Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds
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{"title"=>"Fidelity Variants of RNA Dependent RNA polymerases uncover an indirect, mutagenic activity of amiloride compounds", "type"=>"journal", "authors"=>[{"first_name"=>"Laura I.", "last_name"=>"Levi", "scopus_author_id"=>"56354380400"}, {"first_name"=>"Nina F.", "last_name"=>"Gnädig", "scopus_author_id"=>"57194735878"}, {"first_name"=>"Stéphanie", "last_name"=>"Beaucourt", "scopus_author_id"=>"8747038900"}, {"first_name"=>"Malia J.", "last_name"=>"McPherson", "scopus_author_id"=>"36930873400"}, {"first_name"=>"Bruno", "last_name"=>"Baron", "scopus_author_id"=>"7103355524"}, {"first_name"=>"Jamie J.", "last_name"=>"Arnold", "scopus_author_id"=>"7403060858"}, {"first_name"=>"Marco", "last_name"=>"Vignuzzi", "scopus_author_id"=>"8590949500"}], "year"=>2010, "source"=>"PLoS Pathogens", "identifiers"=>{"sgr"=>"78449244303", "scopus"=>"2-s2.0-78449244303", "doi"=>"10.1371/journal.ppat.1001163", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "pui"=>"359979603", "issn"=>"15537366", "pmid"=>"21060812"}, "id"=>"a0bd69f3-13d0-3d41-9d62-6cbbf265b526", "abstract"=>"In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg²+ and Mn²+, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.", "link"=>"http://www.mendeley.com/research/fidelity-variants-rna-dependent-rna-polymerases-uncover-indirect-mutagenic-activity-amiloride-compou", "reader_count"=>49, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>4, "Researcher"=>17, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>2, "Student > Doctoral Student"=>4, "Researcher"=>17, "Student > Ph. D. Student"=>18, "Student > Postgraduate"=>1, "Student > Master"=>2, "Student > Bachelor"=>4, "Professor"=>1}, "reader_count_by_subject_area"=>{"Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>4, "Agricultural and Biological Sciences"=>35, "Medicine and Dentistry"=>2, "Chemistry"=>4, "Social Sciences"=>1, "Computer Science"=>1, "Immunology and Microbiology"=>1}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Chemistry"=>{"Chemistry"=>4}, "Social Sciences"=>{"Social Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>35}, "Computer Science"=>{"Computer Science"=>1}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>4}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United States"=>2, "Italy"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/409530", "https://ndownloader.figshare.com/files/409552", "https://ndownloader.figshare.com/files/409581", "https://ndownloader.figshare.com/files/409605"], "description"=>"<div><p>In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg<sup>2+</sup> and Mn<sup>2+</sup>, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.</p> </div>", "links"=>[], "tags"=>["fidelity", "variants", "rna", "polymerases", "mutagenic", "amiloride", "compounds"], "article_id"=>140892, "categories"=>["Medicine", "Cancer", "Cell Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.s001", "https://dx.doi.org/10.1371/journal.ppat.1001163.s002", "https://dx.doi.org/10.1371/journal.ppat.1001163.s003", "https://dx.doi.org/10.1371/journal.ppat.1001163.s004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Fidelity_Variants_of_RNA_Dependent_RNA_Polymerases_Uncover_an_Indirect_Mutagenic_Activity_of_Amiloride_Compounds/140892", "title"=>"Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2010-10-28 00:14:52"}
  • {"files"=>["https://ndownloader.figshare.com/files/821582"], "description"=>"<p>(<b>A</b>) Optimization of the RNA mutagen treatment concentrations to select for resistant CVB3 variants. HeLa cells, treated with different concentrations of either ribavirin or AZC were infected with wild type CVB3 at MOI of 0.01. 