Research finds that different strains of hepatitis C virus (HCV) show highly variable rates of replicase complex decline in response to NS5A inhibitors.
In a new article
published in PLOS Pathogens
, lead investigator David McGivern, PhD, from the Lineberger Comprehensive Cancer Center at the University of North Carolina at Chapel Hill, and his colleagues, use both experimental and mathematical approaches to determine the rate of disappearance of viral RNA in cells infected with HCV and treated with NS5A inhibitors.
Direct-acting antiviral (DAA) combination therapy is the current treatment standard for chronic HCV infections, leading to a sustained viral response (SVR) in most patients. DAAs combine drugs that target viral proteins such as the NS3A protease, the NS4A helicase, the NS5B RNA-dependent RNA polymerase, and the NS5A protein, which is a multi-purpose protein involved in multiple stages in the life cycle of the virus. Although the exact mechanism of action of NS5A inhibitors has yet to be elucidated, drugs that target the NS5A protein are necessary components of DAA cocktails used to treat HCV-infected patients. It is thought that NS5A inhibitors work by inhibiting both RNA synthesis as well as viral assembly. NS5A inhibitors have been shown to inhibit RNA synthesis in an indirect manner, through inhibiting the formation of new replicase complexes (RC), which are assemblies of viral and cellular proteins that synthesize HCV RNA genomes. By stopping new RCs from being made, the already existing RCs are eventually degraded and disappear from the cell.
In an effort to better understand the mechanism of NS5A inhibitors, Dr. McGivern and his colleagues sought to determine the half-life of RCs in a panel of HCV. The authors compared different strains of HCV and classified them based on their sensitivity to lipid peroxidation, either assigning them to a lipid peroxidation resistant and rapidly replicating group or a slowly replicating, lipid peroxidation sensitive group.
The authors utilized a reporter luciferase assay to measure the kinetics of viral decline after adding NS5A inhibitors to infected cells. They determined that lipid peroxidation sensitive strains declined with slower kinetics in comparison to lipid peroxidation resistant strains, meaning that the pre-existing RCs disappear faster in lipid peroxidation resistant strains. The authors also examined kinetics of the two groups of viruses in response to NS5B inhibitors and determined no difference in the rate of decline of RCs, which suggests that the difference observed in response to NS5A inhibitors is not just a result of the variability in RNA stability.
Dr. McGivern and his colleagues also performed further studies comparing two genotype 1a viruses, the lipid peroxidation sensitive H77S.3, and the lipid peroxidation resistant H77D; these two strains only differ by 12 amino acids. The H77D strain had an RC half-life of 3.5 hours, while the H77S.3 had an RC half-life of 9.9 hours. The authors used mathematical modeling to show that the difference in RC stability is the cause of the observed discrepancy in kinetics of antiviral decline between lipid peroxidation sensitive and resistant strains.
Overall, the authors sought to further elucidate the mode of action of NS5A inhibitors, and determined that the half-life of RCs in hepatitis C viruses are highly variable.
Samar Mahmoud graduated from Drew University in 2011 with a BA in biochemistry and molecular biology. After two years of working in industry as a quality control technician for a blood bank, she went back to school and graduated from Montclair State University in 2016 with a MS in pharmaceutical biochemistry. She is currently pursuing her PhD in molecular and cellular biology at the University of Massachusetts at Amherst.
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