A Quantitative High-Resolution Genetic Profile Rapidly Identifies Sequence Determinants of Hepatitis C Viral Fitness and Drug Sensitivity

Qi, Hangfei; Olson, C. Anders; Wu, Nicholas C.; Ke, Ruian; Loverdo, Claude; Chu, V.; Truong, Shawna; Remenyi, Roland; Chen, Zugen; Du, Yushen; Su, Sheng; Al‐Mawsawi, Laith Q.; Wu, Ting; Chen, Shu Hua; Lin, Chung Yen; Zhong, Weidong; Lloyd‐Smith, James O.; Sun, Ren

doi:10.1371/journal.ppat.1004064

Cited by 70 publications

(60 citation statements)

References 60 publications

(96 reference statements)

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“…Consistent with this hypothesis, transposon mutagenesis demonstrated that very few insertion sites in domain I were tolerated (in comparison with domains II and III) (Arumugaswami et al, 2008). In agreement with the transposon study, saturation mutagenesis coupled with next-generation sequencing of virus pools derived from multiple rounds of passage has revealed that very few residues in the N terminus of domain I (aa 18-103) can tolerate substitutions (Qi et al, 2014). These data also confirmed the importance of the PTPPL sequence (residues 100-104 of NS5A), which was previously shown to be required for both association of NS5A with lipid droplets and release of infectious virus (Miyanari et al, 2007) (Fig.…”

Section: Phosphorylation Of Ns5asupporting

confidence: 57%

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Ross‐Thriepland

Harris

2015

Journal of General Virology

116

128

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Since one of us co-authored a review on NS5A a decade ago, the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone, which allowed the study of all aspects of the virus life cycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We shall review the recent structural insights -NS5A is predicted to comprise three domains; X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains, which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we shall highlight a few pertinent examples from the everexpanding list of NS5A-binding partners identified over the past decade. Lastly, we shall review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct-acting antivirals (DAAs) are now either licensed, or in the late stages of approval for clinical use both in the USA and in the UK/ Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionizing treatment for HCV infection.

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Section: Phosphorylation Of Ns5asupporting

confidence: 57%

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Ross‐Thriepland

Harris

2015

Journal of General Virology

116

128

View full text Add to dashboard Cite

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“…To profile the HCV genome systematically in an unbiased manner, we constructed a mutant library by in vitro Mu transposonmediated random insertional mutagenesis on a plasmid carrying the HCV genome (pFNX-HCV; a genome that we chemically synthesized based on the chimeric genotype 2a clone, J6/JFH1) (36) (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein

Chu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to downregulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.HCV | innate immune evasion mechanism | IF6-16 antiviral function | high-throughput mutagenesis | p7 ion channel protein W ith an estimated 170 million people persistently infected worldwide, hepatitis C virus (HCV) has emerged as a major cause of human liver diseases, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (1, 2). Despite the recent breakthroughs in the development of HCV direct antiviral agents (DAAs) aiming to cure chronic HCV infection, emerging resistant mutations and drug-resistant polymorphisms at the baseline of treatments remain major challenges to eradicate HCV (3-8). In addition, the high cost of these DAAs limits their accessibility to the majority of patients worldwide. Therefore, HCV eradication is still heavily dependent on the development of an effective preventative vaccine (9). Understanding how the virus evades the immune system, which results in a poor immune response of the infected host against the virus, will provide important information for immune therapy and vaccine development.

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“…High-throughput genetics have been applied to a number of viral, bacterial, and cellular proteins (16, 33–38, 111, 112). Here, point mutations were randomly introduced into segment 2 of influenza A/WSN/33 virus through error-prone PCR.…”

Section: Resultsmentioning

confidence: 99%

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

Jiang

et al. 2016

mBio

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Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

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A Quantitative High-Resolution Genetic Profile Rapidly Identifies Sequence Determinants of Hepatitis C Viral Fitness and Drug Sensitivity

Cited by 70 publications

References 60 publications

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!

Systematic identification of anti-interferon function on hepatitis C virus genome reveals p7 as an immune evasion protein

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

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