Picornavirus Genome Replication
2008; Elsevier BV; Volume: 283; Issue: 45 Linguagem: Inglês
10.1074/jbc.m806101200
ISSN1083-351X
AutoresHarsh B. Pathak, Hyung Suk Oh, Ian Goodfellow, Jamie J. Arnold, Craig E. Cameron,
Tópico(s)Animal Virus Infections Studies
ResumoThe 5′ ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3′ end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3′ end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed. The 5′ ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3′ end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3′ end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed. The picornavirus family of viruses causes a wide variety of diseases in humans and animals (1Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. American Society for Microbiology, Washington, DC2002Crossref Google Scholar). Poliovirus (PV), 3The abbreviations used are: PV, poliovirus; NTR, nontranslated region; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide; CRE, cis-acting replication element. 3The abbreviations used are: PV, poliovirus; NTR, nontranslated region; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MOPS, 4-morpholinepropanesulfonic acid; nt, nucleotide; CRE, cis-acting replication element. the causative agent of poliomyelitis, is the most extensively studied member of this family and has proven to be a useful model system for understanding picornavirus molecular biology, including genome replication (1Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. American Society for Microbiology, Washington, DC2002Crossref Google Scholar). PV is a nonenveloped virus with a single-stranded RNA genome of positive polarity that is ∼7500 nt in length. As shown in Fig. 1A, the genome encodes a single open reading frame flanked by 5′- and 3′-nontranslated regions (NTRs). The genome contains a 22-amino acid peptide (referred to as either VPg, 3B or primer for RNA synthesis) covalently linked to its 5′ end and is polyadenylated at its 3′ end. Translation of the genome is initiated from an internal ribosome entry site located in the 5′-NTR, producing a 247-kDa polyprotein that is co- and post-translationally processed by the virus-encoded 2A, 3C, and 3CD proteases (1Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. American Society for Microbiology, Washington, DC2002Crossref Google Scholar). Although polyprotein processing ultimately yields a set of processed end products, processing intermediates will form during the course of an infection, greatly increasing the functional proteome of the virus. Processing intermediates located in the P2 (e.g. 2ABC, 2BC, etc.) and P3 regions of the polyprotein are likely important for genome replication. Our laboratory has been quite interested in defining the molecular details of VPg attachment to the 5′ end of picornaviral RNAs (2Pathak 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, 3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar, 5Shen M. Reitman Z.J. Zhao Y. Moustafa I. Wang Q. Arnold J.J. Pathak H.B. Cameron C.E. J. Biol. Chem. 2008; 283: 875-888Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). This reaction is thought to occur in two independent half-reactions catalyzed by the viral RNA-dependent RNA polymerase, 3Dpol. First, VPg is uridylylated to produce VPg-pUpU; second, VPg-pUpU serves as a primer for full-length RNA synthesis (1Semler B.L. Wimmer E. Molecular Biology of Picornaviruses. American Society for Microbiology, Washington, DC2002Crossref Google Scholar). VPg uridylylation requires a template. To date, two templates have been described. The first is the poly(rA) tail at the 3′ end of the genome (6Paul A.V. van Boom J.H. Filippov D. Wimmer E. Nature. 1998; 393: 280-284Crossref PubMed Scopus (294) Google Scholar). The second is an RNA stem-loop structure found at different positions in the genomes of different picornaviruses but most often occurring in protein-coding sequence (7Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (233) Google Scholar, 8Gerber K. Wimmer E. Paul A.V. J. Virol. 2001; 75: 10979-10990Crossref PubMed Scopus (81) Google Scholar, 9Yang Y. Rijnbrand R. McKnight K.L. Wimmer E. Paul A. Martin A. Lemon S.M. J. Virol. 2002; 76: 7485-7494Crossref PubMed Scopus (65) Google Scholar, 10Mason P.W. Bezborodova S.V. Henry T.M. J. Virol. 