Mathematical modeling of subgenomic hepatitis C viral replication in Huh-7 cells

Cell-based hepatitis C virus (HCV) replicon systems have provided a means for understanding HCV replication mechanisms and for testing new antiviral agents. Here, we describe a mathematical model of HCV replication that assumes that the translation of the HCV polyprotein occurs in the cytoplasm, that HCV RNA synthesis occurs in vesicularmembrane structures, and that the strategy of replication involves a double-stranded RNA intermediate. Our results shed light on the intracellular dynamics of subgenomic HCV RNA replication from transfection to steady state within Huh-7 cells. We predict the following: (i) about 6 x 10 3 ribosomes are involved in generating millions of HCV NS5B-polymerase molecules in an Huh-7 cell, (ii) the observed 10:1 asymmetry of plusto minus-strand RNA levels can be explained by a higher affinity (200-fold) interaction of HCV NS5B polymerasecontaining replication complexes with HCV minus-strand RNA over HCV plus-strand RNA in order to initiate synthesis, (iii) the latter higher affinity can also account for the observed ~ 6:1 plus-to minus-strand ratio in vesicular-membrane structures, and (iv) introduction of higher numbers of HCV plus-strand RNA by transfection leads to faster attainment of steadystate, but does not change the steady-state HCV RNA level. Fully permissive HCV replication systems have been developed and the model presented here is a first step towards building a comprehensive model for complete HCV replication. Moreover, the model can serve as an important tool in understanding HCV replication mechanisms, and should prove useful in designing and evaluating new antivirals against HCV. AC CE PT ED on July 4, 2017 by gest http/jvi.asm .rg/ D ow nladed fom Dahari et al. Page-3 Introduction About 200 million people, roughly 3% of the human population, are infected with hepatitis C virus (HCV) (61). Chronic HCV infection is the main cause of chronic liver disease and cirrhosis leading to liver transplantation or death (3, 47). State-of-the-art therapy (peg-interferon and ribavirin) elicits long-term responses in only about 50% of treated patients (18, 37, 41), with no effective alternative treatment for non-responders (56). Progress towards developing model systems of HCV infection that could enhance efforts to identify inhibitors of HCV replication has been hampered by HCV’s limited replication in cell culture and the lack of small animal models (32). In 1999 Lohmann et al. (35) engineered a bicistronic subgenomic HCV replicon system in Huh-7 cells. Since then this system, improved substantially both in Huh-7 cells (34) and in other cell lines (64), has become the standard cell-based assay to study HCV replication mechanisms and to evaluate antiviral agents (51). The first studies of positive strand RNA virus replication were done with RNA bacteriophages, e.g., Qβ and MS2 (54). These studies showed that viral RNA amplification depended on an RNA-dependent RNA polymerase-containing RNA replicase that specifically interacts with the incoming viral RNA (plus strand) to synthesize its complementary (minus) strand. Once the minus-strand RNA is synthesized the amplification of the viral RNA by the replicase begins. Based on these systems, Biebricher et al. (6) quantitatively monitored the kinetics of RNA amplification by Qβ replicase, and developed a kinetic model for selfreplication of Qβ RNA in vitro (5). The full life cycle of Qβ has been mathematically modeled (16) and provides an important starting point for developing intracellular HCV replication