated protein kinase Arg-Gly-Gly nuclear factor-κB Rous sarcoma virus RNA helicase A (RHA)1catalyzes the unwinding of duplex RNA and DNA in a process coupled with the hydrolysis of NTPs (1Zhang S.S. Grosse F. J. Biol. Chem. 1991; 266: 20483-20490Abstract Full Text PDF PubMed Google Scholar, 2Zhang S. Grosse F. Biochemistry. 1994; 33: 3906-3912Crossref PubMed Scopus (104) Google Scholar). We have previously shown that RHA mediates association of the CREB-binding protein (CBP) with RNA polymerase II (pol II) (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar) and that RHA links breast cancer-specific tumor suppressor protein (BRCA1) to pol II (4Anderson S.F. Schlegel B.P. Nakajima T. Wolpin E.S. Parvin J.D. Nat. Genet. 1998; 19: 254-256Crossref PubMed Scopus (346) Google Scholar). RHA consists of two type A double-stranded RNA-binding domains (dsRBDs) (5Gibson T.J. Thompson J.D. Nucleic Acids Res. 1994; 22: 2552-2556Crossref PubMed Scopus (90) Google Scholar, 6St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10979-10983Crossref PubMed Scopus (491) Google Scholar), a classical Walker type NTP-binding site, a DEAH/D helicase domain, and a single-stranded nucleic acid-binding domain characterized by Arg-Gly-Gly (RGG) repeats (7Zhang S. Grosse F. J. Biol. Chem. 1997; 272: 11487-11494Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Trypsin-digested RHA, lacking both dsRBDs and the RGG repeat sequence, has reduced helicase activity, implicating these domains in the unwinding function of RHA (7Zhang S. Grosse F. J. Biol. Chem. 1997; 272: 11487-11494Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Amino acids 1–262 and 255–664 of RHA have proved to be CBP- and pol II-binding sites, respectively (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). RHA shuttles between nucleus and cytoplasm with a cis-acting constitutive transport element in simian retroviruses (8Tang H. Gaietta G.M. Fischer W.H. Ellisman M.H. Wong-Staal F. Science. 1997; 276: 1412-1415Crossref PubMed Scopus (133) Google Scholar). It has been proposed that RHA is necessary for releasing both constitutive transport element- and HIV-1 Rev response element (RRE)-containing RNA from spliceosomes prior to the completion of splicing (9Li J. Tang H. Mullen T.M. Westberg C. Reddy T.R. Rose D.W. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 709-714Crossref PubMed Scopus (129) Google Scholar). After integration of HIV-1 into the host genome, the nuclear factor-κB (NF-κB) binds to enhancer elements in the HIV-1 long terminal repeat (LTR) and stimulates the expression of the viral genome in a signal-dependent manner (10Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1580) Google Scholar, 11Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (480) Google Scholar). HIV-1 expression can be divided into two phases (early and late). In the early phase, the majority of viral mRNA is multiply spliced to produce 2-kb transcripts that encode the regulatory proteins, including Tat and Rev, necessary to activate HIV-1 expression. A transition subsequently occurs to accumulate singly spliced (4-kb) and full-length (9-kb) transcripts encoding the viral structural proteins and providing the genomic RNA (12Muesing M.A. Smith D.H. Cabradilla C.D. Benton C.V. Lasky L.A. Capon D.J. Nature. 1985; 313: 450-458Crossref PubMed Scopus (477) Google Scholar, 13Kim S.Y. Byrn R. Groopman J. Baltimore D. J. Virol. 1989; 63: 3708-3713Crossref PubMed Google Scholar, 14Butera S.T. Roberts B.D. Lam L. Hodge T. Folks T.M. J. Virol. 1994; 68: 2726-2730Crossref PubMed Google Scholar). Regulation of the viral RNA-splicing transition mechanism is reported to involve the protection and transport of full-length HIV-1 RNA by Rev (15Felber B.K. Hadzopoulou-Cladaras M. Cladaras C. Copeland T. Pavlakis G.N. