A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity.
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.
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
Hangfei
H
Qi
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
C Anders
CA
Olson
The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America.
Nicholas C
NC
Wu
Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Ruian
R
Ke
Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Claude
C
Loverdo
Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Virginia
V
Chu
Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America.
Shawna
S
Truong
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
Roland
R
Remenyi
Department of Human Genetics, University of California Los Angeles, Los Angeles, California, United States of America.
Zugen
Z
Chen
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
Yushen
Y
Du
Institute of Information Science, Academia Sinica, Taipei, Taiwan; Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan.
Sheng-Yao
SY
Su
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
Laith Q
LQ
Al-Mawsawi
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America.
Ting-Ting
TT
Wu
Institute of Information Science, Academia Sinica, Taipei, Taiwan.
Shu-Hua
SH
Chen
Institute of Information Science, Academia Sinica, Taipei, Taiwan.
Chung-Yen
CY
Lin
Department of Infectious Diseases, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America.
Weidong
W
Zhong
Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America; Fogarty International Center, National Institutes of Health, Bethesda, Maryland, United States of America.
James O
JO
Lloyd-Smith
Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America; The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America; School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.
Ren
R
Sun
D002219:Carbamates; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000187:drug effects / Q000235:genetics; D020869:Gene Expression Profiling; D056084:Genetic Fitness; D016174:Hepacivirus / Q000502:physiology*; D006526:Hepatitis C / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D011759:Pyrrolidines; D014633:Valine / Q000031:analogs & derivatives; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D002219:Carbamates; D007093:Imidazoles; D011759:Pyrrolidines; D014633:Valine; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase; C549273:daclatasvir