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Analysis of the Enzymatic Activity of an NS3 Helicase Genotype 3a Variant Sequence Obtained from a Relapse Patient
Paola J S Provazzi1, Sourav Mukherjee2, Alicia M Hanson2
1São Paulo State University - UNESP, Department of Biology, São José do Rio Preto/SP, CEP: 15054-000, Brazil.
Insights
A mutation in the hepatitis C virus (HCV) NS3 helicase (W501R) severely impairs its RNA unwinding activity and viral replication. This finding is crucial for understanding HCV genotype 3a treatment resistance.
Area of Science:
- Virology
- Molecular Biology
- Hepatology
Background:
- Hepatitis C virus (HCV) infects over 170 million globally, causing liver disease.
- HCV genotype 3a is prevalent in Brazil and shows poor response to direct-acting antivirals.
- The NS3 protein, a target for HCV drugs, possesses protease and RNA helicase activities.
Purpose of the Study:
- To investigate the impact of the W501R mutation on the NS3 helicase activity in HCV genotype 3a.
- To analyze the effect of the W501R mutation on HCV replication in genotype 2a and 3a.
Main Methods:
- Sub-cloning, expression, and purification of wild type and W501R genotype 3a NS3 proteins.
- Biochemical assays to measure helicase activity (RNA/DNA unwinding) and ATP hydrolysis.
- Replication assays using genotype 2a and 3a subgenomic HCV replicons in Huh7.5 cells.
Main Results:
- The W501R substitution reduced the catalytic efficiency of the genotype 3a NS3 helicase by over 600-fold.
- W501R protein showed slightly weaker DNA binding compared to wild type, with similar ATP hydrolysis rates.
- HCV replicons with the W501R allele exhibited severely impaired replication in both genotype 2a and 3a.
Conclusions:
- The W501R mutation significantly compromises NS3 helicase function and HCV replication.
- The poor replication of W501R variants suggests their reliance on wild-type quasispecies for propagation.
- Understanding this mutation's impact may inform strategies against treatment-resistant HCV genotypes.
Abstract:
The hepatitis C virus (HCV) is a species of diverse genotypes that infect over 170 million people worldwide, causing chronic inflammation, cirrhosis and hepatocellular carcinoma. HCV genotype 3a is common in Brazil, and it is associated with a relatively poor response to current direct-acting antiviral therapies. The HCV NS3 protein cleaves part of the HCV polyprotein, and cellular antiviral proteins. It is therefore the target of several HCV drugs. In addition to its protease activity, NS3 is also an RNA helicase. Previously, HCV present in a relapse patient was found to harbor a mutation known to be lethal to HCV genotype 1b. The point mutation encodes the amino acid substitution W501R in the helicase RNA binding site. To examine how the W501R substitution affects NS3 helicase activity in a genotype 3a background, wild type and W501R genotype 3a NS3 alleles were sub-cloned, expressed in E. coli, and the recombinant proteins were purified and characterized. The impact of the W501R allele on genotype 2a and 3a subgenomic replicons was also analyzed. Assays monitoring helicase-catalyzed DNA and RNA unwinding revealed that the catalytic efficiency of wild type genotype 3a NS3 helicase was more than 600 times greater than the W501R protein. Other assays revealed that the W501R protein bound DNA less than 2 times weaker than wild type, and both proteins hydrolyzed ATP at similar rates. In Huh7.5 cells, both genotype 2a and 3a subgenomic HCV replicons harboring the W501R allele showed a severe defect in replication. Since the W501R allele is carried as a minor variant, its replication would therefore need to be attributed to the trans-complementation by other wild type quasispecies.
