MATHEMATICAL MODELING OF HOST CELL DETERMINANTS AND PHARMACOLOGICAL INTERVENTION IN HEPATITIS C VIRUS REPLICATION

Abstract

Hepatitis C virus (HCV) is a blood-borne, enveloped, single-stranded, (+)-oriented RNA virus that mainly infects hepatocytes. Most infections progress into chronicity and eventually lead to severe liver disease. Although effective treatments have been developed, access to diagnosis and treatment is low, particularly in non-developed countries. Thus, eradication of the disease is unlikely without a prophylactic vaccine. Research, therefore, has to continue despite the high cure rates of today’s HCV regimens. We use mathematical modeling to study HCV replication and its intricate connection with the infected host cell. A model that is able to simulate intracellular HCV RNA replication suggested a host factor species (HF), representing a protein (complex) or a host process, to be critically involved in HCV replication. Gene expression profiling revealed several candidates potentially representing this HF. We validated those candidates in two variants of the human hepatoma cell line Huh7 and could confirm that five of them indeed played a role for HCV replication, namely CRAMP1, LBHD1, CRYM, THAP7, and NR0B2. The latter three are nuclear receptors or transcriptional (co )repressors, suggesting they could influence HCV replication indirectly, e.g. through glucose, lipid, or cholesterol metabolism. Follow-up studies will help to understand the implication of those factors in HCV replication and reveal important insights into the metabolic pathways regulating HCV replication. Model analyses also revealed the most sensitive steps in HCV RNA replication that could potentially be targeted by specific intervention. The standard of care for chronic HCV infection has been interferon alpha (IFN α) therapy that elicited a very broad but rather unspecific antiviral response of the host cell and came along with severe side effects. IFN-α activates signaling cascades that lead to the expression of hundreds of interferon stimulated genes that exert antiviral action. Despite its decades-long use, the exact mechanism of the suppression of HCV replication by IFN α treatment remains elusive. We thus combined experimental data with an intracellular model for HCV replication and revealed the steps in the viral replication cycle that are most probably affected by IFN α treatment. The obtained findings were well in line with in vitro data and confirmed the validity of our intracellular model to make such analyses. Recently, direct-acting antivirals (DAAs) have replaced IFN-α-containing regimens as the standard of care for chronic HCV infection. Those DAAs possess much less side effects, can be taken orally, and give extraordinarily high cure rates. Mainly three classes exist: inhibitors of the viral protease, the viral polymerase, and a viral multifunctional phosphoprotein. The latter class constitutes highly potent inhibitors of the HCV NS5A protein, exerting effects in the low picomolar range. However, due to the many roles of NS5A in the HCV life cycle, the exact mechanism of action of those DAAs remains unclear. For the other two classes, the mode of action is distinct and well defined. We, thus, used one representative member of each of these classes to validate the capacity of our model to implement drug effects and predict HCV replication correctly. Model predictions upon a priori fixing of the affected parameters in the model qualitatively resembled HCV replication dynamics under the respective drug treatment. This allowed us to apply our model to HCV replication data under treatment with an NS5A inhibitor in order to gain insights into its mode of action. The model revealed that the translation rate of HCV RNA as well as RNA synthesis steps in the HCV replication compartment are most probably affected by the drug. These findings were reasonable and supported by known roles of NS5A in the HCV life cycle. However, our model was limited to intracellular HCV replication and did not account for steps like particle assembly or infection of target cells. Therefore, we extended our intracellular model to cover the full viral life cycle. Our new full life cycle model could simulate viral (+)- and (-)-strand RNA, viral titers as well as spread of the infection, and was able to correctly predict HCV replication under drug treatment. Our new model will be helpful in further elucidating the mode of action of NS5A inhibitors and IFN α and in deciphering the role of host factors that determine permissiveness for HCV. Hence, this study provides a novel, extended mathematical model of the full HCV life cycle with the proven capacity of simulating and analyzing HCV replication even under pharmacological intervention. It can serve as an invaluable tool to study further molecular details of HCV replication and to devise and test novel therapeutic approaches.