The selection of mutations in vivo
The selection of mutations in vivo with antivirals is a complex process involving, among other things, the frequency of a given mutation prior to treatment, the effect of that mutation on viral protein function, and the interplay of a given mutation pattern with host immune mechanisms. Investigation of the specific effect on viral protein function can be difficult to study in the replicon system because such experiments are best performed independent of viral replication that can confound the impact of individual viral protein activity. Here, we utilized the SUMO-NS3/4A system to specifically study how mutations in NS3/4A might alter protease activity as measured by the ability to cleave a peptide substrate. Interestingly, GT1b PI mutations with D168E and D168V substitutions increase protease activity, while the D168A substitution decreases protease activity, relative to wild-type enzyme. These data suggest that an alanine substitution at this position may have a deleterious effect on protease function and can explain, at least in part, why the E and V variants, as opposed to the A, are more frequently selected with PI treatment in vivo (Table 3) (Dvory-Sobol et al., 2012b, Svarovskaia et al., 2012).
Previously it has been shown that deep-sequencing technology that allows the detection of minor viral subpopulations can detect NS3 protease mutations at amino MK-4827 hydrochloride positions R155, A156, and D168 down to a detection limit of 0.25% in patients treatment with NS3 protease inhibitors (Svarovskaia et al., 2012). This method can reveal diversity of minority drug-resistance NS3 variants that can be potentially associated with resistance. Combining studies of deep sequencing, replicon systems and SUMO-NS3/4a system can allow studying resistance to direct acting antiviral and development of new therapies for HCV patients.
Acknowledgements This work was supported by Gilead Sciences, Inc.
Introduction Hepatitis C virus (HCV) infection, a worldwide public health problem, affects 135 million people, i.e., 3% of the world population . Nearly 80% of infected individuals develop chronic disease, often associated with liver cirrhosis and hepatocellular carcinoma . No vaccine has been developed yet for HCV. Conventional therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV) is effective for only 40% of patients infected with the most prevalent genotype (HCV-1) , . Therefore, direct-acting antiviral (DAAs) agents, particularly protease inhibitors (PI), have emerged as a major advance in hepatitis management, with several DAAs currently under development . Early emergence of viral resistance mutations has been, however, associated with PI monotherapy , , , , with several of these mutations showing cross-resistance to multiple PI agents . Still, PI resistance mutations are widespread and even present in naïve patients , , , , , , indicating that monotherapy with PI may select for resistance mutations already present at baseline of treatment , . Consequently, previous studies have suggested that the presence of resistance mutations before treatment could be a reliable predictor of PI-based HCV therapy efficacy , . Even though routine baseline resistance mutation detection before PI therapy is still prescribed as a prognostic tool , the predictive value of these mutations at baseline remains controversial. In fact, recent research ,  has suggested that resistant variants that emerge during PI therapy might not be the same as those identified at baseline. Moreover, the presence of resistance mutations before treatment could not be associated with therapy outcome in samples of non-cirrhotic patients , . However, it is not yet possible to discriminate the influence of potentially confounding factors on these results, including previous treatment, host-dependent factors, HCV-genotype, and the specific antiviral agent used , . Also, it is not known whether these observations can be generalized to other populations of varying disease severity.