Hepatitis C virus genotype 3 NS5A protein: resistance to direct acting antiviral agents and functional analysis

Hepatitis C virus (HCV) is an important human pathogen and it is estimated that 70 million people worldwide are infected with this virus. Recently a number of very effective antiviral drugs have been developed that can cure infected individuals, however these are expensive and the virus can change to become resistant. Thus there is a need to better understand how the virus grows and causes disease, how the drugs work and ultimately to develop more antivirals.
HCV is a very variable virus and 7 distinct strains (termed genotypes 1-7) have been identified. This project focusses on genotype 3, the second most common genotype worldwide accounting for approximately 30% (approx. 20 million) of HCV cases. Genotype 3 is the most common in low to middle income countries (LMIC), accounting for 44% of cases and in particular, 70% of HCV infections in South Asia (Pakistan, India and Thailand) are genotype 3. Consistent with this, genotype 3 is also prevalent in parts of Western Europe, and accounts for 44% of HCV cases in the UK. This is important as genotype 3 infection is associated with a more rapid progression of liver disease, a higher incidence of diabetes, fatty liver and liver cancer, and a higher mortality rate compared to other genotypes. Genotype 3 patients also exhibit high levels of resistance to the new drugs, limiting the treatment options.
The differences between genotype 3 and other genotypes are not very well understood, mainly because until recently there were no good laboratory systems available to study this genotype. We seek to address this lack of knowledge in particular focusing on one viral protein, NS5A, which has been shown in other genotypes to be required for virus growth and disease, as well as being a target for some of the new drugs. We will use our expertise in studying NS5A in other genotypes, together with new systems developed by us and others to grow genotype 3 virus in cell culture, to ask why it is so resistant to the drugs and how NS5A interacts with the cell. We hope that these studies will shed light on why genotype 3 is so different, and provide opportunities to develop new therapeutic strategies for patients infected with this genotype of HCV.

Hepatitis C virus (HCV) genotype 3 (GT3) represents both a clinical issue and an unique biological question. GT3 isolates exhibit high baseline resistance to direct acting antivirals (DAAs) such that the sustained virological response rates for GT3 patients are significantly lower than for other GTs, particularly for patients with cirrhosis. GT3 is also associated with more rapid progression of liver disease and a direct correlation with metabolic syndrome, leading to a higher incidence of insulin resistance, steatosis (fatty liver), and hepatocellular carcinoma compared to other GTs.
The underlying biological differences between GT3 and other GTs are poorly understood, largely because of a lack of robust in vitro culture systems. However, we and others have recently developed both transiently replicating sub-genomic replicons (SGRs), and infectious viruses that overcome this hurdle. Focussing on the NS5A protein, we will use both SGR and virus to investigate the nature of the baseline resistance to DAAs that target NS5A, using a mutagenic approach but also screening isolates from the STOP-HCV and HCV-Research UK biorepositories which both contain a number of GT3 patients who either failed therapy or relapsed.
We will establish viable SGRs and infectious virus constructs containing in-frame tags within NS5A to address how the GT3 NS5A interactome and phosphorylation pattern differs from other GTs. We will follow these studies with phenotypic analysis of interacting proteins and phosphorylation sites and ask how DAA resistance affects the function of the protein.
Lastly, we will pursue structural analysis of GT3 NS5A - the protein comprises 3 domains, the structure of the N-terminal domain I has been determined for GT1 isolates so we will attempt to achieve a similar level of structural understanding for GT3. This will help to frame the information obtained from the other aspects of the proposal.

The basic scientific discoveries of this proposal will be of interest and value to academic investigators in the fields of hepatitis C virus as outlined in the 'academic beneficiaries' section. Delivery of impact during the grant to non-academic beneficiaries will be pursued in four main areas.
Firstly, the post-doctoral research assistant and technician employed on the grant will be trained to a high level of competency in virological techniques. Importantly this training will include safe handling of Biological Safety Level 3 (BSL3) pathogens and work within a BSL3 laboratory. The need for, and societal impact of, individuals with such training is exemplified by the recent deployment of researchers from the MH group with experience handling BSL3 viruses to help with the response to the Ebola outbreak in Western Africa. In more general terms the UK/global economy will benefit through the development of two highly skilled researchers, with high level scientific and transferable skills equipping them to continue in a research career.
Secondly, the general public (both adults and children) will benefit through a range of public engagement events, leading to improved understanding of viruses in general and hepatitis C virus in particular.
Thirdly, the outcomes of this proposal are also expected to have direct impact for clinicians who are involved in treatment of hepatitis C virus patients. The treatment options for the 44% of patients in the UK with genotype 3 infection are limited and a better understanding of the mechanisms by which genotype 3 is resistant to the direct acting antivirals will enable more informed choice of therapeutic options. For example, by determining which residues in NS5A are associated with resistance to NS5A-targeting direct acting antivirals (DAAs) it will be possible to make informed decisions based on patient-derived sequence information. In this regard it is particularly pertinent to note that genotype 3 is a major issue (70% of hepatitis C virus infections) in LMIC such as Pakistan, India and Thailand where access to new, high cost therapies may be limited.
Although pharmaceutical companies are disengaging from development of new hepatitis C virus DAAs we expect that the inevitable development of resistance (hepatitis C virus is the most highly variable virus known to infect man) will reignite their interest in the not too distant future. This interest may also be fuelled by the continued prohibitive cost of the DAAs and it is possible that smaller companies may seek to develop low-cost alternatives. An understanding of the biology of genotype 3 and the underlying mechanisms of resistance will be beneficial in this process. We will endeavour to exploit translatable applications in follow on 'proof of concept' studies e.g via the MRC's CiC scheme. In this we will be assisted by the Research and Innovation Development Manager in the Faculty of Biological Sciences, whose role is to support the development of research and innovation and assist in matching academics with industrial partners.
Should an Intellectual Property arise from the project it will be managed through the University's Commercial Services team who manage all of the University's Intellectual Property and its exploitation. The Commercial Services team has a pool of existing professional advisors (patent attorneys, market specialists and business analysts) that can be drawn in to provide support for commercialisation. It also maintains a close relationship with IP Group PLC a UK leading venture capital partner who support opportunity evaluation and University spin-out companies.