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1495-1499Crossref PubMed Scopus (541) Google Scholar, 16Malim M.H. Hauber J. Le S.Y. Maizel J.V. Cullen B.R. Nature. 1989; 338: 254-257Crossref PubMed Scopus (1024) Google Scholar). TAR, a nascent viral leader RNA transcribed from the R region of the LTR, plays an important role in HIV-1 gene expression and forms a unique stem and loop structure (17Jeang K.T. Xiao H. Rich E.A. J. Biol. Chem. 1999; 274: 28837-28840Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 18Taube R. Fujinaga K. Wimmer J. Barboric M. Peterlin B.M. Virology. 1999; 264: 245-253Crossref PubMed Scopus (117) Google Scholar). Tat function is mediated by the TAR RNA and requires the recruitment of a complex consisting of Tat and cyclin T1 component of positive transcription elongation factor b (P-TEFb) bound to TAR (19Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1058) Google Scholar). A defect of Tat-induced transactivation in murine cells was attributed to the lack of a functional cyclin T1 (20Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (386) Google Scholar,21Bieniasz P.D. Grdina T.A. Bogerd H.P. Cullen B.R. EMBO J. 1998; 17: 7056-7065Crossref PubMed Scopus (235) Google Scholar). Alternatively, the defect was linked to the reduced abundance of p300 and p300/CBP-associated factor (22Benkirane M. Chun R.F. Xiao H. Ogryzko V.V. Howard B.H. Nakatani Y. Jeang K.T. J. Biol. Chem. 1998; 273: 24898-24905Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). In addition, several cellular cofactors play crucial roles in HIV-1 gene expression through binding to the TAR RNA. For instance, TRBP consists of two type A dsRBDs and another type B dsRBD and activates the HIV-1 LTR (6St Johnston D. Brown N.H. Gall J.G. Jantsch M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10979-10983Crossref PubMed Scopus (491) Google Scholar, 23Gatignol A. Buckler-White A. Berkhout B. Jeang K.T. Science. 1991; 251: 1597-1600Crossref PubMed Scopus (343) Google Scholar) through binding of the second type A dsRBD (dsRBD2) to the TAR RNA (24Gatignol A. Buckler C. Jeang K.T. Mol. Cell. Biol. 1993; 13: 2193-2202Crossref PubMed Scopus (118) Google Scholar). PKR contains two type A dsRBDs and functions as a cellular antiviral factor by inhibiting eukaryotic initiation factor 2 via phosphorylation of its α-subunit (25Chong K.L. Feng L. Schappert K. Meurs E. Donahue T.F. Friesen J.D. Hovanessian A.G. Williams B.R. EMBO J. 1992; 11: 1553-1562Crossref PubMed Scopus (292) Google Scholar). A similar function of PKR is mediated by an interaction with several kinds of RNA, leading to PKR autophosphorylation (26Samuel C.E. Virology. 1991; 183: 1-11Crossref PubMed Scopus (553) Google Scholar). Like TRBP and PKR, RHA possesses dsRBDs and may act as a cellular transcriptional regulator. Of particular interest is whether RHA binds to the TAR RNA and influences HIV-1 gene expression. This study shows that RHA acts as a novel TAR-binding cellular cofactor and enhances HIV-1 LTR-directed gene expression and viral production in vivo. Human embryonic kidney (HEK) 293 (27Graham F.L. Smiley J. Russell W.C. Nairn R. J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3607) Google Scholar) and HeLa cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% fetal bovine serum (Sigma), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. The cells were grown at 37 °C in a humidified 95% air, 5% CO2 atmosphere. Plasmid pHyg LTR-Luc encodes a chimeric gene consisting of the HIV-1 LTR containing two intact κB elements and a luciferase gene (28Koseki S. Ohkawa J. Yamamoto R. Takebe Y. Taira K. J. Controlled Release. 1998; 53: 159-173Crossref PubMed Scopus (34) Google Scholar). The mNFκB LTR-Luc plasmid was constructed by inserting both of the κB element-mutated LTR fragments (14Butera S.T. Roberts B.D. Lam L. Hodge T. Folks T.M. J. Virol. 