Viruses are only capable of replicating within infected cells. This is because viruses typically only encode the minimal additional machinery required for a cell to produce virus particles, and rely on the host cell for the majority of what the virus needs to replicate. Much of my past research has focused on understanding how the virus and infected cell interact, which portions of the host cell are required for the virus to replicate successfully, and how the host can defend itself against infection.
Many viruses (particularly positive-sense RNA viruses) encode at least one protease. Typically this would be used by the virus to cleave viral proteins, but proteins from the host cell can also be targeted in order to help the virus inhibit the immune response or otherwise control host cell processes. I recently showed that the norovirus protease (NS6) cleaves the cellular protein poly(A) binding protein (PABP) in order to inhibit protein synthesis within infected cells and dampen the immune response (Emmott et. al. 2017).
Cleavage of viral proteins is a carefully regulated process. Within positive-sense RNA virus polyproteins, different rates of cleavage at individual cleavage sites result in the production of a variety of intermediate cleavage products termed precursors. I recently developed a FRET-based assay which allowed rates of cleavage within cells to be determined by measuring fluorescence (Emmott et. al. 2015). Through a combination of mutagenesis of the protease and substrate, and viral reverse genetics, I was able to use this system to examine substrate cleavage across human and mouse norovirus proteases, as well as how these protease differ in their substrate preferences. Later, we were able to use this assay to measure the effect of potential antiviral drugs targeting the protease, as well as resistance mutations that developed in the protease during replication in the presence of these drugs (Kitano et. al. 2018).
Much of the research I have done has used mass spectrometry-based quantitative proteomics to look at how viral infection changes the infected cell, or to identify the cellular proteins needed by a virus for infection. The techniques I have used include stable isotope labelling of amino acids in cell culture (SILAC) and tandem mass tagging (TMT) which allows a user to determine the relative abundance of proteins between samples. Amongst other uses these methods can be used to identify cellular proteins binding to a viral protein (e.g. Emmott et. al. 2013), changes in protein subcellular localisation or organelle composition (e.g. Emmott et. al. 2010a, b) or changes in specific cellular complex formation (e.g. the translation initiation complex, Emmott et. al. 2017).
Single cell proteomics
As part of my current research in the Slavov lab, I am involved in helping to refine and further develop the single cell proteomics methods (SCoPE-MS) first developed in the lab. Most cell or virology studies are conducted on many thousands to millions of cells worth of material. These have been an incredibly powerful tool, including for proteomics. However they are limited in that by averaging across a large population, trends evident on the individual or single cell level can be missed or even reversed when measuring only the averaged population. This phenomena is known as ‘Simpsons paradox’. The use of single cell methods such as single cell sequencing or proteomics allows these features to be investigated. SCoPE-MS relies on the use of a carrier channel containing material from 200 cells to provide sufficient material for identification. Material from the individual single cell channels can be distinguished through the use of TMT tags. In the future I hope to use this method to study virus-host interactions and virus replication!
I’ve worked on a number of viruses, some briefly as part of collaborations or shorter work, and some more extensively. The major viruses I have worked on are noroviruses/caliciviruses and coronaviruses (infectious bronchitis virus).
Noroviruses are the causative agent of ‘winter vomiting disease’, which typically involves 24-48h of diarrhoea and vomiting symptoms for infected individuals. Whilst this infection is typically short-lived and individuals recover quickly, in the elderly or immunocompromised infection can be more severe and norovirus does claim >200,000 lives per year.
There is no current vaccine or antiviral to treat norovirus. Research on noroviruses for years was incredibly difficult due to an inability to grow this virus in the laboratory, instead relying on volunteer studies which are expensive (and messy). This situation changed in 2003 with the discovery of a mouse norovirus (MNV) which could be grown in the lab. Whilst no animal model is able to perfectly represent human disease, this was a huge step forward for the field and represents the most straightforward and well-characterised system for studying norovirus infection. More recently in 2015/16, several systems have emerged permitting the growth of norovirus in cell culture. These rely on infection of B cells in the presence of killed gut bacteria, or on use of stem cell-derived gut organoids, which are effectively miniature guts which mimic the environment the virus infects in the body.
Coronaviruses typically cause mild infections in humans and represent a small percentage of the viruses responsible for the common cold. Exceptions include the zoonotic coronaviruses SARS (Severe Acute Respiratory Syndrome) and MERS (Middle Eastern Respiratory Syndrome) coronavirus which emerged in the last 10-20 years. However, whilst these viruses do not generally cause substantial human interest, a number of animal coronaviruses do represent a significant disease/economic burden on the agricultural industry. Examples of these include avian coronavirus and porcine epidemic diarrhoea coronavirus (PEDV).
The major coronavirus I worked on for my PhD was avian coronavirus (also known as infectious bronchitis virus – IBV). Unlike SARS or MERS coronavirus, this virus doesn’t pose a direct risk to human health. However, it is responsible for large economic losses as it can kill large numbers of chickens and spread easily through flocks. Chickens which survive may have permanent effects from the disease such as loss of egg-laying ability.
Whilst vaccines are available for IBV, these do suffer from a poor ability to provide protection against strains that are dissimilar to the vaccine strain. Efforts to improve current or novel vaccines, and generate more resistant chicken breeds, are areas of current research.