48 hours after infection, progeny virus was quantified by TCID<sub>50</sub> assay. The percentage of viruses surviving treatment (y-axis) with 50, 100, 200, 300 or 400 µM of ribavirin or AZC (x-axis) was determined by dividing the virus titers of treated populations by virus titers in untreated controls (0 µM). Mean values ± S.E.M are shown, N = 4, * P<0.01, ** P<0.001, ***P<0.0001. (<b>B</b>) Isolation of RNA mutagen resistant CVB3. CVB3 was passaged 20 times in 50 µM ribavirin, 50 µM AZC or mock treated HeLa cells. Every 5 passages the virus population was sequenced. The emergence of a single point mutation resulting in a A372V change in the RdRp is indicated by a solid circle. (<b>C</b>) A372V is resistant to three different base analog RNA mutagens. HeLa cells treated with 300 µM of ribavirin or AZC, or 150 µM FU, were infected with either wild type CVB3 (black bars) or A372V variant (gray bars) at an MOI of 0.01. Control infections (no drug) were also performed. At 48 hours after infection, progeny virus was titered by TCID<sub>50</sub> assay. Mean virus titers ± S.E.M are shown, N = 8, * P<0.05, ** P<0.01, *** P<0.001. (<b>D</b>) Schematic of the viral RNA genome showing the 5′ untranslated region, structural capsid coding region, P2 and P3 region of non structural proteins, including the RdRp (shaded gray) and the 3′ untranslated region. A 1.3 kb region of the viral capsid was RT-PCR amplified and subcloned for sequencing of individual clones to obtain the observed mutation frequencies presented throughout this work. The total number of clones and nucleotides sequenced in each population is shown in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#ppat.1001163.s004\" target=\"_blank\">Table S1</a>. (<b>E</b>) Average mutation frequencies of each viral population shown as the mean number of mutations per 10<sup>4</sup> nucleotides sequenced. Statistical analysis is described in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#s4\" target=\"_blank\">Methods</a>. * P<0.05 statistical significance of difference between WT and A372V mutation frequencies.</p>", "links"=>[], "tags"=>["rna", "mutagen", "resistant", "cvb3", "fidelity"], "article_id"=>491948, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Isolation_of_an_RNA_mutagen_resistant_CVB3_with_a_high_fidelity_RdRp_/491948", "title"=>"Isolation of an RNA mutagen resistant CVB3 with a high fidelity RdRp.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:32:28"}
  • {"files"=>["https://ndownloader.figshare.com/files/821674"], "description"=>"<p>(<b>A</b>) A radiolabeled primer/template is used to measure the incorporation of bases in vitro using purified RdRp enzymes, conditions detailed in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#s4\" target=\"_blank\">Methods</a>. The templating U (in bold) permits the quantification of incorporation of the correct (ATP) or incorrect (GTP) nucleotide over time. (<b>B</b>) Visualization of GTP mis-incorporation by wild type, A372V and S299T RdRp. The incorporation of GTP is monitored over time (m) as the accumulation of elongation product (n+1) minus blank control. (<b>C</b>) Quantification of relative incorporation product at 2 and 5 minutes reaction time.</p>", "links"=>[], "tags"=>["biochemical", "assays", "higher", "incorporation", "fidelities", "cvb3"], "article_id"=>492037, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_In_vitro_biochemical_assays_confirm_the_higher_A372V_and_lower_S299T_incorporation_fidelities_of_CVB3_variants_/492037", "title"=>"<i>In vitro</i> biochemical assays confirm the higher (A372V) and lower (S299T) incorporation fidelities of CVB3 variants.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:33:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/821749"], "description"=>"<p>(<b>A</b>) A372V is more resistant than wild type in increasing concentrations of amiloride. HeLa cells were treated with different concentrations of amiloride and infected with wild type or A372V virus at an MOI of 0.01. At 48 h, viable progeny virus was quantified by TCID<sub>50</sub> assay. The mean virus titers (TCID<sub>50</sub>/ml) and S.E.M are shown, N = 3, * P<0.05. (<b>B</b>) One-step growth kinetics of wild type and A372V viruses. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID<sub>50</sub> assay. Mean titers (TCID<sub>50</sub>/ml) ± S.E.M are shown, N = 3, no significant difference found. (<b>C</b>) The relative increase in signal from time 0 to indicated time points is shown, values are means of 2 separate northen blots of samples from (B), error bars show range of values (<b>D</b>) One-step growth kinetics of wild type and A372V viruses in the presence of 400 µM amiloride. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID<sub>50</sub> assay. Mean titers (TCID<sub>50</sub>/ml) ± S.E.M are shown, N = 3, no significant differences found except *, p = 0.03. (<b>E</b>) Northern blot analysis of RNA synthesis determined 48 hours after infection with wild type or A372V virus at MOI of 0.01 in the presence or the absence of 400 µM amiloride. Two independent treatment samples per virus are shown.