1994; 68: 2726-2730Crossref PubMed Google Scholar) into theSmaI/HindIII site of PGV-B containing a luciferase gene without a promoter (Toyo-Inki). The pCD-SRα/tat plasmid, a mammalian expression vector for HIV-1 Tat, was constructed as described previously (28Koseki S. Ohkawa J. Yamamoto R. Takebe Y. Taira K. J. Controlled Release. 1998; 53: 159-173Crossref PubMed Scopus (34) Google Scholar). The HIV-1 pNL4–3 proviral DNA clone (30Adachi A. Gendelman H.E. Koenig S. Folks T. Willey R. Rabson A. Martin M.A. J. Virol. 1986; 59: 284-291Crossref PubMed Google Scholar) was obtained from the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, National Institutes of Health) and was contributed by Malcolm Martin. Wild-type (wt) RHA and RHAmATP constructs were prepared as described previously (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). The RHAmATP and RHAW339A 2S. Aratani, R. Fujii, T. Oishi, H. Fujita, T. Amano, T. Ohshima, M. Hagiwara, A. Fukamizu, and T. Nakajima, manuscript in preparation. constructs contain single amino acid substitutions leading to defects in ATPase/helicase activity and pol II binding ability of RHA, respectively. RHAK236E containing the lysine to glutamate substitution at residue 236 was generated by PCR. The wt RHA construct and three RHA mutants were tagged by hemagglutinin (HA) for Western blot assays and immunoprecipitation studies. To obtain the RNA probes by in vitro transcription, the pcTAR and ΔloopTAR plasmids were constructed by inserting PCR-generated cDNA fragments (5′-GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCC-3′ and 5′-GGGTCTCTCTGGTTAGACCAGATCTGAGCGCTCTCTGGCTAACTAGGGAACCC-3′) withHindIII linker into the HindIII site of pcDNA3 (Invitrogen). The plasmids as described above were purified using cesium chloride gradients. A series of RHA polypeptides (1–262, 255–664, 649–1077, and 1064–1270) fused to glutathione S-transferase (GST) was described previously (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). RHA-(1–90), RHA-(76–174), RHA-(160–262), RHA-(1–262/Δ235–249), RHA-(1–262/K236E), and full-length TRBP were created by PCR. These fragments were cloned into pGEX-5X-1 (Amersham Pharmacia Biotech), and all of the PCR-generated plasmids were confirmed by sequence analysis. HEK 293 and HeLa cells were transiently transfected with 100 ng of pHyg LTR-Luc or mNFκB-Luc reporter, 100 ng of RSV-β-gal control plasmid, 2 or 10 ng pCD-SRα/tat, and various amounts of RHA, using the calcium phosphate method as described previously (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.30-16.37Google Scholar). To ensure an equal amount of DNA, "empty plasmids" were added in each transfection. In the reporter gene assays, luciferase activity derived from expression of the pHyg LTR-Luc and mNFκB LTR-Luc reporter plasmids was normalized to β-galactosidase activity from cotransfected Rous sarcoma virus (RSV)-expression plasmid containing the β-galactosidase gene (RSV-β-gal) (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). Luciferase activity was measured with AutoLumat (Berthold). All experiments were performed in triplicate, and all results were obtained from at least three separate experiments. Equivalent expression of RHA constructs was verified by Western blot analysis using the anti-HA antibody 12CA5 (Roche Molecular Biochemicals). Total cellular extract fromEscherichia coli BL21, expressing a series of RHA polypeptides or full-length TRBP fused to GST protein, was electrophoresed on an SDS gel and transferred to a polyvinylidene difluoride membrane (Immobilon P, Millipore). TAR binding assays were performed as reported previously (23Gatignol A. Buckler-White A. Berkhout B. Jeang K.T. Science. 