2002; 76: 9686-9694Crossref PubMed Scopus (120) Google Scholar, 11van 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). We refer to this latter template as oriI (origin of replication internal). PV oriI is located in 2C-coding sequence (Fig. 1A) (7Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (233) Google Scholar). Our current model for oriI-templated VPg uridylylation is shown in Fig. 1B. This model is consistent with much of what is known about this reaction (2Pathak 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, 3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar, 5Shen M. Reitman Z.J. Zhao Y. Moustafa I. Wang Q. Arnold J.J. Pathak H.B. Cameron C.E. J. Biol. Chem. 2008; 283: 875-888Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 7Paul A.V. Rieder E. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10359-10370Crossref PubMed Scopus (233) Google Scholar, 8Gerber K. Wimmer E. Paul A.V. J. Virol. 2001; 75: 10979-10990Crossref PubMed Scopus (81) Google Scholar, 11van 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, 12Rieder E. Paul A.V. Kim D.W. van Boom J.H. Wimmer E. J. Virol. 2000; 74: 10371-10380Crossref PubMed Scopus (127) Google Scholar, 13Paul 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 (69) Google Scholar, 14Yin J. Paul A.V. Wimmer E. Rieder E. J. Virol. 2003; 77: 5152-5166Crossref PubMed Scopus (71) Google Scholar, 15Murray K.E. Barton D.J. J. Virol. 2003; 77: 4739-4750Crossref PubMed Scopus (107) Google Scholar, 16Morasco B.J. Sharma N. Parilla J. Flanegan J.B. J. Virol. 2003; 77: 5136-5144Crossref PubMed Scopus (78) Google Scholar, 17Thiviyanathan V. Yang Y. Kaluarachchi K. Rijnbrand R. Gorenstein D.G. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12688-12693Crossref PubMed Scopus (19) Google Scholar, 18Yang 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, 19Amero C.D. Arnold J.J. Moustafa I.M. Cameron C.E. Foster M.P. J. Virol. 2008; 82: 4363-4370Crossref PubMed Scopus (12) Google Scholar), but the details have emerged from studies performed in vitro on minimal templates and minimal protein domains (2Pathak 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, 3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar, 5Shen M. Reitman Z.J. Zhao Y. Moustafa I. Wang Q. Arnold J.J. Pathak H.B. Cameron C.E. J. Biol. Chem. 2008; 283: 875-888Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Briefly, two molecules of 3C(D) bind to oriI (step 1) (3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). This complex isomerizes, unwinding the stem and extending the loop (step 2) (3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar). 3Dpol associates with the complex, directed and stabilized by an interaction between the back of the “thumb” subdomain of 3Dpol and a convex surface formed by the top of both subunits of the 3C dimer (step 3) (2Pathak 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, 3Pathak H.B. Arnold J.J. Wiegand P.N. Hargittai M.R. Cameron C.E. J. Biol. Chem. 2007; 282: 16202-16213Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar, 5Shen M. Reitman Z.J. Zhao Y. Moustafa I. Wang Q. Arnold J.J. Pathak H.B. Cameron C.E. J. Biol. Chem. 2008; 283: 875-888Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). VPg joins the complex, perhaps by binding to the RNA-primer-binding site of 3Dpol in an extended conformation (step 4) (19Amero C.D. Arnold J.J. Moustafa I.M. Cameron C.E. Foster M.P. J. Virol. 2008; 82: 4363-4370Crossref PubMed Scopus (12) Google Scholar). In the presence of UTP, Tyr-3 hydroxyl of VPg is used as a nucleophile to form VPg-pU, the 3′-OH of which, in turn, serves as the nucleophile to form VPg-pUpU (step 5) (13Paul 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 (69) Google Scholar). Both uridylate residues are templated by a single adenylate residue in the oriI loop by using a slide-back mechanism (13Paul 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 (69) Google Scholar). VPg-pUpU must occur processively as VPg-pU appears to be catalytically incompetent (20Korneeva V.S. Cameron C.E. J. Biol. Chem. 2007; 282: 16135-16145Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). This model provides a clear mechanistic framework for the study of oriI-templated VPg uridylylation in vitro. Importantly, this model explains and predicts biological phenotypes (2Pathak 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, 4Shen M. Wang Q. Yang Y. Pathak H.B. Arnold J.J. Castro C. Lemon S.M. Cameron C.E. J. Virol. 