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

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

Kinetic analyses reveal potent and early blockade of hepatitis C virus assembly by NS5A inhibitors

Background & Aims All-oral regimens combining different classes of direct-acting antivirals (DAA) are highly effective for treatment of patients with chronic hepatitis C. NS5A inhibitors will likely form a component of future interferon-sparing treatment regimens. However, despite their potential, the detailed mechanism of action of NS5A inhibitors is unclear. To study their mechanisms, we compared their kinetics of antiviral suppression with those of other classes of DAA, using the hepatitis C virus genotype 1a cell culture-infectious virus H77S.3. Methods We performed detailed kinetic analyses of specific steps in the hepatitis C virus life cycle using cell cultures incubated with protease inhibitors, polymerase inhibitors, or NS5A inhibitors. Assays were designed to measure active viral RNA synthesis and steady-state RNA abundance, polyprotein synthesis, virion assembly, and infectious virus production. Results Despite their high potency, NS5A inhibitors were slow to inhibit viral RNA synthesis compared with protease or polymerase inhibitors. By 24 hours after addition of an NS5A inhibitor, polyprotein synthesis was reduced <50%, even at micromolar concentrations. In contrast, inhibition of virus release by NS5A inhibitors was potent and rapid, with onset of inhibition as early as 2 hours. Cells incubated with NS5A inhibitors were rapidly depleted of intracellular infectious virus and RNA-containing hepatitis C virus particles, indicating a block in virus assembly. Conclusions DAAs that target NS5A rapidly inhibit intracellular assembly of genotype 1a virions. They also inhibit formation of functional replicase complexes, but have no activity against preformed replicase, thereby resulting in slow shut-off of viral RNA synthesis

Silymarin for HCV infection

Silymarin, an extract of milk thistle seeds, and silymarin-derived compounds have been considered hepatoprotective since the plant was first described in ancient times. Hepatoprotection is defined as several non-mutually exclusive biological activities including antiviral, antioxidant, anti-inflammatory and immunomodulatory functions. Despite clear evidence for silymarin-induced hepatoprotection in cell culture and animal models, evidence for beneficial effects in humans has been equivocal. This review will summarize the current state of knowledge on silymarin in the context of HCV infection. The information was collated from a recent workshop on silibinin in Germany

Developing methods to understand intra-host evolution and the effect of antiviral drugs on RNA viruses

Viral infections are common and are particularly problematic in immunocompromised individuals. However, other than for HIV, Hepatitis B, Hepatitis C, Influenza, and more recently SARS-CoV-2, there have been few approved drugs available for treating viral infections. Instead, repurposed drugs are often used, especially at the beginning of the current pandemic, for treating SARS-CoV-2. It remains unclear how these repurposed drugs act on the viral population and whether the suppression of viral load we observe is attributed to the drug or the immune response or a combination of both. The research presented in this thesis primarily focuses on the study of two RNA viruses, SARS-CoV-2 and Norovirus. A mixture of viral load data and viral genomic data were analysed to understand the course of infection within individuals. First, we presented a meta-analysis on SARS-CoV-2 viral load dynamics where we investigated the changes of viral dynamics over time, with and without the presence of antiviral drugs. Then, we presented an evolutionary model used for reconstructing haplotypes in mixed infections. Finally, we demonstrated the use of viral deep sequencing to study the within-host evolution of RNA viruses. We identified mutagenic signatures and consensus level changes associated with antiviral treatments. We developed unique methods to analyse viral sequences which allow us to understand the within-host genomic variations and hence inform our understanding of the heterogeneous efficacy of a drug between patients. Overall, this thesis provides insights into how the efficacy of a drug can be evaluated by monitoring the within-host viral dynamics and evolution

An Assessment of the Use of Chimpanzees in Hepatitis C Research Past, Present and Future: 1. Validity of the Chimpanzee Model

The USA is the only significant user of chimpanzees in biomedical research in the world, since many countries have banned or limited the practice due to substantial ethical, economic and scientific concerns. Advocates of chimpanzee use cite hepatitis C research as a major reason for its necessity and continuation, in spite of supporting evidence that is scant and often anecdotal. This paper examines the scientific and ethical issues surrounding chimpanzee hepatitis C research, and concludes that claims of the necessity of chimpanzees in historical and future hepatitis C research are exaggerated and unjustifiable, respectively. The chimpanzee model has several major scientific, ethical, economic and practical caveats. It has made a relatively negligible contribution to knowledge of, and tangible progress against, the hepatitis C virus compared to non-chimpanzee research, and must be considered scientifically redundant, given the array of alternative methods of inquiry now available. The continuation of chimpanzee use in hepatitis C research adversely affects scientific progress, as well as chimpanzees and humans in need of treatment. Unfounded claims of its necessity should not discourage changes in public policy regarding the use of chimpanzees in US laboratories