1991; 251: 1597-1600Crossref PubMed Scopus (343) Google Scholar). Filters were incubated in binding buffer (20 mm HEPES, pH 7.3, 40 mm KCl, 1.5 mm MgCl2, and 1 mmdithiothreitol) with 10 μg/ml yeast RNA, 10 μg/ml calf thymus DNA, and 200 fmol/ml gel-purified 32P-labeled TAR RNA probe. Filters were washed with binding buffer and then exposed to x-ray film. Comparable expression of GST-fused protein was determined by Western blotting with an anti-GST antibody (Amersham Pharmacia Biotech) and Coomassie Brilliant Blue staining. Wild-type TAR, Δloop TAR RNA, and ssRNA were obtained by in vitro transcription using BamHI-cleaved pcTAR or ΔloopTAR plasmid and XhoI-cleaved pcDNA3, respectively. GST and RHA-(1–262) polypeptide blots were incubated in binding buffer with 10 μg/ml calf thymus DNA, 1 pmol/ml32P-labeled wt TAR, Δloop TAR RNA or ssRNA probe, and 300 pmol/ml unlabeled RNA or yeast tRNA (Sigma) as a competitor. The filters were washed with binding buffer and exposed to x-ray film. HEK 293 cells were transfected with 3 μg of pHyg LTR-Luc or G5b-Luc (32Yoshida E. Aratani S. Itou H. Miyagishi M. Takiguchi M. Osumu T. Murakami K. Fukamizu A. Biochem. Biophys. Res. Commun. 1997; 241: 664-669Crossref PubMed Scopus (97) Google Scholar), 150 ng of pCD-SRα/tat or 300 ng Gal4-VP16 (33Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (684) Google Scholar), and 3 μg of each RHA construct. After 24 h of transfection, the cells were lysed in 1 ml of 0.65% Nonidet P-40 lysis buffer (20 mm HEPES, pH 7.3, 150 mm KCl, 1.5 mm MgCl2, 0.65% Nonidet P-40). Cell lysates were pre-cleared with 5 μg of normal mouse IgG (Santa Cruz Biotechnology) conjugated to protein G-Sepharose beads (Amersham Pharmacia Biotech) for 1 h at 4 °C. After a brief centrifugation, the lysates were mixed with 5 μg of normal mouse IgG or 5 μg of anti-HA antibody conjugated to protein G-Sepharose beads. After an overnight incubation at 4 °C, the beads were washed three times with lysis buffer. RNA was extracted from the beads with ISOGEN (Nippon Gene) and then blotted onto GeneScreen Plus membrane (PerkinElmer Life Sciences) using a slot blotting apparatus (HYBRI-SLOT MANIFOLD, Life Technologies, Inc.). The membrane was hybridized with a 32P-labeled luciferase gene probe generated by PCR using the primer pair 5′-GGATGGAACCGCTGGAGAG-3′ and 5′-GTTTCATAGCTTCTGCCAACCG-3′ and exposed to x-ray film. Equivalency for immunoprecipitation of HA-tagged RHA was verified by Western blot analysis. HEK 293 cells were transfected with 10 ng of pNL4-3, 20 ng of PGV-C control plasmid containing the SV40-luciferase gene (Toyo-inki, Tokyo Japan), and various amounts of RHA construct. To ensure an equal amount of DNA, empty plasmid was added in each transfection. After 24 and 48 h of transfection, culture supernatants were collected and tested for HIV-1 p24 antigen levels using a p24 antigen detection kit (Retro-tek, Zepto Metrix Corporation, Buffalo, NY). Equivalent transfection efficiency was verified by luciferase activity derived from the cotransfected PGV-C control plasmid. All experiments were performed in triplicate. HEK 293 cells were transfected with 500 ng of pNL4-3, 1 μg of PGV-C control plasmid, and 5 μg of each RHA construct. After 24 h of transfection, polyadenylated RNA was extracted from the transfected cells using an mRNA purification kit (Amersham Pharmacia Biotech), run on a 0.8% agarose gel, and blotted onto GeneScreen Plus membrane. The membrane was hybridized with a32P-labeled HIV-1 LTR probe and then exposed to x-ray film. The probe was amplified by PCR using the primers 5′-AGTGCTCAAAGTAGTGTGTG-3′ and 5′-GATCTCCTCTGGCTTTACTTT-3′ and pNL4-3 as a template. Equivalent transfection efficiency was determined by measuring the amount of luciferase mRNA derived from the cotransfected PGV-C control plasmid. To assess the regulatory effects of RHA on HIV-1 LTR-directed gene expression, several human cell lines were transfected with the pHyg LTR-Luc reporter plasmid. In HEK 293 cells, wt RHA markedly enhanced Tat-induced reporter activity in a dose-dependent manner (Fig. 1 A). In contrast, overexpression of wt RHA did not affect HIV-1 LTR-directed luciferase activity in HeLa cells (Fig. 1 B) as described previously (9Li J. Tang H. Mullen T.M. Westberg C. Reddy T.R. Rose D.W. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 709-714Crossref PubMed Scopus (129) Google Scholar). Northwestern assays were conducted to determine whether RHA binds to the TAR RNA. First, we divided full-length RHA into four fragments (Fig. 2 A) and examined each fragment for their TAR RNA binding activity. Only RHA-(1–262), which contains both dsRBD1 and dsRBD2, could bind to the TAR RNA (Fig. 2 B, left panel). Second, we constructed three deletion mutants of RHA-(1–262) (Fig. 2 A). Unexpectedly, only RHA-(160–262) (containing dsRBD2) and not RHA-(1–90) (containing dsRBD1) bound to the TAR RNA (Fig. 2 B, middle panel). Amino acids 235–249 in the RHA dsRBD2 are well conserved among the dsRNA-binding protein family (Fig. 2 C). In particular, the lysine at residue 211 (Lys-211) in the TRBP dsRBD2 is critical for binding to the TAR RNA (34Erard M. Barker D.G. Amalric F. Jeang K.T. Gatignol A. J. Mol. Biol. 1998; 279: 1085-1099Crossref PubMed Scopus (33) Google Scholar). Therefore, we constructed two mutants (RHA-(1–262/Δ235–249) and RHA-(1–262/K236E)) that contain the intact dsRBD1 and the mutated dsRBD2. RHA-(1–262/Δ235–249) is a deletion mutant missing amino acids 235–249 from RHA-(1–262). RHA-(1–262/K236E) is also an RHA-(1–262) mutant with lysine at residue 236 (Lys-236) substituted with glutamate. The Lys-236 of RHA corresponds to the Lys-211 of TRBP. Both RHA-(1–262) mutants failed to interact with the TAR RNA (Fig. 2 B, right panel), indicating that amino acids 235–249 and in particular residue Lys-236 are essential for TAR binding in vitro. HIV Tat and cyclin T1 recognize the bulge and loop of TAR RNA, respectively (20Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (386) Google Scholar). Similarly, it is important to define the region of TAR that interacts with the RHA dsRBD and to confirm its relative specificity. Presumably, RHA dsRBD binds to the stem region of TAR RNA, since dsRBD binds to dsRNA. The Δloop TAR RNA probe illustrated in Fig. 3 A was therefore constructed. GST-fused RHA-(1–262) and GST alone (negative control) were blotted onto filters, and the filters were incubated with an equivalent amount of 32P-labeled wt TAR, Δloop TAR RNA, or ssRNA. Interestingly, the binding affinity of Δloop TAR RNA to RHA-(1–262) was significantly stronger than that of wt TAR RNA. ssRNA bound weakly to RHA-(1–262). None of the tested probes bound to GST (Fig. 3 B). In competition assay (Fig. 3 C), the binding of wt TAR RNA to RHA-(1–262) was almost completely inhibited by wt TAR or Δloop TAR RNA competitor at a concentration of 300-fold higher than that of the 32P-labeled probe. In contrast, the ssRNA competitor and yeast tRNA slightly reduced the probe binding. These results suggest that the RHA dsRBD preferably recognizes the stem of the TAR RNA. To test for in vivo interaction of the TAR RNA with the RHA complex, coimmunoprecipitation assays were performed on whole cell lysates from HEK 293 cells cotransfected with pHyg LTR-Luc (to express TAR-containing luciferase mRNA) or G5b-Luc (to express TAR-negative luciferase mRNA) and each RHA construct. Except for "no-transfectant," a comparable amount of TAR-containing and TAR-negative mRNA was detected in each cell lysate (Fig.4, middle panel). Equal amounts of wt RHA and RHAK236E were precipitated with anti-HA antibody (Fig. 4, lower panel). The TAR-containing mRNA was present in the wt RHA precipitant (Fig. 4, upper panel). However, although significantly less mRNA was coprecipitated with RHAK236E. No precipitant was identified in the negative controls (no-transfectant and "mock-transfectant"). Furthermore, the association of wt RHA with the TAR-negative luciferase mRNA was