Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus.
OBJECTIVE
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.
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, USA.
Tetsuro
T
Shimakami
Christoph
C
Welsch
Daisuke
D
Yamane
David R
DR
McGivern
Minkyung
M
Yi
Stefan
S
Zeuzem
Stanley M
SM
Lemon
D019943:Amino Acid Substitution; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D024882:Drug Resistance, Viral / Q000235:genetics*; D056084:Genetic Fitness; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D011480:Protease Inhibitors / Q000494:pharmacology*; D011487:Protein Conformation; D012367:RNA, Viral / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D012093:Replicon / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000998:Antiviral Agents; D011480:Protease Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases