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Picornavirus Genome Replication

2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês

10.1074/jbc.m610608200

ISSN

1083-351X

Autores

Harsh B. Pathak, Jamie J. Arnold, P. Wiegand, Michele R.S. Hargittai, Craig E. Cameron,

Tópico(s)

Animal Virus Infections Studies

Resumo

All picornaviruses have a protein, VPg, covalently linked to the 5′-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication. All picornaviruses have a protein, VPg, covalently linked to the 5′-ends of their genomes. Uridylylated VPg (VPg-pUpU) is thought to serve as the protein primer for RNA synthesis. VPg-pUpU can be produced in vitro by the viral polymerase, 3Dpol, in a reaction in which a single adenylate residue of a stem-loop structure, termed oriI, templates processive incorporation of UMP into VPg by using a "slide-back" mechanism. This reaction is greatly stimulated by viral precursor protein 3CD or its processed derivative, 3C; both contain RNA-binding and protease activities. We show that the 3C domain encodes specificity for oriI, and the 3D domain enhances the overall affinity for oriI. Thus, 3C(D) stimulation exhibits an RNA length dependence. By using a minimal system to evaluate the mechanism of VPg uridylylation, we show that the active complex contains polymerase, oriI, and 3C(D) at stoichiometry of 1:1:2. Dimerization of 3C(D) is supported by physical and structural data. Polymerase recruitment to and retention in this complex require a protein-protein interaction between the polymerase and 3C(D). Physical and functional data for this interaction are provided for three picornaviruses. VPg association with this complex is weak, suggesting that formation of a complex containing all necessary components of the reaction is rate-limiting for the reaction. We suggest that assembly of this complex in vivo would be facilitated by use of precursor proteins instead of processed proteins. These data provide a glimpse into the organization of the ribonucleoprotein complex that catalyzes this key step in picornavirus genome replication. Picornaviruses are the etiologic agent of numerous diseases of medical and veterinary importance. Poliomyelitis, the common cold, summer flu, hepatitis, and foot-and-mouth disease can all be caused by picornaviruses (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). These viruses have a single-stranded RNA genome of positive polarity that is on the order of 7500 nt 3The abbreviations used are: nt, nucleotide(s); PV, poliovirus; CVB3, coxsackievirus B3; HRV14, human rhinovirus type 14; HRV16, human rhinovirus type 16; SLd, stem-loop d; β-ME, β-mercaptoethanol; ATPαS, adenosine 5′-O-(1-thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.3The abbreviations used are: nt, nucleotide(s); PV, poliovirus; CVB3, coxsackievirus B3; HRV14, human rhinovirus type 14; HRV16, human rhinovirus type 16; SLd, stem-loop d; β-ME, β-mercaptoethanol; ATPαS, adenosine 5′-O-(1-thiotriphosphate); Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. in length (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). A protein, VPg (virion protein genome-linked), is covalently linked to the 5′-end of the viral genome, the so-called plus-strand, and a poly(rA) tail is present at the 3′-end (1.Racaniello V.R. Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. 