as weak as that of RHAK236E and the TAR-containing luciferase mRNA. These results indicate that the TAR-containing mRNA existed in the RHA complex in vivoand that the complex was formed primarily via the binding of RHA dsRBD2 to the TAR RNA. Although RHA-(1–262/K236E) could not bind to the TAR RNA in vitro (Fig. 2 B), the TAR-containing mRNA was weakly associated with RHAK236E complexin vivo. Thus, it cannot be excluded that RHA also binds to mRNA in a TAR-independent fashion, as in the case of RHA binding to ssRNA through the RGG motif (7Zhang S. Grosse F. J. Biol. Chem. 1997; 272: 11487-11494Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The finding that RHA binds to the TAR RNA in vivoprompted a study of the role of RHA in TAR-mediated gene expression. Transient transfections of the pHyg LTR-Luc reporter plasmid were conducted to analyze the effect of RHAK236E on HIV-1 LTR-directed gene expression. RHAK236E, but neither RHAmATP nor RHAW339A, enhanced the reporter activity (Fig. 5, lanes 7–12), although the effect of RHAK236E was significantly lower than that of wt RHA (∼40%). This reduced effect of RHAK236E may be due to the lack of TAR-binding ability. Alternatively, RHA may be functionally bound to the CBP·NF-κB complex on the κB elements of HIV-1 LTR (35Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (668) Google Scholar,36Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (724) Google Scholar). To exclude this possibility, transient transfection assays with the mNFκB LTR-Luc reporter plasmid that contained two mutated κB elements were also performed. Mutation of the κB elements strongly reduced luciferase activity (Fig. 5, lane 4 versus 16), as described previously (11Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (480) Google Scholar). Wild-type RHA markedly enhanced the Tat-induced reporter activity in a dose-dependent manner (Fig. 5, lanes 16–18), and although both RHAmATP and RHAW339A slightly increased the luciferase activity, RHAK236E had no effect (Fig. 5, lanes 19–24). These results indicate that the association of RHA with the TAR RNA is required for the RHA-induced transactivation in the mutated κB LTR. Conversely, the higher activity of RHAK236E in intact κB LTR compared with mutated κB LTR suggests that RHA can also interact with HIV-1 LTR through the intact κB elements in a TAR-independent fashion. To elucidate the role of TAR-binding ability of RHA in HIV-1 viral replication, HEK 293 cells were cotransfected with pNL4-3 and different amounts of wt RHA or RHAK236E (Fig.6 A). After 24 h of transfection, HIV-1 p24 production was enhanced ∼5-fold in the wt RHA transfectants in a dose-dependent manner (Fig. 6 A, left panel), as previously demonstrated in HeLa cells (9Li J. Tang H. Mullen T.M. Westberg C. Reddy T.R. Rose D.W. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 709-714Crossref PubMed Scopus (129) Google Scholar). The effect of RHAK236E on HIV-1 p24 production was significantly less than that of wt RHA (∼30%), which was consistent with the results in the reporter gene assays (Fig. 5). Similar results were also obtained after 48 h of transfection, except in the cells expressing for higher p24 antigen levels (Fig. 6 A,right panel). These results suggest that the association of RHA with the TAR RNA is partly required for the RHA-enhanced HIV-1 gene expression. Finally, to elucidate the role of RHA in transcriptional regulation of HIV-1, HEK 293 cells were transfected with pNL4-3 and each RHA construct. Fig. 6 B demonstrates that three different lengths of HIV-1 mRNA (∼2-, 4-, and 9-kb transcripts) were detected by Northern blot assay. Equivalent transfection efficiency was confirmed by the amount of luciferase mRNA derived from each cotransfected PGV-C control plasmid (data not shown). The amounts of all HIV-1 mRNA transcripts were increased by wt RHA coexpression (Fig.6 B). No other RHA mutants affected HIV-1 mRNA synthesis or p24 production. These results strongly support a functional role of RHA in the transcriptional activation of HIV-1. RHA belongs to the dsRNA-binding protein family, which includes TRBP and PKR. These proteins display variable RNA-binding characteristics. For instance, the PKR dsRBD1 displays higher affinity than the PKR dsRBD2 for several kinds of RNA, including the TAR RNA (37Green S.R. Mathews M.B. Genes Dev. 1992; 6: 2478-2490Crossref PubMed Scopus (220) Google Scholar, 38McCormack S.J. Thomis D.C. Samuel C.E. Virology. 1992; 188: 47-56Crossref PubMed Scopus (160) Google Scholar). The TRBP dsRBD2 alone can bind to TAR RNA independent of other dsRBDs (39Daviet L. Erard M. Dorin D. Duarte M. Vaquero C. Gatignol A. Eur. J. Biochem. 2000; 267: 2419-2431Crossref PubMed Scopus (45) Google Scholar). Two polypeptides containing each of the RHA dsRBDs bind to poly(rI·rC) dsRNA with similar affinity, and it has been considered that the two RHA dsRBDs cooperate to interact with dsRNA, such as with poly(rI·rC) (7Zhang S. Grosse F. J. Biol. Chem. 1997; 272: 11487-11494Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The results presented here show that only dsRBD2, and not dsRBD 1, could bind to TAR RNA in vitro(Fig. 2 B). This RNA-binding property of RHA is similar to that of TRBP. Gel mobility shift assays with polypeptides containing each or both RHA dsRBD were used to confirm the contribution of dsRBD1 in binding to the TAR RNA. This approach was unsuccessful due to aggregation of purified dsRBD polypeptide (data not shown). The dsRBDs interact with highly structured RNA and dsRNA, generally without obvious RNA sequence specificity (40Fierro-Monti I. Mathews M.B. Trends Biochem. Sci. 2000; 25: 241-246Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar). In fact, RHA could bind to various RNA species, including ssRNA in vitro. Like TRBP (24Gatignol A. Buckler C. Jeang K.T. Mol. Cell. Biol. 1993; 13: 2193-2202Crossref PubMed Scopus (118) Google Scholar), RHA bound to the TAR RNA with higher affinity than to ssRNA or yeast tRNA and preferably bound to TAR-containing mRNA rather than TAR-negative mRNA. Thus, it appears that TAR RNA is one of the binding targets of RHA dsRBDs. These findings further demonstrate that the conserved amino acids 235–249 in the RHA dsRBD2 are essential for TAR binding. It was previously reported that the Lys-211 residue of TRBP dsRBD2 is important for interaction with TAR RNA (34Erard M. Barker D.G. Amalric F. Jeang K.T. Gatignol A. J. Mol. Biol. 1998; 279: 1085-1099Crossref PubMed Scopus (33) Google Scholar). It was confirmed by x-ray crystallography that the corresponding residue of Xlrbpa, the Xenopus homologue of TRBP, directly interacts with dsRNA (41Ryter J.M. Schultz S.C. EMBO J. 1998; 17: 7505-7513Crossref PubMed Scopus (397) Google Scholar). These residues correspond to the Lys-236 residue of RHA dsRBD2, and the importance of Lys-236 was demonstrated here the using a RHA-(1–262/K236E) mutant. Together, the results confer in that the characteristics of RHA for TAR-binding are similar to those of TRBP. It is known that transcription factor levels differ between cell lines. For instance, murine NIH3T3 cells express a small amount of p300 compared with HeLa cells. Thus, the failure of HIV-1 LTR-directed transactivation by Tat in the murine cells could be attributed to the small amount of p300 (22Benkirane M. Chun R.F. Xiao H. Ogryzko V.V. Howard B.H. Nakatani Y. Jeang K.T. J. Biol. Chem. 1998; 273: 24898-24905Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). Western blot analysis showed that the amount of RHA in HEK 293 cells was less than 50% that in HeLa cells, although an equivalent amount of CBP or CREB was identified in both cell lines. 