The roles of Dicer and TRBP in HCV replication

MicroRNAs (miRNAs) are non-coding small RNAs that regulate eukaryotic gene activity at the post-transcriptional level by a process termed miRNA gene suppression. MicroRNA-122 (miR-122) is predominantly expressed in human liver cells and recent studies indicated that miR-122 promotes Hepatitis C Virus (HCV) replication and translation through physical interaction with two tandem binding sites located in the 5’ untranslated region (5’UTR) of the HCV genome (Jopling, et al., 2006; Jopling, et al., 2008). It has been reported that host genes that are also implicated in the miRNA gene suppression pathway are key regulators of HCV replication (Randall, et al., 2007). Two proteins, Dicer, a key RNaseIII enzyme, and its binding partner TRBP are essential proteins for miRNA activity. They are part of a protein complex called the RNA induced silencing complex (RISC) which also includes Argonaute proteins, and function in miRNA biogenesis loading the miRNA into RISC. As such, they are intriguing targets to study host-viral interplay during HCV replication. In our study, we designed siRNAs to knock down Dicer and TRBP and then observed the effects of gene knockdown on full length J6/JFH-1-RLuc HCV (genotype 2a chimeric genome) replication and translation. The results showed that knocking down Dicer and TRBP reduced wild type (wt) J6/JFH-1-RLuc replication but had almost no effects on HCV translation in human liver cells. However, since knocking down Dicer and TRBP did not significantly alter miR-122 levels in the cell, it appears that the role of Dicer and TRBP was not solely the biogenesis of miR-122. This was confirmed by an experiment in which we observed that knocking down Dicer and TRBP also attenuated replication of a mutant virus in which replication is dependent on a exogenously supplied miRNA instead of endogenous miR-122. Taken together, the results supported the hypotheses that Dicer and TRBP facilitate HCV infection mainly through HCV replication but not translation. The effects of Dicer and TRBP on HCV replication are not solely due to miR-122 biogenesis, and may be due to RISC loading functions in steps of miRNA gene suppression. This study has set some essential groundwork for investigating potential roles of host factors in the RNAi machinery modulating HCV replication/translation and exploring novel antiviral targets

Interfering with interferon: developing a reporter system to study the interaction between hepatitus C viral proteins and the interferon signalling pathway

PhDThe aim of the project was to investigate the mechanism by which HCV evades therapeutic IFN treatment. This involved the development of novel testing systems and their application to patient samples. Initial experiments focused on flavivirus replicons and novel observations on effects of one of these replicons (dengue virus) on interferon signalling were made. The dengue replicon system was demonstrated to inhibit IFNa signalling by reducing the expression of STAT2, an essential component of the type I IFN signalling pathway. This phenomenom was then further examined in dengue virus infected human cells and again it was observed that the expression of STAT2 was reduced. The mechanism of STAT2 degradation was further explored and STAT2 expression was found to be restored using a proteasomal inhibitor. A second flavivirus replicon system involving BVDV was also developed as a reporter system, again with novel observations. The BVDV replicon system was shown to be sensitive to the antiviral effects of I FNa and was not shown to inhibit the IFNa signalling pathway. The BVDV replicon was tested as a reporter system using a well-known viral inhibitor of I FNa. The viral inhibitor, inhibited the antiviral action of IFNa on the BVDV reporter. Having developed and validated this system, the effects of a small number of patient derived samples were assessed and it was demonstrated that NS5a derived from a patient who failed to respond to IFNa treatment inhibited the effects of IFNa on the BVDV reporter. To increase the senstitivity of the assay the reporter cassette was then changed to a destabilised GFP for use in a FACS based assay

New strategies for the treatment of hepatitis C virus infection and implications of resistance to new direct-acting antiviral agents

Persistent hepatitis C virus (HCV) infection is a leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma and the major indication for liver transplantation in adults. Current standard of care treatment (SOC) with pegylated-interferon-α 2 and ribavirin (RBV) has a limited efficacy and is associated with significant side effects frequently associated with poor compliance or treatment discontinuation, requiring specialized and frequent monitoring. To overcome the limited efficacy of SOC, more than 50 direct-acting antiviral agents (DAA) designed to target viral-encoded proteins essential in the HCV life cycle are currently under development. The rapid selection of resistant mutants associated with the quasispecies nature of HCV with high mutation and replication rates is one of the main challenges for the new HCV therapies. Predictive host and viral factors together with combination of DAAs with or without IFN and/or RBV need to be accurately evaluated to design the most effective individualized treatment strategy within the shortest time interval and with minimum side effects