4th Ed. Vol. 1. Lippincott-Raven Publishers, Philadelphia, PA2001: 685-722Google Scholar). Genome replication occurs in a process that uses the plus-strand as a template for minus-strand synthesis, which, in turn, is used as a template for production of an excess of plus-strands (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). Initiation of both plus- and minus-strand RNA synthesis is thought to be primed by a uridylylated form of VPg, VPg-pUpU (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). Several years ago, Paul and Wimmer made the paradigm-shifting observation that an RNA stem-loop structure in the 2C-coding region of the poliovirus (PV) genome was capable of templating production of VPg-pUpU (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (236) Google Scholar, 4.Rieder E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10371-10380Crossref PubMed Scopus (127) Google Scholar) much more efficiently than the poly(rA) tail (5.Paul A.V. van Boom J.H. Filippov D. Wimmer E. Nature. 1998; 393: 280-284Crossref PubMed Scopus (297) Google Scholar). Since this time, it has become clear that all picornaviruses appear to use a similar strategy for production of VP-pUpU (6.Gerber K. Wimmer E. Paul A.V. J. Virol. 2001; 75: 10979-10990Crossref PubMed Scopus (81) Google Scholar, 7.Mason P.W. Bezborodova S.V. Henry T.M. J. Virol. 2002; 76: 9686-9694Crossref PubMed Scopus (120) Google Scholar, 8.Yang Y. Rijnbrand R. McKnight K.L. Wimmer E. Paul A. Martin A. Lemon S.M. J. Virol. 2002; 76: 7485-7494Crossref PubMed Scopus (67) Google Scholar, 9.Paul A.V. Yin J. Mugavero J. Rieder E. Liu Y. Wimmer E. J. Biol. Chem. 2003; 278: 43951-43960Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 10.Yang Y. Rijnbrand R. Watowich S. Lemon S.M. J. Biol. Chem. 2004; 279: 12659-12667Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 11.Nayak A. Goodfellow I.G. Belsham G.J. J. Virol. 2005; 79: 7698-7706Crossref PubMed Scopus (70) Google Scholar, 12.van Ooij M.J. Vogt D.A. Paul A. Castro C. Kuijpers J. van Kuppeveld F.J. Cameron C.E. Wimmer E. Andino R. Melchers W.J. J. Gen. Virol. 2006; 87: 103-113Crossref PubMed Scopus (71) Google Scholar). All picornaviruses have a cis-acting RNA element capable of templating the production of VPg-pUpU, although the position of this element in the genome varies. This element has been termed oriI (2.Paul A.V. Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. 1st Ed. American Society for Microbiology Press, Washington, D. C.2002: 227-246Google Scholar). Although it is generally accepted that oriI is essential for genome replication (6.Gerber K. Wimmer E. Paul A.V. J. Virol. 2001; 75: 10979-10990Crossref PubMed Scopus (81) Google Scholar, 7.Mason P.W. Bezborodova S.V. Henry T.M. J. Virol. 2002; 76: 9686-9694Crossref PubMed Scopus (120) Google Scholar, 8.Yang Y. Rijnbrand R. McKnight K.L. Wimmer E. Paul A. Martin A. Lemon S.M. J. Virol. 2002; 76: 7485-7494Crossref PubMed Scopus (67) Google Scholar, 13.McKnight K.L. Lemon S.M. RNA. 1998; 4: 1569-1584Crossref PubMed Scopus (136) Google Scholar, 14.Lobert P.E. Escriou N. Ruelle J. Michiels T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11560-11565Crossref PubMed Scopus (98) Google Scholar, 15.Goodfellow I. Chaudhry Y. Richardson A. Meredith J. Almond J.W. Barclay W. Evans D.J. J. Virol. 2000; 74: 4590-4600Crossref PubMed Scopus (199) Google Scholar), some suggest that it is only required for production of primers for plus-strand synthesis (16.Murray K.E. Barton D.J. J. Virol. 2003; 77: 4739-4750Crossref PubMed Scopus (108) Google Scholar, 17.Morasco B.J. Sharma N. Parilla J. Flanegan J.B. J. Virol. 2003; 77: 5136-5144Crossref PubMed Scopus (79) Google Scholar, 18.Goodfellow I.G. Polacek C. Andino R. Evans D.J. J. Gen. Virol. 2003; 84: 2359-2363Crossref PubMed Scopus (45) Google Scholar), whereas others suggest that oriI-derived primers are required for both plus- and minus-strand synthesis (12.van Ooij M.J. Vogt D.A. Paul A. Castro C. Kuijpers J. van Kuppeveld F.J. Cameron C.E. Wimmer E. Andino R. Melchers W.J. J. Gen. Virol. 