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• W2023355401 abstract "The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication. The primary oligomerization domain of poliovirus polymerase, 3Dpol, is stabilized by the interaction of the back of the thumb subdomain of one molecule with the back of the palm subdomain of a second molecule, thus permitting the head-to-tail assembly of 3Dpol monomers into long fibers. The interaction of Arg-455 and Arg-456 of the thumb with Asp-339, Ser-341, and Asp-349 of the palm is key to the stability of this interface. We show that mutations predicted to completely disrupt this interface do not produce equivalent growth phenotypes. Virus encoding a polymerase with changes of both residues of the thumb to alanine is not viable; however, virus encoding a polymerase with changes of all three residues of the palm to alanine is viable. Biochemical analysis of 3Dpol derivatives containing the thumb or palm substitutions revealed that these derivatives are both incapable of forming long fibers, suggesting that polymerase fibers are not essential for virus viability. The RNA binding activity, polymerase activity, and thermal stability of these derivatives were equivalent to that of the wild-type enzyme. The two significant differences observed for the thumb mutant were a modest reduction in the ability of the altered 3CD proteinase to process the VP0/VP3 capsid precursor and a substantial reduction in the ability of the altered 3Dpol to catalyze oriI-templated uridylylation of VPg. The defect to uridylylation was a result of the inability of 3CD to stimulate this reaction. Because 3C alone can substitute for 3CD in this reaction, we conclude that the lethal replication phenotype associated with the thumb mutant is caused, in part, by the disruption of an interaction between the back of the thumb of 3Dpol and some undefined domain of 3C. We speculate that this interaction may also be critical for assembly of other complexes required for poliovirus genome replication. RNA-dependent RNA polymerase Dulbecco's modified Eagle's medium N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 4-morpholinepropanesulfonic acid 4-morpholineethanesulfonic acid oligonucleotide symmetrical substrate polyethyleneimine phosphate-buffered saline nucleotide(s) guanidine HCl The RNA-dependent RNA polymerase (RdRP)1 is the key component of the replication machinery of RNA viruses. The RdRP from poliovirus (3Dpol) serves as a paradigm for this class of nucleic acid polymerases. The crystal structure for 3Dpol (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar) revealed that this polymerase has the typical topology observed for other nucleic acid polymerases and can be compared with a cupped, right hand with fingers, palm, and thumb subdomains (Fig. 1A). A unique feature of this polymerase, however, is the presence of two extensive regions of polymerase-polymerase interactions, referred to as interface I and interface II (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). Polymerase molecules interact in a “head-to-tail” fashion to form long, extended fibers via interface I, and these fibers interact with each other via interface II. Interface I is formed by an interaction between the back of the thumb of one polymerase molecule and the back of the palm of a second polymerase molecule (Fig.1B). A few of the critical interactions required for integrity of interface I are shown in Fig. 1C. Specifically, Arg-455 and Arg-456 of the thumb subdomain of one polymerase molecule interact with Asp-339, Ser-341, and Asp-349 of the palm subdomain of the second polymerase molecule (Fig. 1C). A variety of biochemical studies have provided additional evidence for oligomerization of 3Dpol. For example, filter-binding studies, performed in a manner that separates oligomers greater than 100 molecules from smaller assemblies, demonstrate a concentration-dependent increase in formation of large 3Dpol oligomers at low pH values (2Pata J.D. Schultz