A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.


Abstract

Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.

Submission Details

ID: VUPsxLdA4

Submitter: Shu-Ching Ou

Submission Date: July 31, 2018, 4:43 p.m.

Version: 1

Publication Details
Qi H;Olson CA;Wu NC;Ke R;Loverdo C;Chu V;Truong S;Remenyi R;Chen Z;Du Y;Su SY;Al-Mawsawi LQ;Wu TT;Chen SH;Lin CY;Zhong W;Lloyd-Smith JO;Sun R,PLoS Pathog (2014) A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity. PMID:24722365
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2LIF 2012-07-11 Solution Structure of KKGF
2LVG 2013-06-19 NMR structure of HCV Non-structural protein AB, NS4B(1-40)
2KZQ 2011-03-02 s34r Structure
2XYM 2011-01-12 1.77 HCV-JFH1 NS5B T385A mutant
4AEP 2012-05-02 1.8 HCV-JFH1 NS5B POLYMERASE STRUCTURE AT 1.8 ANGSTROM
2XWH 2011-01-12 1.8 HCV-J6 NS5B polymerase structure at 1.8 Angstrom
2XXD 2011-01-12 1.88 HCV-JFH1 NS5B polymerase structure at 1.9 angstrom
4ADP 2012-05-02 1.9 HCV-J6 NS5B POLYMERASE V405I MUTANT
5NPJ 2017-05-24 1.9 Structure of the Hepatitis C virus strain JFH1 glycoprotein E2 antigenic region 532-540 bound to the single chain variable fragment of the non-neutralizing antibody DAO5
1YUY 2005-03-22 1.9 HEPATITIS C VIRUS NS5B RNA-DEPENDENT RNA POLYMERASE GENOTYPE 2a
4J1V 2014-08-06 1.95 Functional and structural studies of MOBKL1B, a Salvador/Warts/Hippo tumor suppressor pathway, in HCV replication
5TWM 2017-03-15 1.97 CRYSTAL STRUCTURE OF THE HEPATITIS C VIRUS GENOTYPE 2A STRAIN JFH1 L30S NS5B RNA-DEPENDENT RNA POLYMERASE IN COMPLEX WITH 5-[3-(tert-butylcarbamoyl)phenyl]-6-(ethylamino)-2-(4-fluorophenyl)-N-methylfuro[2,3-b]pyridine-3-carboxamide
1YVX 2005-03-22 2.0 Hepatitis C Virus RNA Polymerase Genotype 2a In Complex With Non- Nucleoside Analogue Inhibitor
4WTL 2015-02-11 2.0 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-UACC, RNA PRIMER 5'-PGG, MN2+, AND UDP
5QJ0 2018-11-21 2.08 CRYSTAL STRUCTURE OF THE HEPATITIS C VIRUS GENOTYPE 2A STRAIN JFH1 NS5B RNA-DEPENDENT RNA POLYMERASE IN COMPLEX WITH 6-[ethyl(methylsulfonyl)amino]-2-(4-fluorophenyl)-N-methyl-5-(3-{[1-(pyrimidin-2-yl)cyclopropyl]carbamoyl}phenyl)-1-benzofuran-3-carboxamide
4WTM 2015-02-11 2.15 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-UAGG, RNA PRIMER 5'-PCC, MN2+, AND UDP
5QJ1 2018-11-21 2.17 CRYSTAL STRUCTURE OF THE HEPATITIS C VIRUS GENOTYPE 2A STRAIN JFH1 L30S NS5B RNA-DEPENDENT RNA POLYMERASE IN COMPLEX WITH 6-(ethylamino)-2-(4-fluorophenyl)-5-(3-{[1-(5-fluoropyrimidin-2-yl)cyclopropyl]carbamoyl}-4-methoxyphenyl)-N-methyl-1-benzofuran-3-carboxamide
3I5K 2009-09-22 2.2 Crystal structure of the NS5B polymerase from Hepatitis C Virus (HCV) strain JFH1
1YVZ 2005-03-22 2.2 Hepatitis C Virus RNA Polymerase Genotype 2a In Complex With Non- Nucleoside Analogue Inhibitor
4WTJ 2015-02-11 2.2 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-AUCC, RNA PRIMER 5'-PGG, MN2+, AND ADP
4AEX 2012-05-02 2.41 HCV-JFH1 NS5B POLYMERASE STRUCTURE AT 2.4 ANGSTROM in a primitive orthorhombic space group
4WT9 2015-02-11 2.5 APO CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH E86Q E87Q S15G C223H V321I AND DELTA8 MUTATIONS
1YV2 2005-03-22 2.5 Hepatitis C virus NS5B RNA-dependent RNA Polymerase genotype 2a
4WTK 2015-02-11 2.5 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-AGCC, RNA PRIMER 5'-PGG, MN2+, AND CDP
4OBC 2014-09-10 2.5 Crystal structure of HCV polymerase NS5b genotype 2a JFH-1 isolate with the S15G, C223H, V321I resistance mutations against the guanosine analog GS-0938 (PSI-3529238)
4E76 2012-04-18 2.5 Apo crystal structure of HCV NS5B genotype 2A JFH-1 isolate with beta hairpin loop deletion
4WTF 2015-02-11 2.65 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH GS-639475, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-CAAAAUUU
4WTD 2015-02-11 2.7 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH ADP, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-AUAAAUUU
4WTC 2015-02-11 2.75 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH CDP, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-AGAAAUUU
4WTI 2015-02-11 2.8 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS IN COMPLEX WITH RNA TEMPLATE 5'-ACGG, RNA PRIMER 5'-PCC, MN2+, AND GDP
4WTA 2015-02-11 2.8 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH UDP, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-CAAAAUUU
4WTG 2015-02-11 2.9 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH SOFOSBUVIR DIPHOSPHATE GS-607596, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-CAAAAUUU
4WTE 2015-02-11 2.9 CRYSTAL STRUCTURE OF HCV NS5B GENOTYPE 2A JFH-1 ISOLATE WITH S15G E86Q E87Q C223H V321I MUTATIONS AND DELTA8 BETA HAIRPIN LOOP DELETION IN COMPLEX WITH GDP, MN2+ AND SYMMETRICAL PRIMER TEMPLATE 5'-ACAAAUUU
5UJ2 2017-03-22 2.9 Crystal structure of HCV NS5B genotype 2A JFH-1 isolate with S15G E86Q E87Q C223H V321I mutations and Delta8 neta hairpoin loop deletion in complex with GS-639476 (diphsohate version of GS-9813), Mn2+ and symmetrical primer template 5'-AUAAAUUU
4E78 2012-04-18 2.9 Crystal structure of a product state assembly of HCV NS5B genotype 2a JFH-1 isolate with beta hairpin loop deletion bound to primer-template RNA with 3'-dG
4E7A 2012-04-18 3.0 Crystal structure of a product state assembly of HCV NS5B genotype 2a JFH-1 isolate with beta hairpin deletion bound to primer-template RNA with a 2',3'-ddC

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.7 Genome polyprotein P27960 POLG_HCVJ5
91.0 Genome polyprotein P26660 POLG_HCVJ6
100.0 Genome polyprotein Q99IB8 POLG_HCVJF