Common host proteins required for replication organelle function across coronaviruses

Coronaviruses are an important group of pathogens that infect both humans and livestock animals. Human coronaviruses mainly cause common cold-type illnesses and are responsible for approximately 10% of hospitalizations due to respiratory infections. In addition, an estimated 34 million working days were lost as a result of coughs and colds in 2016 meaning coronaviruses contribute to a major source of lost productivity. Coronaviruses are also responsible for economically important livestock diseases. The UK poultry industry contributes £3.4 billion annually to the economy and the most economically damaging infectious disease is caused by the coronavirus infectious bronchitis virus, with an estimated £23 million lost per year due to the disease. In pigs, an outbreak of another coronavirus, porcine epidemic diarrhoea virus, in the USA in 2013-2014 is estimated to have resulted in the death of 8 million pigs and is associated with estimated losses of $900 million - $1.8 billion across the whole economy. Finally, coronaviruses are able to jump between species resulting in new diseases, which are often highly pathogenic. This has happened twice since 2003 resulting in the emergence of SARS- and MERS-coronaviruses into the human population. There are currently no antiviral treatments available for coronaviruses and livestock industries require more effective vaccines that protect against multiple strains of virus. In order to develop new approaches to tackle coronaviruses, in particular ways to treat or prevent future, as yet unknown emerging coronaviruses, it is important to understand commonalities in how all coronaviruses behave and interact with their host cells.

The synthesis of viral RNA is one stage of the coronavirus life cycle that is highly conserved and is critical for the propagation of new virus particles. This is required to direct the production of viral proteins that make up new particles and to produce copies of the viral genome to be packaged into new particles. Coronavirus RNA synthesis takes place at specialized sites called replication organelles (ROs). Our findings have shown that the appearance of ROs is conserved across all coronaviruses. However, little is known about how ROs form and which viral and cellular proteins are required. We propose that due to their central role in virus replication, and the fact that their appearance is highly conserved, all coronaviruses will hijack or use the same core set of host proteins to allow optimal RO assembly and function. An understanding of which cellular proteins all coronaviruses need to complete this critical stage of virus replication will underpin the development of universal anti-coronaviral drugs, development of vaccine viruses and even resistant livestock animals. In this project, we aim to identify and compare the viral and cellular proteins that are present in the ROs of a panel of five coronaviruses to identify the core set of host proteins required for this critical step of the coronavirus life cycle.

Coronaviruses pose a significant threat to human health and food security. Members of this virus family cause economically damaging diseases in poultry and swine globally. In addition, coronaviruses have a proven ability to cross the species a barrier with incursions of highly pathogenic viruses into both human and swine populations in recent years. All coronaviruses induce the rearrangement of cellular membranes during replication to form the replication organelle (RO), the site of viral RNA synthesis. This is a critical step in the virus life cycle and our recent work has shown that RO appearance is highly conserved across all coronaviruses. However, how ROs form and the viral and cellular proteins that play a role in this are largely unknown. The aim of this proposal is to identify host proteins or host pathways that are required by coronaviruses for RO assembly or function and are therefore critical for the replication of all coronaviruses.

Using proteomics informed by transcriptomics (PIT), a unique approach combining mass spectrometry and RNAseq, the proteomes of ROs from a panel of diverse coronaviruses will be characterised and compared. PIT enables analysis of non-model organisms which often have poorly annotated genomes, including livestock pathogens in their natural host. The panel of viruses will represent each of the four coronavirus genera, including both human and livestock viruses as well as a recently emerged zoonotic virus. Host proteins or host pathways present in the ROs from all or combinations of the coronavirus will be identified through comparison of the panel.

Subsequently functional involvement of the identified host proteins in RO assembly or function will be investigated by combining virology and molecular biology techniques with cutting edge microscopy techniques, including super-resolution confocal microscopy, correlative light electron microscopy and electron tomography.

Meat provides an important source of protein for humans. An estimated 55 billion chickens and 781 million pigs are reared globally per year. In the UK, the poultry industry contributes approximately £3.4 billion to the economy and pig meat production had a worth of £1.25 billion in 2018. However, livestock industries are constantly under threat from infectious diseases. Infectious bronchitis virus, a coronavirus infecting chickens, results in losses of £23 million per year in the UK. A recently emerged strain of porcine epidemic diarrhoea virus, a coronavirus infecting pigs, resulted in the death of 8 million pigs in the USA in 2013-14 and estimated losses of $900 million - $1.8 billion to the US economy. Coronaviruses also pose a significant threat to human health with the recent emergence of zoonotic viruses. SARS-coronavirus emerged in 2003, infecting 8096 people with a 10% mortality rate. MERS-coronavirus emerged in 2012 and continues to circulate. As of May 2019, there have been a total of 2428 laboratory confirmed cases since the outbreak began with a 34.5% mortality rate. It is essential that novel strategies are developed to protect livestock and humans against coronaviruses. The work proposed here will identify cellular proteins that are essential for coronaviruses replication, opening up new possibilities to design vaccines and antivirals for diverse coronaviruses by interrupting the function of these cellular proteins in the virus life cycle.

Human and livestock health: Coronaviruses are significant causes of disease in livestock and humans and have huge zoonotic potential. The step of virus replication studied in this proposal is critical and common among all coronaviruses. The comparison between viruses, as outlined here, provides a powerful way to identify similarities and differences in how coronaviruses interact with their host cells. Valuable knowledge will be gained that will inform scientific research and underpin and facilitate work to design vaccines and particularly antiviral therapies for economically important viruses or those posing a threat to human health.

BBSRC: Food security, animal welfare and One Health are BBSRC research strategic priorities. Results from this work will provide the fundamental knowledge that underpins the ability to design novel prevention and therapeutic strategies required to secure UK and global food security and to provide preparedness for emerging zoonotic viruses.

TPI: Understanding molecular virus-host interactions and the molecular biology of replication of important livestock and zoonotic viruses including understanding viral replication organelles are strategic aims of TPI. Therefore this proposal will help fulfil TPIs strategic aims and will also benefit the academic reputation of TPI.

Students: Data generated in this project will have general interest for students. Information will be disseminated via STEM outreach events and through lectures and seminars presented to BSc and MSc students.

Public: Members of the public including veterinarians and farmers will have a general interest in the outputs of this proposal. Information will be shared at public engagement events, including at agricultural shows, as well as through TPIs website and press releases, where appropriate.

Training and development: The project will directly impact the career development and training of the PDRA, who will gain training in several transferable scientific skills including molecular biology and virology techniques. The PDRA will also be trained in transferable yet highly specialized state-of-the-art bioimaging techniques including correlative light electron microscopy and electron tomography. The outputs of this project will directly benefit HM as a recently independent researcher. The project will formalize collaborations between HM, AD and DM and generate data required for scientific development and for submission of future grant applications.