S.C. Kirkegaard K. RNA. 1995; 1: 466-477PubMed Google Scholar, 3Beckman M.T. Kirkegaard K. J. Biol. Chem. 1998; 273: 6724-6730Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 4Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar). In addition, glutaraldehyde cross-linking studies provide evidence for the existence of 3Dpol multimers (2Pata J.D. Schultz S.C. Kirkegaard K. RNA. 1995; 1: 466-477PubMed Google Scholar). Finally, 3Dpol exhibits cooperativity with respect to polymerase activity (2Pata J.D. Schultz S.C. Kirkegaard K. RNA. 1995; 1: 466-477PubMed Google Scholar). However, it should be noted that the observed cooperative nature of polymerase activity is not observed in all cases (5Arnold J.J. Cameron C.E. J. Biol. Chem. 1999; 274: 2706-2716Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and may either be substrate-dependent (5Arnold J.J. Cameron C.E. J. Biol. Chem. 1999; 274: 2706-2716Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) or a reflection of the absence or presence of certain divalent cations, in particular Zn2+ ions (4Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar). It is worth noting that the ability of 3Dpol molecules to interact has also been demonstrated by using the yeast two-hybrid system (6Hope D.A. Diamond S.E. Kirkegaard K. J. Virol. 1997; 71: 9490-9498Crossref PubMed Google Scholar, 7Xiang W. Cuconati A. Hope D. Kirkegaard K. Wimmer E. J. Virol. 1998; 72: 6732-6741Crossref PubMed Google Scholar). Clustered charged-to-alanine mutagenesis of 3Dpol identified residues Arg-455 and Arg-456 as residues essential for virus viability (8Diamond S.E. Kirkegaard K. J. Virol. 1994; 68: 863-876Crossref PubMed Google Scholar). Given the role of these residues in the stability of interface I (Fig.1C) (1Hansen J.L. Long A.M. Schultz S.C. Structure. 1997; 5: 1109-1122Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar), these data provided the first evidence that oligomerization of 3Dpol might play a significant role in some step of the virus multiplication cycle. Subsequent studies of additional 3Dpol derivatives with modifications of residues involved in forming interface I provided additional support for the importance of this interface for 3Dpol function and virus viability (4Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar). However, this study also presented an interesting paradox. Mutation of the residue that interacts with Arg-455, Asp-349, does not produce a lethal phenotype (4Hobson S.D. Rosenblum E.S. Richards O.C. Richmond K. Kirkegaard K. Schultz S.C. EMBO J. 2001; 20: 1153-1163Crossref PubMed Scopus (121) Google Scholar). In this study, we test the hypothesis that the lethal phenotype associated with mutations on the back of the thumb of 3Dpol arises from a requirement for this subdomain that is independent of oligomerization. We find that 1) oligomerization via interface I may not be essential for virus multiplication and 2) the back of the thumb of 3Dpol may interact with host and viral factors to modulate capsid protein processing and initiation of protein-primed RNA synthesis, respectively. The implications of heteromeric interactions between 3Dpol and another viral factor on the mechanism for negative-strand RNA synthesis will be discussed. DNA oligonucleotides were from Invitrogen and Integrated DNA Technologies, Inc.; T4 polynucleotide kinase, Deep Vent DNA polymerase, and restriction enzymes were from New England Biolabs, Inc.; shrimp alkaline phosphatase was from United States Biochemical Corp.; T4 DNA ligase and NZCYM were from Invitrogen; QIAEX was from Qiagen; Sephadex G-25 and RNase A were from Sigma; phosphocellulose (P-11) and DE-81 filter paper were from Whatman; all nucleotides (ultrapure solutions) and Q-Sepharose fast flow were from AmershamBiosciences; RNA oligonucleotides were from Dharmacon Research, Inc. (Boulder, CO); [α-32P]UTP (6000 Ci/mmol) was from PerkinElmer Life Sciences; [γ-32P]ATP (>7000 Ci/mmol) was from ICN; synthetic VPg peptide was from Alpha Diagnostic International (San Antonio, TX); all other reagents were available through Fisher or VWR. Oligos 15 and 16 (Table I lists all oligonucleotides used in this study) were used to amplify the region encoding 3CD by using PCR and the viral cDNA (pMoRA, also known as pXpA-rib+polyAlong (Ref. 9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar)) as template. The 3CD-coding region was cloned into pET26Ub-3D plasmid (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) by using theSacII and AflII sites to give pET26Ub-3CD. To inactivate the protease activity of 3CD, histidine 40 was changed to a glycine by using overlap-extension PCR with oligos 15–18 and pET26Ub-3CD plasmid as template. The product was cloned into pET26Ub-3CD to yield pET26Ub-3CD-H40G.Table IOligonucleotides used in this studya An NheI site was introduced via a silent mutation.b The T7 promoter is underlined. a An NheI site was introduced via a silent mutation. b The T7 promoter is underlined. Cloning of the 3C gene was achieved by using oligos 15 and 19–21 and the viral cDNA (pMoRA (Ref. 9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar)) as template in overlap-extension PCR. The region was cloned into pET26Ub-Chis plasmid by using theSacII and BamHI sites to yield pET26Ub-3C-C147G-Chis. “C147G” designates mutation of cysteine 147 to a glycine to inactivate protease activity. The pET26Ub-Chis plasmid is designed to produce a C-terminal GSSG-His6 tag for any protein cloned in by using the 3′ BamHI site. The pET26Ub expression plasmid for 3Dpol (pET26Ub-3D-BPKN) was previously described (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). DNA sequencing at the Pennsylvania State University Nucleic Acid Facility was used to verify the integrity of all clones. The thumb mutations of Arg-455 and Arg-456 in 3Dpol were introduced by using reverse oligonucleotides encoding the Ala (oligo 8), Ser (oligo 9), or Asp (oligo 10) substitutions and the forward oligonucleotide (oligo 7). PCR products were cloned into pET26Ub-3D-BPKN plasmid (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) using the NheI and EcoRI sites. Palm mutations of Asp-339, Ser-341, and Asp-349 to alanine in 3Dpol were introduced by using overlap extension PCR using oligos 11–14. The PCR product was cloned into pET26Ub-3D-BPKN plasmid (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar) using the KpnI and EcoRI sites. The thumb (R455A,R456A) and palm (D339A,S341A,D349A) mutations were introduced into 3CD by digesting the region containing the mutations in the pET26-Ub-3D-BPKN plasmid and ligating into the pET26Ub-3CD-H40G plasmid. The BstBI and EcoRI sites were used to introduce the thumb mutations into 3CD; theBstBI and MfeI sites were used to introduce the palm mutations into 3CD. DNA sequencing confirmed the integrity of all clones. All proteins expressed from the pET26Ub-based plasmids constructed above are N-terminally fused to yeast ubiquitin. Overexpression of protein in this system is performed in the BL21(DE3)pCG1 strain of Escherichia coli; this strain carries the pCG1 plasmid, which constitutively expresses a yeast ubiquitin protease that processes the ubiquitin fusion protein to produce the authentic N terminus (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). Expression, lysis, polyethyleneimine (PEI) precipitation, and ammonium sulfate precipitation for all 3Dpol and 3CD derivatives were performed as previously described (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar). After suspension of the ammonium sulfate pellets in buffer A (50 mm Tris, pH 8.0, 20% glycerol, 1 mm dithiothreitol, 0.1% Nonidet P-40, and 60 μm ZnCl2), the samples were dialyzed overnight against buffer A containing 10 mm NaCl. Spectra/Por dialysis tubing (Spectrum Laboratories Inc.) with a molecular mass cut-off of 12,000–14,000 Da was used. All of the steps of the purification were performed either on ice or at 4 °C. After dialysis, the conductivity of the protein was adjusted to 50 mm NaCl and the protein was loaded at 1 ml/min