Preexisting drug-resistance mutations reveal unique barriers to resistance for distinct antivirals

Abstract

Clinical trials of direct-acting antiviral agents in patients chronically infected with hepatitis C virus (HCV) have demonstrated that viral resistance is detected rapidly during monotherapy. In patients, HCV does not exist as a single, genetically homogenous virus but rather as a population of variants termed "quasispecies." Preexisting variants resistant to specific antiviral drugs, overlooked in traditional hit-to-lead discovery efforts, may be responsible for these poor clinical outcomes. To enable real-time studies of resistance emergence in live cells, we established fluorescent protein-labeled HCV replicon cell lines. We validated these cell lines by demonstrating that antiviral susceptibility and the selection of signature resistance mutations for various drug classes are similar to traditional replicon cell lines. By quantifying the kinetics and uniformity of replication within colonies of drug-resistant fluorescent replicon cells, we showed that resistance emerged from a single cell and preexisted in a treatment-naive replicon population. Within this population, we determined the relative frequency of preexisting replicons capable of establishing foci during treatment with distinct antivirals. By measuring relative frequency as a function of dose, we quantitatively ranked distinct antiviral molecules on the basis of their distinct barriers to resistance. These insights into RNA virus quasispecies structure provide guidance for selecting clinical drug concentrations and selecting antiviral drug combinations most likely to suppress resistance.

Fig. 1.

Creation of fluorescent protein reporter replicon cell lines. (A) GFP-1b (Upper) and RFP-1a (Lower) replicon constructs. Replicons with fluorescent proteins (eGFP or tdTomato) in the first cistron were constructed. (B) Isolation of Lunet-GFP-1b (Upper) and 51C-RFP-1a (Lower) cell lines. Genotype 1b and 1a replicon constructs were transfected in Lunet or 51C cells, respectively, and were enriched by iterative rounds of FACS.

Fig. 2.

Validation of fluorescent protein reporter replicon cell lines. (A) Assessing the HCV replicon response to HCV-796 by fluorescent microscopy. Images of the replicon levels (RFP expression, Upper) and nuclei (Hoechst staining, Lower) are acquired at increasing concentrations of HCV-796. (B) Determination of HCV-796 potency by quantitative image analysis. The percentage of fluorescent cells in each image is quantified (n = 4), and data are fit by nonlinear regression to a sigmoidal curve. (C) Fluorescent protein reporter levels accurately reflect replication levels. EC₅₀ values for a number of known antivirals targeting the NS3 protease (black), NS5B polymerase (red), or CypA/NS5A (blue) correlate with our historical values obtained using luciferase-labeled replicons.

Fig. 3.

Kinetic evidence of preexisting drug resistance. (A) A kinetic model to determine if drug-resistance mutations preexist. A cell harboring at least one replicon copy encoding the drug-resistant mutation (R) that existed before the first cell division would expand to uniformly replicating (i.e., uniformly fluorescent) drug-resistant colonies. S, cells containing only drug-sensitive variants of the HCV replicon. (B) Mutations that arise after the first division would yield resistant colonies in which half, or fewer, of the cells are fluorescent. (C) Replication kinetics indicates preexisting drug resistance. A resistant 51C-RFP-1a colony that emerged in the presence of a high dose (200 nM) of HCV-796 exhibits replication kinetics that closely follows the theoretical model for preexisting resistance described in A.

Fig. 4.

Uniform replication across drug-resistant colonies supports preexisting drug resistance. (A) Quantifying fluorescence of each cell in a drug-resistant colony. By locating isolated clusters of DAPI-stained nuclei 5 d after drug addition, we identified colonies likely to have arisen from a single cell (Left). Replication subsequently was visualized, by overlaying the RFP signal with the Hoechst signal, to determine if that colony was capable of replicating in the presence of HCV-796 (Center). By quantifying the fluorescence intensity of each cell within the colony, we determined the uniformity of replication in that drug-resistant colony (Right). (B) Uniformity of replication levels across 12 distinct colonies. Each vertical cluster represents a single colony, and each data point represents a single cell. By plotting the fluorescence of each cell in each colony, we show that all identified colonies fit the model of preexisting resistance. As controls, a colony in the absence of drug (red) has uniformly high replication levels, and a colony in the presence of a 500-fold excess of INTM-191 (blue) has uniformly low replication.

Fig. 5.

Quantification of the relative frequencies of preexisting drug-resistance mutations. (A) A pool of drug-naïve RFP-labeled replicons, isolated from stable replicon cells to generate genetic heterogeneity, is transfected into 51 Cured cells in the presence or absence of drug. The resulting foci are counted. (B) In the absence of drug, an average of 5,993 fluorescent replicon foci is observed. An average of 66 (1.1%) of these foci are resistant to 150 nM HCV-796, and 96 (1.5%) are resistant to 2,400 nM A-782759 (n = 2).

Fig. 6.

Antivirals with similar potencies present distinct barriers to resistance. The number of foci (n = 2) is plotted as a function of drug concentration. (A) Much higher concentrations of A-782759 (red) than of CsA (blue) are required to suppress resistant foci. (B) NS3 protease inhibitors. At concentrations normalized to the EC₅₀, the acyclic VX-950 (black) suppresses resistance more effectively than the macrocyclic BILN-2061 (green), MK-7009 (blue), or INTM-191 (red) (n = 2). (C) NS5B polymerase, NS5A, and CypA inhibitors. Normalized to potency, the nucleoside analog MK-608 (tan) suppresses resistance more effectively than the nonnucleoside NS5B inhibitors A-782759 (red) and HCV-796 (green). Normalized to potency, CsA (blue) suppresses resistance more effectively than the NS5A inhibitor BMS-790052 (black) (n = 2). To avoid jackpot effects, replicates were initiated from distinct pools of replicon cells.