Protease Inhibitor–Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus

GastroenterologyVolume 140, Issue 2 , Pages 667-675, February 2011

Protease Inhibitor–Resistant Hepatitis C Virus Mutants With Reduced Fitness From Impaired Production of Infectious Virus

Background & Aims

Several small molecule inhibitors of the hepatitis C virus (HCV) nonstructural protein (NS) 3/4A protease have advanced successfully to clinical trials. However, the selection of drug-resistant mutants is a significant issue with protease inhibitors (PIs). A variety of amino acid substitutions in the protease domain of NS3 can lead to PI resistance. Many of these significantly impair the replication fitness of HCV RNA replicons. However, it is not known whether these mutations also adversely affect infectious virus assembly and release, processes in which NS3 also participates.

Methods
We studied the impact of 25 previously identified PI-resistance mutations on the capacity of genotype 1a H77S RNA to replicate in cell culture and produce infectious virus.

Results
Most PI-resistance mutations resulted in moderate loss of replication competence, although several (V36A/L/M, R109K, and D168E) showed fitness comparable to wild type, whereas others (S138T and A156V) were severely impaired both in RNA replication and infectious virus production. Although reductions in RNA replication capacity correlated with decreased yields of infectious virus for most mutations, a subset of mutants (Q41R, F43S, R155T, A156S, and I170A/T) showed greater impairment in their ability to produce virus than predicted from reductions in RNA replication capacity. Detailed examination of the I170A mutant showed no defect in release of virus from cells and no significant difference in specific infectivity of extracellular virus particles.

Conclusions
Replicon-based assays might underestimate the loss of fitness caused by PI-resistance mutations, because some mutations in the NS3 protease domain specifically impair late steps in the viral life cycle that involve intracellular assembly of infectious virus.

Tetsuro Shimakami
Affiliations Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, Christoph Welsch
Affiliations Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Department of Internal Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany Max Planck Institute for Informatics, Computational Biology, and Applied Algorithmics, Saarbrücken, Germany, Daisuke Yamane
Affiliations Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, David R. McGivern
Affiliations Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, MinKyung Yi
Affiliations Institute for Human Infections & Immunity and Department of Microbiology & Immunology, University of Texas Medical Branch, Galveston, Texas, Stefan Zeuzem
Affiliations Department of Internal Medicine, Johann Wolfgang Goethe-University, Frankfurt, Germany, Stanley M. Lemon
Affiliations
Division of Infectious Diseases, Department of Medicine, Inflammatory Diseases Institute, and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Reprint requests Address requests for reprints to: Stanley M. Lemon, MD, Inflammatory Diseases Institute CB#7292, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7292. fax (919) 843-7240 Received 11 June 2010; accepted 28 October 2010. published online 08 November 2010. Abstract