</p>", "links"=>[], "tags"=>["amiloride"], "article_id"=>492116, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_A372V_resistance_to_amiloride_does_not_involve_improved_replication_/492116", "title"=>"A372V resistance to amiloride does not involve improved replication.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:35:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/821899"], "description"=>"<p>(<b>A</b>) The mutation frequencies of wild type and A372V viruses grown in the presence of 400 µM amiloride were determined, shown as the mean number of mutations per 10<sup>4</sup> nucleotides sequenced. * P<0.05 statistically significant difference between WT and A372V. Asterisks in white indicate statistically significant difference between amiloride-treated virus and the untreated parental population, *** P<0.001. (<b>B</b>) Mutations frequencies of wild type and A372V viruses in the presence of other amiloride compounds. HeLa cells were treated with 40 µM of EIPA, 25 µM of MIA or 20 µM of benzamil and were infected with wild type CVB3 virus or A372V variant (EIPA only). Statistical significance of differences in mutation frequencies are indicated. ns = not significant, * P<0.05, ** P<0.005, **** P<0.0001. (<b>C</b>) Mutation profiles of wild type and A372V populations grown in the absence of drug or presence of 400 µm ribavirin or amiloride in (A). The most commonly occurring mutations are indicated in bold. (<b>D</b>) Mutation frequency of wild type and G64S polioviruses. Viral RNA genomes were extracted following infection of HeLa cells, grown in standard conditions or treated with 400 µM amiloride. A 1.0 kb region of the viral capsid was RT-PCR amplified and subcloned for sequencing of individual clones to obtain the observed mutation frequencies (<a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#ppat.1001163.s004\" target=\"_blank\">Table S1</a>). Asterisks in white indicate statistical significance between drug treated populations and the same untreated parental virus. * P<0.05, ** P<0.005. (<b>E</b>) The percentage of polioviruses surviving amiloride treatment relative to untreated control populations was determined by plaque assay. The mean values ± S.E.M are shown, N = 3. ** P<0.005.</p>", "links"=>[], "tags"=>["rna", "mutagenic", "fidelity", "rdrp", "variants", "picornaviruses"], "article_id"=>492264, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Amiloride_has_RNA_mutagenic_activity_to_which_high_fidelity_RdRp_variants_of_picornaviruses_resist_/492264", "title"=>"Amiloride has RNA mutagenic activity to which high fidelity RdRp variants of picornaviruses resist.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:37:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/821998"], "description"=>"<p>(<b>A</b>) S299T resists amiloride's antiviral activity better than wild type virus. HeLa cells were treated with different concentrations of amiloride and infected with wild type or S299T virus at an MOI of 0.01. At 48 hours, viable progeny virus was quantified by TCID<sub>50</sub> assay. The mean virus titers (TCID<sub>50</sub>/ml) and S.E.M are shown, N = 3, * P<0.05. (<b>B</b>) One-step growth kinetics of wild type and S299T viruses. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID<sub>50</sub> assay. Mean titers (TCID<sub>50</sub>/ml) ± S.E.M are shown, N = 3, no significant difference found. (<b>C</b>) The relative increase in signal from time 0 to indicated time points is shown, values are means of 2 separate northern blots of samples from (B), error bars show range of values (<b>D</b>) One-step growth kinetics of wild type and S299T viruses in the presence of 400 µM amiloride. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID<sub>50</sub> assay. Mean titers (TCID<sub>50</sub>/ml) ± S.E.M are shown, N = 3, no significant differences found. (<b>E</b>) Northern blot analysis of RNA synthesis determined 48 hours after infection with wild type or S299T (MOI = 0.01) virus in the presence or the absence of 400 µM amiloride. Two independent treatment samples per virus are shown. (<b>F</b>) Average mutation frequencies of S299T untreated population and treated with ribavirin, amiloride or EIPA. *** P<0.0001; ns = no statistically significant difference. NB: All data from <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#ppat-1001163-g005\" target=\"_blank\">Figure 5</a> was obtained simultaneously with that for <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001163#ppat-1001163-g003\" target=\"_blank\">Figure 3</a> and separated for clarity of presentation, hence, wild type and A372V data is the same for both figures, except for (C).