onto a phosphocellulose (P-11) column that was equilibrated with buffer A containing 50 mm NaCl. Approximately 1 ml of resin was used per 20 mg of total protein. Protein concentration was measured by using the Bio-Rad protein assay. The column was washed to baseline with buffer A containing 50 mm NaCl and protein was eluted by using a six-column volume, linear gradient from 50 to 350 mm NaCl in buffer A. Fractions (0.1 bed volume of the column) were collected and assayed for purity by SDS-PAGE. Conductivity of the pooled fractions was adjusted to 50 mm NaCl by using buffer A and the pooled fractions were loaded at 1 ml/min onto a Q-Sepharose column equilibrated with buffer A containing 50 mm NaCl. Again, 1 ml of resin was used per 20 mg of total protein. Washing was the same as for the P-11 column; protein was eluted using a six-column volume, linear gradient from 50 to 500 mm NaCl in buffer A. Fractions (0.1 bed volume of the column) were collected and assayed for purity by SDS-PAGE. Conductivity of the pooled fractions was adjusted to 50 mm NaCl using buffer A. This pool was loaded at 1 ml/min onto a 0.5-ml Q column equilibrated as described above. The column was washed to baseline using buffer B (50 mm HEPES, pH 7.5, 20% glycerol, 10 mm 2-mercaptoethanol, 0.1% Nonidet P-40, and 60 μm ZnCl2) containing 50 mm NaCl. Protein was eluted from this Q column by using buffer B containing 500 mm NaCl. Fractions (0.5 ml) were collected until the concentration of the eluted protein fell below the desired value. Protein concentration was determined by using the following extinction coefficients: 0.071830 μm−1·cm−1 (3Dpol) and 0.079510 μm−1·cm−1 (3CD). These values were determined by using the protein parameters tool on the ExPASy site (us.expasy.org/tools/protparam.html). The absorbance values were measured at 280 nm in 6 m guanidine HCl (GdnHCl), pH 6.5. The conductivity of the individual fractions was measured; the fractions were aliquoted and stored at −80 °C. 3C was expressed essentially as described for 3Dpol and 3CD (10Gohara D.W., Ha, C.S. Kumar S. Ghosh B. Arnold J.J. Wisniewski T.J. Cameron C.E. Protein Expr. Purif. 1999; 17: 128-138Crossref PubMed Scopus (116) Google Scholar); however, a different lysis buffer was employed (50 mmHEPES, pH 7.5, 20% glycerol, 10 mm 2-mercaptoethanol, 5.6 μg/ml pepstatin A, 4 μg/ml leupeptin, 0.1 mm EDTA, and 500 mm NaCl). Phenylmethylsulfonyl fluoride and Nonidet P-40 were added after lysis to a final concentration of 1 mm and 0.1% (v/v), respectively. PEI was added to a final concentration of 0.25% (v/v); the lysate was stirred slowly at 4 °C for 30 min and then centrifuged in a Beckman Ti-60 rotor for 30 min at 30,000 rpm at 4 °C. The conductivity of the PEI supernatant was adjusted to 50 mm NaCl, and the supernatant was passed through a Q-Sepharose column followed in tandem by a P-11 column. Both columns were equilibrated with buffer C (50 mm HEPES, pH 7.5, 20% glycerol, 10 mm 2-mercaptoethanol, and 0.1% Nonidet P-40) containing 50 mm NaCl prior to loading. Approximately 1 ml of resin/80 mg of total protein was used for the Q column, and ∼1 ml of resin/500 mg of total protein was used for the P-11 column. After all of the protein passed through the Q column, the P-11 column was detached and washed with 10 column volumes of buffer C containing 50 mm NaCl. 3C was eluted from the P-11 column by using buffer C containing 2 m NaCl. Fractions (1 ml) were eluted until the concentration of the eluted protein fell below the desired value. The high NaCl concentration was reduced from the fractions by dialysis against buffer C containing 250 mm NaCl using Spectra/Por dialysis tubing with a molecular mass cut-off of 6000–8000 Da. SDS-PAGE was used to assess the purity of the eluted fractions. Protein concentration was determined as described for 3Dpol and 3CD; the extinction coefficient used was 0.007680 μm−1·cm−1. The conductivity of the individual fractions was measured; the fractions were aliquoted and stored at −80 °C. Introduction of