2007; 81: 12485-12495Crossref PubMed Scopus (19) Google Scholar, 5Shen M. Reitman Z.J. Zhao Y. Moustafa I. Wang Q. Arnold J.J. Pathak H.B. Cameron C.E. J. Biol. Chem. 2008; 283: 875-888Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 20Korneeva V.S. Cameron C.E. J. Biol. Chem. 2007; 282: 16135-16145Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). However, VPg uridylylation occurs 10–50-fold slower in vitro than necessary to support the rate constant (∼0.1/s) for initiation calculated from biological data (see Ref. 21Arnold J.J. Cameron C.E. J. Biol. Chem. 2000; 275: 5329-5336Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In addition, this model assumes that the processed VPg peptide is the primer employed in vivo, which may not be the case. Finally, this model does not provide any hints of how a VPg-pUpU molecule produced in the middle of the genome could be transferred to the 3′ end of plus- and/or minus-strand RNA for production of the complementary RNA strand. Because the end products of polyprotein processing may not be the forms of the protein employed for assembly and/or function of the genome-replication complexes in vivo, it is possible that the use of polyprotein processing intermediates may be essential to recapitulate VPg uridylylation in vitro that occurs on a biologically relevant time scale, providing additional insight into the mechanism of this reaction that is absolutely essential for picornavirus genome replication. In this study, we show that P3 precursor proteins containing VPg at the amino terminus (3BC and 3BCD) can be uridylylated in vitro. 3BC-containing precursors bind to oriI more efficiently than processed proteins and serve as VPg donors that can be recruited to and/or retained in the uridylylation complex better than processed VPg. We demonstrate an optimal affinity of precursor binding to oriI for maximal accumulation of uridylylated product in the steady state, suggesting formation and/or collapse as a rate-limiting step for uridylylation in vitro. The use of precursor proteins during replication in cells was queried by preventing the production of processed VPg. Preventing VPg formation in cells is not lethal and leads to production of 3BC-linked RNA. Therefore, processed VPg is not essential for uridylylation and/or full-length plus- and/or minus-strand RNA synthesis in vivo. Analysis of the polyprotein processing profile of the VPg-processing-defective mutant in cell-free extracts uncovered two pathways of P3 precursor processing, major and minor, and only the major pathway was perturbed. We propose that the minor pathway is responsible for production of proteins (processing intermediates) required for uridylylation and RNA synthesis. These studies provide new insight into the mechanism of VPg uridylylation and suggest mechanisms for transfer of the diuridylylated protein primer from the middle of the genome to the 3′ end of plus- and/or minus-strand RNA. Materials—Deep Vent DNA polymerase and restriction enzymes were from New England Biolabs; shrimp alkaline phosphatase was from U. S. Biochemical Corp.; T4 DNA ligase was from Invitrogen; Difco-NZCYM was from BD Biosciences; QIAEX beads were from Qiagen; RNases A and T1 were from Sigma; Ultrapure UTP solution was from GE Healthcare; [α-32P]UTP (6000 Ci/mmol) was from PerkinElmer Life Sciences; synthetic VPg peptide was from Alpha Diagnostic International (San Antonio, Texas); all other reagents and apparatuses were available through Fisher, VWR, or as indicated. Construction of Expression Plasmids for 3BC, 3BCD, 3AB, and 3Cpro—Standard PCR and cloning procedures were used to generate expression plasmids for 3BC, 3BCD, 3AB, and 3Cpro. 3Cpro refers to 3C protein with an active protease. Oligonucleotides used in PCRs for this study were purchased from Invitrogen or Integrated DNA Technologies, Inc; sequences are provided in **supplemental Table S1. Clones were verified by sequencing at the Pennsylvania State Nucleic Acid Facility. A detailed description of the cloning is provided in the supplemental material. Bacterial Expression and Purification of 3BC, 3BC-Y3F, 3BCD, 3Cpro, 3AB, 3Dpol, and 3C—3Dpol and 3C were purified as described previously (2Pathak 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 procedures for wild-type 3BC, the 3BC Y3F mutant, 3BCD, 3Cpro, and 3AB represented modifications of our published protocol. A detailed description of the purification protocols is provided in the supplemental material. Transcription and Purification of 61-nt oriI— oriI for the VPg uridylylation reactions and for the filter-binding assays was transcribed from the pUC18–61-nt oriI plasmid (2Pathak 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) linearized using the BstZ17I site. A complete protocol is provided in the supplemental material. VPg Uridylylation Assays—Reactions were performed essentially as described previously (2Pathak 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), with slight modifications. Reaction mixtures contained 1 μm 3Dpol, 1 μm 61-nt oriI, in reaction buffer (50 mm HEPES, pH 7.5, 10% glycerol, 5 mm magnesium acetate, 10 mm β-mercaptoethanol, 10 μm UTP, and 0.04 μm [α-32P]UTP (6000 Ci/mmol)). The concentrations of 3BC, VPg, and 3C were 1 μm unless these were being titrated. All reactions were adjusted to a final NaCl concentration of 20 mm. All components were diluted to working concentrations immediately prior to use. Reactions were assembled on ice with oriI, 3C, and VPg and/or 3BC, or 3BCD in reaction buffer. The reactions were then transferred to 30 °C for 5 min and initiated by addition of 3Dpol. Reactions were incubated at 30 °C for 20 min or the indicated amount of time during a time course and quenched with an equal volume of 100 mm EDTA in 75% formamide containing 0.05% bromphenol blue dye. Quenched reactions were analyzed by using Tris-Tricine SDS-PAGE as described previously (2Pathak 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). 3BC Cleavage by Using 3Cpro—Processing of 3BC by 3Cpro was performed as follows. PV 3Cpro was freshly diluted to 6 μm in dilution buffer (50 mm HEPES, pH 7.5, 10% glycerol, 5 mm magnesium acetate, 10 mm β-mercaptoethanol). Following uridylylation of 3BC for 20 min as described above, 3Cpro (60 pmol, 10 μl of 6 μm) was added to a 10-μl aliquot of the uridylylation reaction. This was then incubated at 30 °C for 60 min. The reaction was quenched with an equal volume of quench dye (100 mm EDTA in 75% formamide containing 0.05% bromphenol blue), and the sample was analyzed by using Tris-Tricine SDS-PAGE as described previously (2Pathak 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). RNA Filter-binding Assays—Reaction mixtures (20 μl) contained 10 nm oriI and varying concentrations of 3BC, 3C, or 3C and VPg in reaction buffer (50 mm HEPES, pH 7.5, 10% glycerol, 5 mm magnesium acetate, 10 mm β-mercaptoethanol). Binding reactions were initiated by the addition of freshly diluted 3BC (3C or 3C and VPg) to the RNA in reaction buffer. Reactions were incubated at 30 °C for 15–20 min. Membranes and Whatman 3MM paper were presoaked in equilibration buffer (50 mm HEPES, pH 7.5, and 5 mm magnesium acetate, and 10% glycerol) for 2 min and assembled, in order from top to bottom, nitrocellulose, nylon, and Whatman paper, in a slot blotter (GE Healthcare). After assembly, the binding reactions (20 μl) were loaded into the slot blotter, and vacuum was applied for 2 min at 200 mm Hg. Membranes were air-dried and visualized by using a Typhoon 8600 scanner in the storage phosphor mode and quantified by using ImageQuant software. Construction of Mutated Replicons—Standard PCR and cloning procedures were used to generate poliovirus subgenomic replicons containing the mutation of the Gln-Gly cleavage site between 3B and 3C to Gly-Gly and for construction of the Y3F mutant with a Gly-Gly mutation between 3B and 3C. A complete description of the cloning is provided in the supplemental material. Transcription of Subgenomic Replicons and Luciferase Assays—RNA transcripts for performing luciferase assays were generated from the pRLucRA plasmids after linearization with ApaI. Luciferase assays were performed as described previously (2Pathak 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) with slight modifications. A complete description of the transcription reactions and the luciferase assays is provided in the supplemental material. Western Blot Analysis of Replicon Proteins—Cells were transfected with replicon RNA and incubated at 34 °C for 20 h. Cells were harvested and lysed, and Western blot analysis was performed as described in the supplemental material. Northern Blot Analysis—RNA isolation for Northern blot analysis was performed as follows. HeLa cells (6 × 106) were transfected by using electroporation as described above with 25 μg of the indicated subgenomic RNAs (5 μg/1.2 × 106 cells). Cells were suspended in 30 ml of normal growth media and incubated at 34 °C. Total RNA was then isolated from the cells at the indicated times post-transfection using TRIzol reagent (Invitrogen). Briefly, cells were pelleted by centrifugation at 1000 × g for 4 min at 4 °C, washed with phosphate-buffered saline (10 ml), and lysed in TRIzol reagent (1 ml). Lysed cells were incubated at room temperature for 5 min, and chloroform (0.2 ml) was added. The samples were vortexed vigorously (15 s) and incubated at room temperature for 3 min. Samples were centrifuged at 12,000 × g for 15 min at 4 °C. Isopropyl alcohol (0.5 ml) was added to the aqueous phase for each sample and incubated at room temperature for 10 min. Following centrifugation at 12,000 × g for 10 min at 4 °C, the RNA pellet was washed with 75% ethyl alcohol (1 ml) and then dissolved in water (50 μl). Concentration was determined by measuring the absorbance at 260 nm. The quality of