2006; 87: 103-113Crossref PubMed Scopus (71) Google Scholar). The VPg uridylylation reaction can be mimicked in vitro by using purified components: the viral RNA-dependent RNA polymerase (3Dpol); the VPg peptide; (bio)synthetic oriI RNA; UTP; and Mg2+ or Mn2+ (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (236) Google Scholar, 4.Rieder E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10371-10380Crossref PubMed Scopus (127) Google Scholar, 6.Gerber K. Wimmer E. Paul A.V. J. Virol. 2001; 75: 10979-10990Crossref PubMed Scopus (81) Google Scholar, 11.Nayak A. Goodfellow I.G. Belsham G.J. J. Virol. 2005; 79: 7698-7706Crossref PubMed Scopus (70) Google Scholar, 12.van Ooij M.J. Vogt D.A. Paul A. Castro C. Kuijpers J. van Kuppeveld F.J. Cameron C.E. Wimmer E. Andino R. Melchers W.J. J. Gen. Virol. 2006; 87: 103-113Crossref PubMed Scopus (71) Google Scholar). The reaction is also stimulated by a viral precursor protein, 3CD. Protein 3CD has both RNA-binding and protease activities, but only the RNA-binding activity is required for stimulation of the VPg uridylylation reaction (3.Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (236) Google Scholar, 4.Rieder E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10371-10380Crossref PubMed Scopus (127) Google Scholar). We have shown that the fully processed viral protein 3C also stimulates VPg uridylylation (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The mechanism for 3C(D) stimulation is not known. Our previous studies of PV support a model in which 3C(D) binds to oriI and recruits polymerase to oriI (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Moreover, we suggested that this recruitment was mediated by an interaction between the thumb subdomain of 3Dpol and some undefined subdomain of 3C (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). This model was based on the identification of 3Dpol derivatives that contained wild-type polymerase activity on primed RNA templates and retained basal VPg uridylylation activity that could not be stimulated by 3C(D) (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Recently, the suggestion was made that our interpretation of a 3Dpol-3C(D) interaction could also be explained as an allosteric effect unique to the 3Dpol derivatives studied (20.Boerner J.E. Lyle J.M. Daijogo S. Semler B.L. Schultz S.C. Kirkegaard K. Richards O.C. J. Virol. 2005; 79: 7803-7811Crossref PubMed Scopus (20) Google Scholar). In this report, we describe the establishment of a minimal VPg uridylylation system for PV that we use to evaluate the composition, stoichiometry, and functional and structural organization of the active VPg uridylylation complex. Consistent with previous studies (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), PV polymerase is recruited to and retained in the VPg uridylylation complex by a direct, physical interaction with 3C(D) bound to oriI. This conclusion is the same for two other picornaviruses: coxsackievirus B3 (CVB3) and human rhinovirus type 14 (HRV14). Targeting of 3C(D) to oriI is mediated solely by the 3C subdomain, the 3D subdomain serves only to increase affinity on oriI and, in doing so, increases the observed specificity of 3CD relative to 3C when oriI is placed in the context of longer RNA sequence. Protein 3C(D) binds to oriI at a 2:1 stoichiometry; 3C dimerizes in solution in the presence and absence of oriI. One of the 3C subdomains of 3C(D) binds to oriI contacting the stem in a position near the loop that would facilitate appropriate positioning of the polymerase. 