</p>", "links"=>[], "tags"=>["polymerase", "resistant", "inhibitory", "amiloride", "rna", "synthesis", "mutagenic"], "article_id"=>492353, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Another_polymerase_variant_S299T_is_resistant_to_both_the_inhibitory_effect_of_amiloride_on_RNA_synthesis_as_well_as_its_mutagenic_activity_/492353", "title"=>"Another polymerase variant, S299T, is resistant to both the inhibitory effect of amiloride on RNA synthesis as well as its mutagenic activity.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:39:13"}
  • {"files"=>["https://ndownloader.figshare.com/files/822095"], "description"=>"<p>(<b>A</b>) Dose dependence of virus titer decrease and mutation frequency increase with ribavirin treatment. The reduction in wild type virus yield (solid lines, left y axis) is shown as a function of the percentage of viruses surviving treatment with different concentrations of ribavirin. The increase in mutation frequency (dashed lines, right y-axis) of the starting population at these same drug concentrations is shown. (<b>B</b>) Dose dependence of amiloride treatment, as in (A). (<b>C</b>) Inhibition of RNA synthesis by amiloride is dose dependent. Northern blot analysis of RNA synthesis of wild type virus treated with different concentrations of amiloride. Cells were infected at MOI of 0.01 and RNA was extracted 48 hours after infection. (<b>D</b>–<b>F</b>) Treatment with Mg<sup>2+</sup> and Mn<sup>2+</sup>, but not Na<sup>+</sup> or Ca<sup>2+</sup>, increase the mutation frequency of wild type (D) and A372V (E), but not S299T (F). Cells were infected at MOI of 0.01 with virus in media supplemented with the indicated concentrations of salts and mutation frequencies were determined in progeny populations 48 hours after infection. * P<0.05, ** P<0.01, *** P<0.0001; ns = no statistically significant difference. (<b>G</b>) Passage of wild type virus in high concentrations of Mg<sup>2+</sup> and Mn<sup>2+</sup> selects for high fidelity A372V. Virus was passaged 20 times in 5 mM MgCl<sub>2</sub>, 1 mM MnCl<sub>2</sub> or regular media. At the indicated passage numbers the virus population was sequenced. The emergence of a single point mutation resulting in a A372V change is indicated by a solid circle.</p>", "links"=>[], "tags"=>["mutagenic", "amiloride", "antiviral", "correlates", "increases", "intracellular", "divalent", "cation"], "article_id"=>492455, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_The_mutagenic_activity_of_amiloride_is_an_indirect_secondary_antiviral_effect_that_correlates_with_increases_in_intracellular_divalent_cation_Mg_2_or_Mn_2_concentrations_/492455", "title"=>"The mutagenic activity of amiloride is an indirect, secondary antiviral effect that correlates with increases in intracellular divalent cation (Mg<sup>2+</sup> or Mn<sup>2+</sup>) concentrations.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2010-10-28 00:40:55"}
  • {"files"=>["https://ndownloader.figshare.com/files/822177"], "description"=>"a<p>1 mM ATP, determined from stopped-flow fluorescence assay for AMP incorporation.</p>b<p>5 mM GTP, determined from <sup>32</sup>P-labeled primer-extension assay for GMP misincorporation.</p>c<p>k<sub>obs correct</sub>/k<sub>obs incorrect</sub>.</p>", "links"=>[], "tags"=>["kinetics", "fidelity", "cvb3", "rdrp"], "article_id"=>492539, "categories"=>["Virology", "Medicine", "Plant Biology"], "users"=>["Laura I. Levi", "Nina F. Gnädig", "Stéphanie Beaucourt", "Malia J. McPherson", "Bruno Baron", "Jamie J. Arnold", "Marco Vignuzzi"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.1001163.t001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Incorporation_kinetics_and_relative_fidelity_of_CVB3_RdRp_variants_/492539", "title"=>"Incorporation kinetics and relative fidelity of CVB3 RdRp variants.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2010-10-28 00:42:19"}

PMC Usage Stats | Further Information

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