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The impact of human defence GTPases on poxvirus infection

Primary Supervisor: Professor Jason Mercer, School of Biosciences

Secondary supervisor: Dr Eva Frickel, School of Biosciences

PhD project title: The impact of human defence GTPases on poxvirus infection

University of Registration: University of Birmingham

Project outline:

Poxviruses are a family of dsDNA viruses characterised by their large size, complex composition, and exclusively cytoplasmic lifestyle. Members of the poxvirus family include variola virus, the causative agent of smallpox (the deadliest disease in human history), and vaccinia virus (VACV), the laboratory model poxvirus used for the global eradication of smallpox.

With over 200 genes at their disposal poxviruses dedicate nearly 50% of their genome to encoding immunomodulating proteins, thereby displaying the most diverse range of immune evasion strategies employed by viruses.

Human GBPs are a family of seven ~65kDa GTPases whose expression is upregulated by interferons, for example during an infection. All mammalian GBPs contain a GTPase domain and a C-terminal helical region. GBP1, GBP2 and GBP5 have a C-terminal CXXX motif that is modified by lipidation which then can anchor them to membranes. GBPs can traffic to microbial compartments destroying these membranes, enhance inflammation by activating the inflammasome and limit pathogen replication. These pathways happen inside the infected cell with the goal of controlling the infection and thus this process is called cell-intrinsic host defence.

GBPs play a role in pathogen defence for viruses, bacteria and protozoa. Initially GBPs were identified as defence proteins against viral infection and recently GBP2 and 5 were found to drive retroviral defence through regulating viral glycoprotein processing [1, 2]. However, most mechanistic molecular work has so far focused on GBPs in host defence to bacterial and protozoan pathogens [3]. Thus, we currently lack understanding of how GBPs drive host defence mechanisms against viruses.

For bacterial and protozoan pathogens, the prerequisite for GBP-mediated control is targeting of the GTPase to the pathogen-containing compartment or the pathogen directly, with the consequence being the disruption of the compartment or the co-recruitment of signalling molecules. Whether this is the case for GBP-mediated control of viruses is not fully understood. We can analyse vaccinia [replication and spread by performing viral plaque assays] and host cell death in human macrophages employing both cell lines (THP-1) or primary cells (MDMs, monocyte-derived macrophages) [4, 5]. We can overexpress GBPs and downregulate them using siRNA in both cell systems or employ CRISPR technologies in THP-1s to analyse the behaviour of GBP mutants reconstituted in the corresponding GBPKO background. We have set up high-content image analysis using a battery of fluorescent recombinant VACV viruses that allow us to measure infection parameters such as binding, entry, genome uncoating and replication, as well as virus assembly [6]. This enables us to assess quantitatively and reproducibly the effect of many GBP mutants throughout many timepoints of infection. We will be able to pair functional infection control results with imaging-based localisation studies of the virus and the GBPs.

In addition to the GTPase and lipidation mutants described above, we will analyse mutants of GBPs that fail to be ubiquitinated or phosphorylated to define the post-translational regulatory mechanisms active in vaccinia virus control. Using these GBP mutants and the knowledge acquired in vaccinia infection control dynamics, we will employ bioinformatics, protein biochemistry, proximity-labelling and mass spectrometry to discover novel cellular host factors participating in this pathway. These novel players will be characterised for their ability to control vaccinia virus. In summary, this project will define the mechanisms of GBP-driven control of VACV and discover novel regulatory mechanisms of GBP-driven pathogen control in general.

References:

  1. Krapp, C., et al., Guanylate Binding Protein (GBP) 5 Is an Interferon-Inducible Inhibitor of HIV-1 Infectivity. Cell Host Microbe, 2016. 19(4): p. 504-14.
  2. Braun, E., et al., Guanylate-Binding Proteins 2 and 5 Exert Broad Antiviral Activity by Inhibiting Furin-Mediated Processing of Viral Envelope Proteins. Cell Rep, 2019. 27(7): p. 2092-2104 e10.
  3. Huang, S., et al., Cell-autonomous immunity by IFN-induced GBPs in animals and plants. Curr Opin Immunol, 2019. 60: p. 71-80.
  4. Fisch, D., et al., Human GBP1 is a microbe-specific gatekeeper of macrophage apoptosis and pyroptosis. EMBO J, 2019. 38(13): p. e100926.
  5. Fisch, D., et al., Human GBP1 Differentially Targets Salmonella and Toxoplasma to License Recognition of Microbial Ligands and Caspase-Mediated Death. Cell Rep, 2020. 32(6): p. 108008.
  6. Kilcher, S., et al., siRNA screen of early poxvirus genes identifies the AAA+ ATPase D5 as the virus genome-uncoating factor. Cell Host Microbe, 2014. 15(1): p. 103-12.

BBSRC Strategic Research Priority: Understanding the Rules of Life: Immunology & Microbiology

Techniques that will be undertaken during the project:

  • Construction, propagation and purification of vaccinia virus
  • Basic virology assays (plaque assays, 24-hour yields, gene expression assays)
  • Culturing of human cells
  • Fluorescent microscopy: super-resolution, confocal, widefield and high content imaging of viruses and cells
  • Artificial intelligence driven image analysis
  • Genetic manipulation of tissue culture cells: siRNA, CRISPR
  • Genetic manipulation of viruses: gene tagging/depletion or deletion
  • Immunoblotting
  • Immunoprecipitation from cellular lysates
  • ELISA and cell death assays
  • Preparation of cellular lysates for mass spectrometry analysis
  • Protein expression and purification

Contact: Professor Jason Mercer, University of Birmingham