3C binds to both single- and double-stranded RNA. In the context of oriI, affinity for the two single strands of the stem individually is higher than that for the annealed stem, suggesting an isomerization step after 3C(D)-oriI complex formation. Finally, association of VPg with this complex is weak and is probably rate-limiting for uridylylation. This study provides the most complete view to date of assembly and organization of the picornavirus VPg uridylylation complex. Materials−Deep Vent DNA polymerase and restriction enzymes were from New England Biolabs, Inc.; shrimp alkaline phosphatase was from USB; T4 DNA ligase was from Invitrogen; Difco-NZCYM was from BD Biosciences; QIAEX beads were from Qiagen; RNase A was from Sigma; Ultrapure UTP solution was from GE Healthcare; [α-32P]UTP (6000 Ci/mmol) was from PerkinElmer Life Sciences; synthetic PV and HRV14 VPg peptides were purchased from Alpha Diagnostic International (San Antonio, TX); synthetic CVB3 VPg was a gift from Willem Melchers; all other reagents and apparatuses were available through Fisher or VWR or as indicated. Construction of Expression Plasmids−The CVB3 3C coding region was amplified by using oligonucleotides 1–4 (Table 1 lists all oligonucleotides used in this study; oligonucleotides were from Invitrogen or Integrated DNA Technologies, Inc.) to perform overlap-extension PCR using the Knowlton CVB3/H3 cDNA (21.Knowlton K.U. Jeon E.S. Berkley N. Wessely R. Huber S. J. Virol. 1996; 70: 7811-7818Crossref PubMed Google Scholar) as template. The 3C-coding region was cloned into the pET26Ub plasmid (22.Gohara D.W. Ha C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) using SacII and EcoRI sites to give the pET26Ub-CVB3-3C-C147G-CHIS plasmid. The HRV16 3C-coding region was amplified by using oligonucleotides 5–8 to perform overlap-extension PCR using the HRV16 cDNA (23.Cheney I.W. Naim S. Shim J.H. Reinhardt M. Pai B. Wu J.Z. Hong Z. Zhong W. J. Virol. 2003; 77: 7434-7443Crossref PubMed Scopus (14) Google Scholar) as template. The amplified fragment was cloned into the pET26Ub-CHIS plasmid (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) using the SacII and BamHI sites to give the pET26Ub-HRV16-3C-CHIS plasmid. Cloning of the CVB3 3D is described by van Ooij et al. (12.van Ooij M.J. Vogt D.A. Paul A. Castro C. Kuijpers J. van Kuppeveld F.J. Cameron C.E. Wimmer E. Andino R. Melchers W.J. J. Gen. Virol. 2006; 87: 103-113Crossref PubMed Scopus (71) Google Scholar); cloning of HRV14 3C and 3D will be described by Shen et al. 4M. Shen, Q. Wang, Y. Yang, H. B. Pathak, J. J. Arnold, S. M. Lemon, and C. E. Cameron, manuscript in preparation. PV 3C without a His tag was made by PCR amplification using oligonucleotides 17 and 18 and pET26Ub-3C-C147G-CHIS (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) as template. The amplified fragment was cloned into pET26Ub (22.Gohara D.W. Ha C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). DNA sequencing at the Penn State Nucleic Acid Facility was used to verify the integrity of the above clones.TABLE 1Oligonucleotides used in this studyNumberNameSequence1CVB3-3CD-SacII-f5′-GCG ACT AGT CCG CGG TGG AGG TCC TGC ATT TGA ATT TGC-3′2CVB3-3C-Chis-EcoRI-r5′-GCG GAA TTC GCG GCC GCT TAC TAA TGG TGG TGA TGG TGG TGA CCA GAG GAT CCT TGT TCA TCA TTG AAA TAG TG-3′3CVB3-3C-C147G-f5′-ACG AGA GCA GGT CAA GGT GGC GGA GTA CTC ATG-3′4CVB3-3C-C147G-r5′-CAT GAG TAC TCC GCC ACC TTG ACC TGC TCT CGT-3′5HRV16-3C-SacII-f5′-GCG CCG CGG TGG AGG TCC AGA AGA AGA ATT TGG AAT G-3′6HRV16-3C-BamHI-r5′-GCG GGA TCC TTG TTG TTC AGT GAA GTA TGA TCT CAA TAG-3′7HRV16-3C-C147G-f5′-ACA AAA TCT GGG TAT GGT GGT GGT GTG TTA TAC-3′8HRV16-3C-C147G-r5′-GTA TAA CAC ACC ACC ACC ATA CCC AGA TTT TGT-3′9BamHI-T7-51ntCRE-f5′-CGG GAT CCT AAT ACG ACT CAC TAT AGG GAC AAC TAC ATA CAG TTC A-3′10EcoRI-NcoI-51ntCRE-r5′-GGA ATT CCC ATG GCA AAC ATA CTG GTT CAA T-3′11BamHI-T7-CVB3/H3-CRE-f5′-CGG GAT CCT AAT ACG ACT CAC TAT AGG GAG TAA TTA CAT ACA GTT C-3′12EcoRI-XhoI-CVB3/H3-CRE-r5′-GGC GAA TTC CTC GAG CAG ACA TAC AGG C-3′13PV-3C-SacII-f5′-GCG GAA TTC CTC CGC GGT GGA GGA CCA GGG TTC GAT T-3′143C-Authentic-Cterm-r5′-GCG GAA TTC GTT TAA ACT TAC TAT TGA CTC TGA GTG AAG TA-3′ Open table in a new tab Expression and Purification of 3C Proteins−All of the 3C proteins were expressed using the ubiquitin fusion system described previously for 3Dpol (22.Gohara D.W. Ha C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expression Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). BL21(DE3)pCG1 cells were transformed with either the pET26Ub-PV-3C-CHIS, pET26Ub-CVB3-3C-CHIS, pET26Ub-HRV14-3C-CHIS, or pET26Ub-HRV16-3C-CHIS plasmids and plated (10%) onto NZCYM plates containing kanamycin at 25 μg/ml (K25), chloramphenicol at 20 μg/ml (C20), and dextrose at 0.4%. Ten colonies were then used to seed 100 ml of NZCYM medium supplemented with K25, C20, and dextrose at 0.1%. The culture was grown at 37 °C until to an A600 of 1. Cells were chilled to 25 °C and induced by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 500 μm. Cells were grown for 4 h at 25 °C and harvested. Cell pellets were weighed and stored at –80 °C. Frozen cell pellets were thawed on ice and suspended in lysis buffer (100 mm potassium phosphate, pH 8.0, 20% glycerol, 10 mm 2-mercaptoethanol (β-ME), 5.6 μg/ml pepstatin A, 4 μg/ml leupeptin) at a concentration of 4 ml of lysis buffer/g of cell pellet. Cells were homogenized using a Dounce homogenizer; cells were lysed by passing through a French pressure cell at a pressure per square inch of 20,000. Phenylmethylsulfonyl fluoride and Nonidet P-40 were added after lysis to final concentrations of 2 mm and 0.1% (v/v), respectively. The lysates were clarified by centrifugation in a Beckman JA-30.50 Ti rotor for 30 min at 24,000 rpm at 4 °C. The clarified lysate was passed over Ni2+-nitrilotriacetic acid spin columns (Qiagen). The columns were equilibrated prior to loading and washed after loading with buffer A (50 mm HEPES, pH 7.5, 20% glycerol, 10 mm β-ME, and 0.1% Nonidet P-40) containing 500 mm NaCl according to the manufacturer's protocol. Protein was eluted in two 100-μl fractions using buffer A containing 500 mm NaCl and 500 mm imidazole. Protein concentration was determined by using the Bio-Rad protein assay. Conductivities of the fractions were measured, and the fractions were then aliquoted and stored at –80 °C. Purification of PV 3D and PV 3CD-His was described previously (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Purification of CVB3 3D was described by van Ooij et al. (12.van Ooij M.J. Vogt D.A. Paul A. Castro C. Kuijpers J. van Kuppeveld F.J. Cameron C.E. Wimmer E. Andino R. Melchers W.J. J. Gen. Virol. 2006; 87: 103-113Crossref PubMed Scopus (71) Google Scholar); purification of HRV14 3D will be described by Shen et al.4; Purified PV 3C containing an authentic carboxyl terminus was prepared by Dr. Jamie Arnold as a part of another study. 5J. J. Arnold and C. E. Cameron, unpublished results. Cloning and Transcription of oriIs−The region corresponding to the 51-nt oriI presented in Fig. 3 was amplified from an expression vector encoding the 2C gene 6C. E. Cameron, unpublished results. using oligonucleotides 9 and 10. The purified 51-nt oriI PCR product was linearized using NcoI, and a transcription reaction (40 mm HEPES, pH 7.5, 32 mm magnesium acetate, 40 mm dithiothreitol, 2 mm spermidine, 28 mm NTPs, 25 μg/ml linearized template, and 25 μg/ml T7 RNA polymerase) was performed at 37 °C for 2 h. Magnesium pyrophosphate was removed by centrifugation for 2 min. The supernatant was treated