mutations into the thumb subdomain in the viral cDNA required overlap extension PCR by using oligos 1–4; the viral cDNA, pMoRA (9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar), was used as the template. The PCR product was digested with MfeI and EcoRI and subcloned into the intermediate plasmid, pUC18-BglII-EcoRI-3CD (referred to as pUC-3CD in Ref. 11Gohara D.W. Crotty S. Arnold J.J. Yoder J.D. Andino R. Cameron C.E. J. Biol. Chem. 2000; 275: 25523-25532Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). From this subclone, the fragment between BglII and EcoRI was cloned into the viral cDNA (pMoRA (Ref. 9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar)) to yield the thumb mutant viral cDNA (pMoRA-R455A,R456A). For the introduction of mutations to the palm subdomain, PCR amplification from the expression vector containing the palm mutations (pET26Ub-3D-BPKN-D339A,S341A,D349A) was performed using oligos 5 and 6; the fragment between BstBI and MfeI was subcloned into the pUC18-BglII-EcoRI-3CD intermediate and from there to the viral cDNA using the BglII and EcoRI sites to yield the palm mutant viral cDNA (pMoRA-D339A,S341A,D349A). To introduce mutations into the replicon, the fragment between the BglII and ApaI sites from the mutated viral cDNAs was cloned into pRLucRA (also known as pRLuc31-rib+polyAlong (Refs. 9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar and 12Andino R. Rieckhof G.E. Achacoso P.L. Baltimore D. EMBO J. 1993; 12: 3587-3598Crossref PubMed Scopus (407) Google Scholar)) to yield pRLucRA-R455A,R456A and pRLucRA-D339A,S341A,D349A. DNA sequencing was used to verify the integrity of all clones. RNA transcripts were generated according to the instructions of the manufacturer for the T7-MEGAscript kit (Ambion, Inc.) after linearization with EcoRI. DNase treatment was used to remove the template; lithium chloride precipitation was used to remove unincorporated nucleotides. RNA concentration was calculated by measuring absorbance at 260 nm, assuming an A260 of 1 was equivalent to 40 μg/ml. HeLa cells were propagated in DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), always keeping the cultures between 20 and 80% confluence. Subconfluent monolayers of HeLa cells were detached from the culture flasks by trypsin treatment, washed with 1× phosphate-buffered saline (PBS), and cell number adjusted to 3 × 106 cells/ml in PBS. Cell suspension (400 μl) was mixed with 10 μg of RNA (wild-type (pMoRA), thumb mutant (pMoRA-R455A,R456A), or palm mutant (pMoRA-D339A,S341A,D349A) viral transcripts) in a microcentrifuge tube, transferred to an electroporation cuvette (0.2-cm gap width; Bio-Rad) and subjected to an electric pulse at 500 microfarads and 130 V using a Gene Pulser system (Bio-Rad). Electroporated cells were diluted either 10- or 100-fold in PBS, and 100 μl of each dilution were plated on 2 × 105 HeLa cells (prepared 1 day in advance) in six-well dishes; 400 μl of DMEM/F-12 were added to each well. Undiluted electroporated cells (100 μl) were also plated using the same procedure. Cells were allowed to adsorb to the plate for 1 h at 37 °C, and then the medium/PBS was aspirated; the cells were covered with 3 ml of a mixture of 1× DMEM/F-12 plus 10% fetal bovine serum and 1% low melting point agarose (American Bioanalytical). Plates were then incubated at 37 °C for 3 days. The agarose overlay was removed by using a spatula. Wells were stained with crystal violet, and viral plaques were counted. RNA transcripts were generated as described for the viral genomes from ApaI-linearized plasmids encoding the wild-type (pRLucRA (Refs. 9Herold J. Andino R. J. Virol. 