the RNA was assayed by agarose gel electrophoresis, and the concentration of all of the RNAs was normalized to the 18 S rRNA band by using the Typhoon 8600 scanner in the fluorescence mode. For Northern blot analysis, total RNA (5 μg) was separated on a 0.6% agarose gel containing 0.8 m formaldehyde. The gel was then washed twice in water for 30 min each and then in 20× SSC (3 m sodium chloride, 0.3 m sodium citrate, pH 7.2) for 30 min. RNA was transferred to nylon membrane (Hybond XL, GE Healthcare) by using overnight capillary blotting using 10× SSC as the transfer buffer. RNA was cross-linked to the membrane by using a Stratalinker 2400 UV cross-linker (Stratagene). The membrane was dried and washed twice in wash buffer (1× SSC, 0.1% SDS) at 65 °C for 30 min each. Prehybridization was performed in modified Church buffer (0.5 m sodium phosphate, pH 7.2, 7% SDS, 1 mm EDTA) for 4 h at 65°C. Hybridization probes were denatured at 95 °C for 5 min and chilled on ice for 1 min prior to addition to the membrane (1 × 107 cpm). Hybridization was performed in the modified Church buffer for 16 h at 65 °C. The membrane was washed twice in wash buffer for 20 min at 65 °C and one time at room temperature. The membrane was dried, wrapped in plastic wrap, and exposed to a phosphor screen and scanned on a Typhoon 8600 scanner in the storage phosphor mode and quantified by using ImageQuant software. Hybridization probes were made by PCR using oligonucleotides 16 and 18 (supplemental Table S1) and pRLuc as template. [α-32P]dATP (1 mCi/ml and 3000 Ci/mmol; GE Healthcare) was included in the PCR; the concentration of cold dNTPs was 3 mm for dCTP, dGTP, and dTTP and 500 μm for dATP. The quality of the PCR product was assayed by agarose gel electrophoresis. Scintillation counting was performed to determine the counts/min for the probe. Immunoprecipitation of RNA—Total RNA was prepared, and concentration was measured as described above from HeLa cells transfected with the wild-type, Gly-Gly mutant, or Y3F/Gly-Gly mutant RNA. Total RNA was aliquoted equally into three microcentrifuge tubes (30 μg per aliquot), and IP buffer (50 mm Tris, pH 7.4, 0.5 m NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) was added to a final volume of 500 μl. Purified polyclonal anti-VPg, anti-3C, or anti-NS5A sera were added to each tube. The mixtures were rotated at room temperature for 45 min. Protein A magnetic beads (New England Biolabs, 50 μl) were conditioned by washing twice in IP buffer and then suspended in IP buffer (50 μl). These conditioned beads were added to total RNA and anti-sera mixture and incubated for 1.5 h at room temperature. The beads were collected by a magnetic stand (Dynal, Oslo, Norway) and washed four times with IP buffer followed by two times with Tris buffer (50 mm Tris, pH 7.4, 150 mm NaCl). For Northern blotting, 5 μl of water and 15 μl of formaldehyde loading dye (20 mm MOPS, 63.3% formamide, 23.3% formaldehyde, 5 mm sodium acetate, 4.3 mm EDTA, 0.066% bromphenol blue, 0.066% xylene cyanol) were added to the beads and incubated for 15 min at 65 °C. The beads were then pelleted and the entire volume loaded on a denaturing gel for Northern blot analysis as described above. Generation and Purification of Polyclonal PV VPg, PV 3C, PV 3D, and HCV NS5A Antisera—Polyclonal antibodies raised in rabbits against viral proteins PV VPg, PV 3C-His, PV 3D, and HCV NS5A-His were purified using ammonium sulfate precipitation and DEAE-Affi-Gel blue (Bio-Rad) column purification as described in detail in the supplemental material. HeLa/S10 Translation/Replication Reactions—Reactions were performed as described previously (22Barton D.J. Black E.P. Flanegan J.B. J. Virol. 1995; 69: 5516-5527Crossref PubMed Google Scholar) (method 3) with the following modifications. Nuclease-treated rabbit reticulocyte lysate (10% v/v) was used rather than initiation factors for both the translation and replication reactions. [α-32P]UTP rather than [α-32P]CTP was used for the replication reactions. Radioimmunoprecipitations were performed as follows. A 100-μl in vitro translation reaction set up in the presence of 1 mm guanidine HCl containing 50 μCi of [35S]methionine was allowed to proceed at 30 °C for 3 h after which 8 μl were removed into 80 μl of 1× SDS-PAGE sample buffer to serve as the translation control. 1 ml of RIPA buffer (50 mm Tris, pH 8, 250 mm NaCl, 1% Triton X-100, 0.1% SDS) was then added to the remainder of the reaction, and 100 μl of a 50% slurry of protein-G-agarose in RIPA buffer was added to preclear the lysate. This was rotated at room temperature for 15–30 min. Th
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