BACKGROUND: Hepatitis C virus (HCV) NS5A replication complex inhibitors (RCIs) 
have been shown to exhibit picomolar antiviral activity against genotype 1 (GT1) 
in vitro. This has translated into rapid and robust declines in HCV RNA in GT1 
patients. Less is known about the susceptibility of other genotypes such as GT3 
to inhibition by NS5A RCIs.
OBJECTIVES: To detect and phenotype naturally occurring HCVGT3 NS5A 
polymorphisms against two NS5A RCIs (daclatasvir [DCV] and GS-5885) currently in 
clinical development.
STUDY DESIGN: The NS5A region from 96 HCV GT3 treatment-naive patients spanning 
North America, Europe and Australia was determined.
RESULTS: Phylogenetic analysis revealed a broad distribution with no significant 
geographic clustering. GT1 DCV resistance-associated variants (RAVs) were 
observed in GT3 subjects; variants (and their frequencies) included 28M/V (1%), 
30A/K/S/T/V (10%), 31L/M (1%), E92A (1%) and Y93H (8.3%). A consensus sequence 
was used to generate a JFH1/3a-NS5A hybrid replicon and employed to assess 
susceptibility to NS5A RCIs. Against JFH1/3a-NS5A, DCV was more potent (EC(50) = 
0.52 nM) than GS-5885 (EC(50) = 141 nM). DCV sensitivity was increased against 
JFH1/3a-NS5A-M28V (EC50 = 0.006 nM), A30V (EC(50) = 0.012 nM), and E92A (EC(50) 
= 0.004 nM) while the NS5A-A30K and -Y93H variants exhibited reduced sensitivity 
to DCV (EC50 values of 23 nM and 1120 nM, respectively) and to GS-5885 (EC50 
values of 1770 nM and 4300 nM, respectively).
CONCLUSIONS: Substitutions conferring resistance to NS5A RCIs pre-existed in 
treatment-naive patients infected with HCV GT3. The effectiveness of these NS5A 
RCIs to exert efficacy in the clinic may depend on which inhibitor is used in 
combination with other antivirals.