2000; 74: 6394-6400Crossref PubMed Scopus (122) Google Scholar and 12Andino R. Rieckhof G.E. Achacoso P.L. Baltimore D. EMBO J. 1993; 12: 3587-3598Crossref PubMed Scopus (407) Google Scholar)), the thumb mutant (pRLucRA-R455A,R456A) or the palm mutant (pRLucRA-D339A,S341A, D349A) subgenomic replicons. HeLa cells were propagated as described for the infectious center assays. HeLa cells were transfected with subgenomic replicons (20 μg) by using electroporation. Electroporated cells were immediately transferred to prewarmed (37 °C) DMEM/F-12 as follows. The volume of the electroporated cells added was calculated by multiplying 33 μl byn + 1 (where n is the number of time points to be measured). The volume of DMEM/F-12 to which the electroporated cells were added was calculated by multiplying 500 μl by n + 1 (where n is the number of time points to be measured). After mixing the appropriate volume of electroporated cells with the appropriate volume of medium, 500-μl aliquots were prepared in microcentrifuge tubes for each time point to be measured. These aliquots were then incubated in a water bath at 37 °C without any agitation. At fixed time points, cells were pelleted by centrifugation at 14,000 × g for 2 min in an Eppendorf microcentrifuge. Lysis was performed by using 100 μl of 1× cell culture lysis reagent (Promega) and placed on ice for 2 min before removal of cellular debris and nuclei by centrifugation at 14,000 × g for 1 min. Lysates were left on ice at 4 °C until all time points were collected. Lysates were assayed for luciferase activity by mixing 10 μl of lysate with 10 μl of luciferase assay substrate (Promega) and quantifying light production by using a Lumat LB 9501 luminometer (Berthold). HeLa cell S10 extract (S10) and HeLa cell translation initiation factors were prepared as previously described (14Barton D.J. Morasco B.J. Flanegan J.B. Methods Enzymol. 1996; 275: 35-57Crossref PubMed Scopus (59) Google Scholar). HeLa S10 translation-replication reaction mixtures contained 50% by volume S10, 20% by volume initiation factors, 10% by volume 10× nucleotide reaction mix (10 mm ATP, 2.5 mm GTP, 2.5 mm UTP, 600 mm KCH3CO2, 300 mmcreatine phosphate, 4 mg/ml creatine kinase, and 155 mmHEPES-KOH, pH 7.4), 2 mm GdnHCl, and viral mRNA at 50 μg/ml. Poliovirus mRNA translation was assayed by including [35S]methionine (1.2 mCi/ml;Amersham Biosciences) in HeLa S10 translation-replication reaction mixtures. After 3 h of incubation at 34 °C, samples (4 μl) of the HeLa S10 translation-replication reaction mixtures containing [35S]methionine were mixed with 100 μl of SDS-PAGE sample buffer (2% SDS (Sigma), 62.5 mm Tris-HCl, pH 6.8, 0.5% 2-mercaptoethanol, 0.1% bromphenol blue, 20% glycerol). The samples were heated at 100 °C for 5 min, and 25-μl portions of each sample were loaded onto a 0.75-mm-thick polyacrylamide gel (29:1, acrylamide:bisacrylamide) consisting of a 4% stacking gel and a 9–18% gradient separating gel. The gels were fixed and dried; radiolabeled proteins were detected by phosphorimaging. Poliovirus RNA synthesis was assayed using preinitiation RNA replication complexes formed in HeLa S10 translation-replication reaction mixtures as previously described (15Barton D.J. Flanegan J.B. J. Virol. 1997; 71: 8482-8489Crossref PubMed Google Scholar). Briefly, viral RNAs (50 μg/ml) were incubated in S10 reaction mixtures containing 2 mm GdnHCl for 3 h at 34 °C. Preinitiation complexes were isolated from the reactions by centrifugation at 13,000 × g for 15 min at 4 °C. Pellets containing preinitiation complexes were then suspended in 50-μl labeling reaction mixtures containing [α-32P]CTP and incubated at 37 °C for 60 min as previously described (method 4 in Ref. 15Barton D.J. Flanegan J.B. J. Virol. 1997; 71: 8482-8489Crossref PubMed Google Scholar). Under these conditions, radiolabel was incorporated into nascent negative- and positive-strand RNA as it was synthesized. The reactions were centrifuged at 13,000 × g to pellet the viral RNA replication complexes. Radiolabeled viral RNA remained in the replication complexes. 2D. J. Barton, unpublished results. The supernatant, containing unincorporated radiolabel, was discarded. The pellets were solubilized in SDS sample buffer. The products of the reaction were phenol/chloroform-extracted, ethanol-precipitated, and separated by electrophoresis in a 1% agarose MOPS formaldehyde gel. RNA in the gels was stained with ethidium bromide and visualized by UV light. 