with RQ1DNase (1 unit/μg of template; Promega) for 30 min to remove the template. The RNA was spun through a Micropure-EZ centrifugal filter (Amicon Bioseparations; Millipore) to remove any protein from the RNA according to the manufacturer's instructions. Two G25 Sephadex (Sigma) spin columns were then used to remove any free nucleotides. The RNA was precipitated with ammonium acetate and suspended in TE (10 mm Tris·HCl, pH 8.0, 1 mm EDTA), and RNA concentration was calculated by measuring absorbance at 260 nm. The extinction coefficient was calculated for the 51-nt oriI plus three guanosine residues that are transcribed at the 5′-end of oriI to be 0.627000 μm–1·cm–1 (24.Carroll S.S. Benseler F. Olsen D.B. Methods Enzymol. 1996; 275: 365-382Crossref PubMed Scopus (19) Google Scholar). These extra nucleotides do not alter the fold of the 51-nt oriI as predicted by the mfold RNA folding server (available on the World Wide Web at www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi) (25.Zuker M. Nucleic Acids Res. 2003; 31: 3406-3415Crossref PubMed Scopus (10253) Google Scholar, 26.Mathews D.H. Sabina J. Zuker M. Turner D.H. J. Mol. Biol. 1999; 288: 911-940Crossref PubMed Scopus (3211) Google Scholar). The region corresponding to the CVB3 oriI was amplified using oligonucleotides 11 and 12 and the Knowlton CVB3/H3 cDNA as template. The purified oriI PCR product was linearized using XhoI and RNA transcribed as described above. The concentration was measured as described above using the calculated extinction coefficient (0.665700 μm–1·cm–1) that includes two guanosine residues at the 5′-end and a guanosine and adenosine at the 3′-end, a consequence of the XhoI digestion. These extra nucleotides do not alter the fold of the CVB3/H3 oriI as predicted by mfold. The cloning of the HRV14 97-nt oriI is described elsewhere4; cloning of the PV 61-nt oriI was described previously (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Transcription and determination of the concentration for the HRV14 97-nt oriI and the PV 61-nt oriI were done as described above. RNA transcripts from the PV subgenomic replicon, pRLucRA (27.Andino R. Rieckhof G.E. Achacoso P.L. Baltimore D. EMBO J. 1993; 12: 3587-3598Crossref PubMed Scopus (407) Google Scholar, 28.Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (121) Google Scholar), were generated after linearization with ApaI. Transcription reactions, typically 20 μl, consisted of 350 mm HEPES, pH 7.5, 32 mm magnesium acetate, 40 mm dithiothreitol, 2 mm spermidine, 28 mm NTPs, 0.5 μg of template, and 0.5 μg of T7 RNA polymerase. Reactions were incubated at 37 °C for 3 h followed by removal of magnesium pyrophosphate. DNase treatment with RQ1DNase (Promega) was used to remove the template; lithium chloride precipitation of the RNA was used to remove unincorporated nucleotides. RNA concentration was calculated by measuring absorbance at 260 nm, assuming that an A260 of 1 was equivalent to 40 μg/ml. RNA transcripts of the PV 2C gene were generated after linearization of pET26Ub-2C plasmid with HindIII. Transcription reactions and RNA quantification were performed as described above. The 29-, 22-, and 14-nt oriIs presented in Fig. 3 were chemically synthesized by Dharmacon, Inc. (Lafayette, CO). Each RNA was deprotected prior to use in the VPg uridylylation assays. Deprotection was done by suspension of the RNA in 500 mm acetic acid and incubation at 65 °C for 15 min, followed by the addition of an equal volume of 660 mm Tris, pH 8.0, and incubation at 65 °C for 15 min. Concentrations were measured as described above. The extinction coefficients used for each are 0.378500 μm–1·cm–1 (29 nt), 0.261100 μm–1·cm–1 (22 nt), and 0.166400 