Title: Exploiting and advancing single-cell proteomics to identify novel pathways to disrupt RNA virus infection.
Funder: The Academy of Medical Sciences

Abstract: Single-cell studies have been transformative in biology, dominated by single-cell transcriptomics. Recently, methods for single-cell proteomics by mass spectrometry (SCoPE-MS/SCoPE2) have emerged permitting quantitative analysis of the single-cell proteome. I will apply SCoPE2 to study RNA virus replication and virus-host interactions with the goal of identifying targets for antiviral intervention. My preliminary data demonstrates the method has the sensitivity to identify the full range of viral proteins expressed during infection. To achieve this goal, I propose to advance methods for single-cell proteomics by adapting SCoPE2 for targeted analysis of specific peptides of interest, permitting reproducible sampling of viral peptides. This proposal has two aims: 1: Develop single-cell proteomics for the analysis of specific viral targets of interest. 2: Employ targeted single-cell proteomics to track changes in viral gene expression at single-cell level. The first aim of this proposal relies on the SCoPE2 method that I helped develop during my postdoctoral research. By altering the mass spectrometer settings from data-dependent (abundance-based) peptide sampling it is possible to target specific peptides of interest, ensuring reproducibility across multiple experiments. I will optimise SCoPE2 for targeted analysis of RNA virus proteins from single-cell samples. For aim 2, I will apply the optimised SCoPE2 method developed in aim 1 to perform targeted SCoPE2 analysis of murine norovirus and SARS-CoV-2 infection. Both of these viruses replicate using post-translational cleavage of a long polyprotein. Targeted protein-level measurements are necessary to track such cleavage at single-cell level, identifying mechanisms of viral replication, and potential targets for antiviral intervention.

Lay abstract: RNA viruses are responsible for common endemic disease such as norovirus, as well as the COVID-19 pandemic. How infection progresses differs between individuals with some getting severe disease while others may not have any symptoms. Even within an individual the results of infection can vary, with some cells getting infected and producing high levels of virus, while in others infection fails. Single-cell studies allow us to investigate this variation between individual cells. By identifying how cells differ in their ability to allow the virus to replicate, we can identify weak points in a virus’s growth, which we can target to treat disease. Most current single-cell studies look at mRNA from different cells. However, RNA virus replication is highly regulated at the protein level, and it is at the protein level that most drugs act. In my past research, I helped develop a method to study how thousands of the proteins in our cells can differ between single-cells, a method called SCoPE2. For this research, I will be improving SCoPE2, to enhance its ability to detect specific portions of viral proteins which can tell us about viral replication. I will then use this improved method to study how norovirus, and SARS-CoV-2, the virus responsible for COVID-19 replicate in single-cells. The goal of this research is to identify weaknesses which we can target with antivirals to treat infection in patients.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION