Identifying and Characterizing Antiretroviral Interferon Stimulated Genes (ISGs)

Viral infections often have negative consequences. These can range from common colds that interfere with our daily lives, right through to debilitating infections that can lead to disability (such as polio), cancer (such as Hepatitis C) and death (such as HIV). It is likely that all life on Earth has been continually plagued by viruses for hundreds of millions of years. In order to survive, humans and animals have developed new and ever more inventive ways to resist infections. These antiviral defences have accumulated over time, such that an arsenal of antiviral defences now exists within our cells. These defences are potent and organized. As soon as our body senses an infection, interferons (proteins that "interfere" with viral replication) are released that increase the expression of our antiviral defences. Viruses, however, have developed their own ingenious ways of overcoming these antiviral defences. Thus, the extraordinary diversity of viruses we observe on Earth today represent those that have adapted to thrive in this hostile host environment.

Not everybody is equally susceptible to viral infection. Some individuals are more readily infected than others, and different people, infected with the same virus, can have different clinical outcomes. Many factors, such as nutrition, underlying health conditions, or previous viral exposure, can influence the clinical course of a viral-disease. However, if we are to ever truly understand how hosts and viruses interact, we must understand a cell's built-in antiviral defences. A better understanding of these interactions might help explain how viral epidemics occur and why some people die from an infection and others do not.

In this project I will investigate the interactions between viruses and the humans and animals they infect. Initially, I will focus on finding the novel antiviral factors that we humans have developed in order to resist infection by human immunodeficiency virus (HIV-1). Previous studies have shown that our in-built immunity to virus infection is often conferred by a single factor. Expression of such factors can be increased by interferons, and so they are referred to as interferon stimulated genes (ISGs). The identity and function of the vast majority of these ISGs remains unknown and so I believe that many more antiviral factors are yet to be identified.

I have previously assembled a collection of human and macaque ISGs, most of which have unknown functions. In preparation for this project, I have examined the ability of hundreds of different ISGs to inhibit HIV-1. Now I will extend this approach to a wider range of retroviruses in an attempt to identify new antiviral factors. Crucially, human viruses have adapted to replicate in the presence of human antiviral defences so examining animal viruses increases the likelihood of identifying human antiviral genes. Conversely, human viruses are not adapted to replicate in the presence of macaque antiviral defences so screening human viruses using macaque ISGs increases the likelihood of identifying genes active against human viruses. I have already successfully used this screening approach to identify a new protein called CNP, which is abundantly expressed in the human brain and inhibits HIV-1 replication.

During the course of this fellowship, I hope to be able to find more antiviral factors and explain how their mechanism of action helps humans and animals resist virus infection. It is my long-term ambition to use the information I produce to help design new drugs and treatments to reduce the impact of viral disease in humans and animals. This ambition will not be realised within the timeframe of this fellowship, but I believe that the work I will undertake here will lay the foundations for these future benefits

The hypothesis underlying this proposal is that more antiviral factors are yet to be identified. Over the last ten years, the work of many research groups has led to the characterization of several factors with antiviral activity, including APOBEC3, TRIM5, tetherin and SAMHD1, all of which target HIV-1. Despite arising independently, these factors share several traits, including that they are all interferon stimulated genes (ISGs). In many cases, human retroviruses, like HIV-1, have adapted to overcome the human ISG, but not the macaque orthologue. Using arrayed ISG-expression libraries of human and macaque origin, I will examine the ability of ~800 individual ISGs for their ability to inhibit the generation of a divergent array of retroviruses.

I was previously part of a collaboration that reported the first use of large-scale ISG-expression screening. This approach dissected the ability of the interferon response to protect cells from different viruses (Schoggins et al., Nature 2011). Here I will utilize a different approach to examine the ability of ISGs to inhibit the generation of new infectious particles (rather than protect cells from incoming virions). This powerful method utilizes a 96-well plate format to coexpress individual ISGs along with plasmids encoding a particular retrovirus. Subsequent measurement of infectious particle production then rapidly reveals the presence of any antiviral activity amongst hundreds of ISGs.

Additionally, I have already conducted extensive pilot screening to identify ISGs that might underlie the requirement for Nef to generate maximally infectious HIV-1 virions. This work has already identified a candidate factor whose action could be responsible for this phenotype. This ISG will be vigorously pursued using a variety of virology and cell biology methodologies that will focus on the relationships between this factor and Nef, viral envelope, and particle-infectivity.

This fellowship proposal encapsulates the type of basic research that the MRC proactively supports. Not only will the results provide immediate benefit to academics through the acquisition of new knowledge, but they may provide the wider public with a means to understand more about viral emergence, epidemics and pandemics. In the longer term, this work may have the potential to inform the design of new and improved antiviral treatments, and will therefore be of interest to healthcare professionals and pharmaceutical companies. Ultimately, the results of the work proposed has the potential to open entirely new research directions that, over the timescale of decades, could deliver tangible human and animal health benefits, as well as economic benefits.

Stratification of phenotype: Understanding antiviral defences and their diversity in human populations will eventually help healthcare professionals identify individuals who are at greater risk of infection with particular viruses, or who may have a poorer prognosis following infection. Such predictive power is already becoming a reality with the insight that human polymorphisms in IFITM3 alter disease progression following infection with pandemic influenza H1N1/09 virus. It is possible that antiviral factors I identify may also contribute to human susceptibility to viruses. These observations may one day inform the application of 'personalised medicine' approaches in clinical settings.

Novel therapies: The identification of antiviral factors could eventually lead to new gene therapy approaches to tackle viral infection. For example, multiple laboratories are investigating the ability of human TRIM5-CypA fusions to protect T-cells from HIV-1 infection. Moreover, improving our understanding of how host defences are evaded by viral pathogens could uncover novel drug targets. Specifically, if the mechanism of viral antagonism is inhibited, natural antiviral defences become active. In this way, understanding how Nef enhances HIV-1 particle infectivity could lead to new therapies, as could discovery of the antiviral activities of another ISG.

Biotechnologies: Improved understanding of intrinsic immunity could lead to the generation of GM livestock that are immune to specific viral infections. This would not only improve animal health, but would also provide economic and food security benefits to at-risk populations. Similarly, arthropod vectors could be genetically modified to contain mammalian antiviral factors, rendering them resistant to arbovirus infection and breaking the transmission cycle. This again could reduce the disease burden, caused by these viruses, in human and livestock populations. Ultimately, any protein with broad-spectrum antiviral activity I identify could potentially be used with technologies of this type.

Vaccine development: The identification and characterization of antiviral factors could assist in vaccine development strategies. Most notably, a better understanding of intrinsic immunity will lead to the development of new animal models of viral infection, this is especially important for viruses that naturally have a very narrow host range (such as HIV-1). These animal models would facilitate the development and testing of improved vaccines for eventual use in humans.

It is unlikely that all the potential benefits described above will be fulfilled by translational approaches derived directly from the work proposed herein. However, it should be stressed that when all the investment in the basic research of host-virus interactions is considered, it will almost certainly lead to new therapies, improved prognoses, better management of epidemics and reduced incidence of viral diseases. Thus, continued investment in basic research of this kind will undoubtedly lead to substantially improved health and wellbeing in the UK within this century.