32P-Labeled poliovirus RNA was detected and quantified by using a PhosphorImager (Bio-Rad). VPg uridylylation was assayed using preinitiation RNA replication complexes. Preinitiation RNA replication complexes were isolated from HeLa S10 translation-replication reaction material using the same procedure as described above for poliovirus RNA synthesis. The preinitiation RNA replication complexes were suspended in 50 μl of labeling reaction mixture containing [α-32P]UTP rather than [α-32P]CTP. The reaction mixtures were incubated for 60 min at 37 °C. Following incubation, the reaction mixtures were centrifuged at 13,000 × g to pellet the viral RNA replication complexes. Radiolabeled VPgpUpU and radiolabeled viral RNA remained in the replication complexes and were not released into the soluble portion of the reaction mixtures.2 The supernatant, containing unincorporated radiolabel, was discarded. The pellets, containing radiolabeled viral RNA and uridylylated VPg, were solubilized in SDS sample buffer. The samples were fractionated by electrophoresis in a 10% polyacrylamide Tris-Tricine gel, and radiolabeled VPgpUpU was detected by phosphorimaging. The RNA oligonucleotide was end-labeled by using [γ-32P]ATP and T4 polynucleotide kinase essentially as specified by the manufacturer. Reactions, typically 20 μl, contained 0.1 μm[γ-32P]ATP, 10 μm RNA oligonucleotide (rU30), 1× kinase buffer, and 0.4 units/μl T4 polynucleotide kinase. Reactions were incubated at 37 °C for 60 min and quenched by incubation at 65 °C for 5 min. Reaction mixtures, typically 50 μl, contained 10 nm trace-labeled rU30 and wild-type or mutant 3Dpol derivatives of varying concentrations in reaction buffer (25 mm MES-NaOH, pH 5.5, 60 mmNaCl, 5 mm MgCl2, 0.1 mmZnSO4 5 mm dithiothreitol, 0.25 mmATP, 20% glycerol). 3Dpol was diluted immediately prior to use in enzyme dilution buffer (50 mm HEPES, pH 7.5, 10 mm 2-mercaptoethanol, 60 μm ZnCl2and 20% glycerol). Binding reactions were initiated by the addition of 5 μl of the diluted 3Dpol to the remaining components. Reactions were incubated on ice for 30 min. This assay employs three filters: polysulfone membrane, which traps any protein-nucleic acid complexes larger than 0.45 μm; a nitrocellulose membrane, which traps all other protein-nucleic acid complexes; and a nylon membrane, which binds all unbound nucleic acid. Membranes were presoaked in wash buffer (50 mm HEPES, pH 7.5, and 5 mm MgCl2) and assembled, in order from top to bottom, polysulfone, nitrocellulose, and nylon, in a slot blotter (Invitrogen). After assembly," @default.
• W2023355401 created "2016-06-24" @default.
• W2023355401 creator A5014881760 @default.
• W2023355401 creator A5015247627 @default.
• W2023355401 creator A5021526153 @default.
• W2023355401 creator A5026543837 @default.
• W2023355401 creator A5034083570 @default.
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• W2023355401 creator A5052829441 @default.
• W2023355401 creator A5063693441 @default.
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• W2023355401 date "2002-08-01" @default.
• W2023355401 modified "2023-09-28" @default.
• W2023355401 title "Structure-Function Relationships of the RNA-dependent RNA Polymerase from Poliovirus (3Dpol)" @default.
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• W2023355401 cites W1973711348 @default.
• W2023355401 cites W197507937 @default.
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• W2023355401 cites W2030009404 @default.
• W2023355401 cites W2031536871 @default.
• W2023355401 cites W2031926002 @default.
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• W2023355401 cites W2071460423 @default.
• W2023355401 cites W2092307225 @default.
• W2023355401 cites W2093191777 @default.
• W2023355401 cites W2095425492 @default.
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• W2023355401 cites W2118848130 @default.
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