3S. Aratani and T. Nakajima, unpublished observations. The HIV-1 LTR-directed luciferase activity was enhanced by exogenous RHA in HEK 293 cells but not in HeLa cells. HEK 293 cells are widely used in transfection experiments with HIV-1 proviral DNA clones (29Ulich C. Dunne A. Parry E. Hooker C.W. Gaynor R.B. Harrich D. J. Virol. 1999; 73: 2499-2508Crossref PubMed Google Scholar, 42Natarajan V. Radjendirane V. Salzman N.P. Virology. 1993; 196: 122-129Crossref PubMed Scopus (4) Google Scholar). Taken together, the results indicate HEK 293 cells to be a favorable host cell system to assess the functions of RHA in HIV-1 gene expression by transient transfection. RHA was reported to bind weakly to HIV-1 RRE and be involved in post-transcriptional regulation of HIV-1 (9Li J. Tang H. Mullen T.M. Westberg C. Reddy T.R. Rose D.W. Wong-Staal F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 709-714Crossref PubMed Scopus (129) Google Scholar). However, the reporter gene constructs used in this study do not contain RRE, and the results in the reporter gene assays accorded well with those in the p24 assays, suggesting that the reduced activity of RHAK236E in the p24 assays was due primarily to the lack of the TAR-binding ability of RHA in HEK 293 cells. This discrepancy may be due to either the different cell types used or to the distinct roles of RHA in transcription and post-transcriptional regulation in HIV-1 gene expression. We have previously suggested that both the ATPase/helicase activity and pol II binding ability of RHA contribute to the CREB-dependent transcription (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). It can be proposed from the current studies that these functions of RHA may also be important for HIV-1 transcription. Moreover, the data demonstrate that RHA enhances HIV-1 LTR-directed gene expression in a κB element-dependent and -independent fashion. NF-κB functionally binds to the κB elements in the HIV-1 LTR (10Nabel G. Baltimore D. Nature. 1987; 326: 711-713Crossref PubMed Scopus (1580) Google Scholar, 11Griffin G.E. Leung K. Folks T.M. Kunkel S. Nabel G.J. Nature. 1989; 339: 70-73Crossref PubMed Scopus (480) Google Scholar) and associates with CBP (35Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (668) Google Scholar, 36Gerritsen M.E. Williams A.J. Neish A.S. Moore S. Shi Y. Collins T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2927-2932Crossref PubMed Scopus (724) Google Scholar). Furthermore, RHA was shown to mediate the association of CBP with pol II (3Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (468) Google Scholar). The RHAK236E mutant was capable of binding to CBP in GST pull-down assays (data not shown). Therefore, the certain activity of RHAK236E in the reporter assays using intact κB elements but not in the assays using the mutated κB elements may indicate that RHA also mediates the association of CBP with pol II on the κB-dependent HIV-1 preinitiation complex. Conversely, it was shown that RHA enhances HIV-1 gene expression, at least in part, through its TAR binding. TAR RNA is required for the recruitment of a complex consisting of Tat and the cyclin T1 component of P-TEFb (19Wei P. Garber M.E. Fang S.M. Fischer W.H. Jones K.A. Cell. 1998; 92: 451-462Abstract Full Text Full Text PDF PubMed Scopus (1058) Google Scholar, 20Garber M.E. Wei P. KewalRamani V.N. Mayall T.P. Herrmann C.H. Rice A.P. Littman D.R. Jones K.A. Genes Dev. 1998; 12: 3512-3527Crossref PubMed Scopus (386) Google Scholar, 21Bieniasz P.D. Grdina T.A. Bogerd H.P. Cullen B.R. EMBO J. 1998; 17: 7056-7065Crossref PubMed Scopus (235) Google Scholar). It is possible that both the ATPase/helicase activity and pol II binding ability of RHA may be required for the unwinding of highly structured RNA, such as TAR RNA, and following HIV-1 transcription. We thank Yukiko Okada and Megumi Fujita for technical assistance.

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