μm–1·cm–1 (14 nt). Footprint Analysis Using an Iodine Cleavage Assay−oriI containing phosphorothioated ATP (ATPαS) and a 5′-OH to be used for footprinting by the iodine cleavage assay was obtained by performing a 1-ml transcription reaction (40 mm HEPES, pH 7.5, 32 mm magnesium acetate, 40 mm dithiothreitol, 2 mm spermidine, 12 mm NTPs, 0.3 mm ATPαS (10% of the total ATP concentration), 8 mm guanosine, 25 μg/ml linearized template, and 25 μg/ml T7 RNA polymerase) at 37 °C for 2 h. Magnesium pyrophosphate was removed by centrifugation for 2 min. The supernatant was treated with RQ1DNase (1 unit/μg of template; Promega) for 30 min to remove the template; two phenol/chloroform extractions followed by a chloroform extraction were performed to deproteinate the RNA. Next, the RNA was precipitated with ammonium acetate and washed with 70% ethanol, and the pellet was suspended in 50% formamide. This provided the starting material for gel purification. The entire volume was loaded onto a 10% acrylamide, 50% formamide gel (18 cm × 24 cm × 2 mm). The gel was run at 25 mA for ∼4 h (the band corresponding to the full-length oriI had migrated to the middle of the gel by this time, as determined by the migration of bromphenol blue and xylene cyanol indicator dyes). The oriI band was excised from the gel by using UV-shadowing using a TLC plate with a fluorescent indicator (PEI Cellulose F; EM Science). The gel piece was cut into tiny squares and placed in an Elutrap electrophoresis chamber (Schleicher & Schuell). The eluted RNA was precipitated with ammonium acetate, washed with 70% ethanol, and suspended in TE. This was then passed over two Sephadex G-25 (Sigma) spin columns. The gel-purified RNA was radiolabeled using [γ-32P]ATP and polynucleotide kinase (New England Biolabs, Inc.). The labeled RNA was gel-purified again following the same procedure described above. RNA quality was assessed by 15% denaturing PAGE. RNA concentration was calculated by measuring absorbance at 260 nm. The extinction coefficient was calculated for the 61-nt oriI plus three guanidine residues that are transcribed at the 5′-end of oriI (0.749400 μm–1·cm–1) as described previously (19.Pathak H.B. Ghosh S.K. Roberts A.W. Sharma S.D. Yoder J.D. Arnold J.J. Gohara D.W. Barton D.J. Paul A.V. Cameron C.E. J. Biol. Chem. 2002; 277: 31551-31562Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). The 5′-end-labeled phosphorothioate oriI (3.3 μm) was incubated at 80 °C for 5 min in folding buffer (166 mm HEPES, pH 7.5, 66.6 mm NaCl) and then gradually cooled to room temperature for 1 h. At 55 °C, MgCl2 (16.6 mm) was added. The cleavage reaction proceeded as follows. The folded RNA was incubated with varying concentrations of PV 3C-His and incubated at 30 °C for 5 min. To initiate cleavage, 2 μl of 500 μm iodine dissolved in ethanol was added. After incubation at 30 °C for 2 min, the cleavage was quenched with 2 μl of 20 mm dithiothreitol. The final reaction mixture contained 50 mm HEPES, pH 7.5, 5 mm MgCl2, 20 mm NaCl, 1 μm RNA, 0–10 μm protein, 100 μm iodine, and 20% ethanol. An alkaline hydrolysis ladder was generated by incubating the RNA in 50 mm sodium bicarbonate and 3 mm EDTA, pH 8.0, at 90 °C for 5 min. The samples were placed on ice to quench the cleavage reaction. Prior to analysis, an equal volume of 100% formamide was added to each sample. The RNA was denatured at 90 °C for 5 min and placed on ice. The cleavage products were separated on a 15% denaturing polyacrylamide gel run at constant power of 90 W. The gel was visualized by using a Typhoon 8600 scanner in the storage phosphor mode and quantified by using ImageQuant software. Fluorescence P

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