Viral evasion of immune recognition
We study how viruses evade the human immune system. As the ultimate intracellular parasites, viruses target the host cellular machinery to enable their replication and avoid elimination by the immune system. Our research goals and themes are to use functional genetic and proteomic technologies to identify novel cellular receptors manipulated by viruses, understand why these receptors are targeted and elucidate the mechanisms used by viruses to manipulate the cellular immunoreceptors.
Why is the study of viral evasion mechanisms of interest?
Identifying host cellular receptors targeted by viruses is important for several reasons: viruses are assiduous cell biologists. Studying the critical host receptors targeted by viruses and understanding the mechanisms they use to manipulate cellular processes provides unique insights into fundamental cell biological pathways, teaches us about viral pathogenesis and has the capacity to offer novel therapeutic approaches, for instance by targeting these newly identified receptors.
Viral regulation of cell surface receptors:
Cell surface receptors are modulated by all intracellular pathogens. Our work on viral evasion of the MHC class I antigen presentation pathway has driven our interest in the role of ubiquitin in immunoreceptor regulation.
Functional proteomic approaches:
A major goal has been to develop techniques to establish an unbiased view of cellular receptors whose expression is altered upon viral infection. We recently developed ‘Plasma Membrane Profiling’ (PMP), a SILAC- or TMT-based proteomics approach which allows us to compare the relative abundance of >1000 cell surface receptors between infected and non-infected cells, and therefore identify receptors whose expression is altered by viral infection. This technology has identified immune evasion as well as metabolic receptors whose expression is dramatically altered following infection. Using genetic, biochemical and cell biological approaches we then determine how newly identified cell surface proteins are compromised by intracellular pathogens. This approach has led to a method for identifying cells that are latently infected in human cytomegalovirus, providing a potential strategy for their removal prior to transplantation surgery (Weekes et al. Science 340, 199-202; 2013).
Forward genetic screens in haploid human cells assigns function to new genes:
We combine functional proteomic technologies together with novel insertional mutagenesis-based genetic screens in human haploid cells to identify the critical pathway components required for immunoreceptor regulation. For example, this screening strategy has revealed a role for the MARVEL domain protein PLP2 (proteolipid protein 2) in downregulation of plasma membrane MHC-I immunoreceptors through its effects on viral ubiquitin E3 ligase activity (Timms et al Plos Pathogens 9, e1003772; 2013).
Figure 1. A haploid genetic screen identifies genes required for cell surface expression of MHC-I. a. Schematic of screen. b. Selecting MHC-Ilow cells by FACS. Mutagenised KBM7 cells were labelled for surface MHC-I and cells defective for MHC-I presentation enriched by two sequential rounds of cell sorting. c. The genetic screen identifies multiple genes of the MHC-I pathway. Important genes within the MHC-I antigen presentation pathway are targeted by multiple independent retroviral integrations (red triangles). d. Schematic representation of the β2m, tapasin and TAP2 knockout clones identified by PCR from 96 single cell clones from the HLA-Blow selected population. e. The gene-trap insertions result in a loss of gene expression. The knockout clones were analysed for HLA-A2, β2m, tapasin and TAP2 expression by RT-PCR. f. Knockout of genes involved in the MHC-I pathway impairs cell surface expression of MHC-I molecules. The β2m, HLA-A2, tapasin and TAP2 knockout clones were labelled for the indicated proteins and analysed by flow cytometry.
Tchasovnikarova IA, Timms RT, Matheson NJ, Wals K, Antrobus R, Göttgens B, Dougan G, Dawson MA, Lehner PJ. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science doi: 10.1126/science.aaa7227 PMID: 26022416 (2015). (abstract) (reprints).
Hsu J-L, van den Boomen, DJH, Tomasec P, Weekes MP, Antrobus R, Stanton RJ, Ruckova E, Sugrue D, Wilkie GS, Davison AJ, Wilkinson GWG, Lehner PJ. Plasma Membrane Profiling Defines an Expanded Class of Cell Surface Proteins Selectively Targeted for Degradation by HCMV US2 in Cooperation with UL141. PLoS Pathogens DOI:10. 1371/journal.ppat.1004811 (2015).
van den Boomen DJH, Timms RT, Grice G, Stagg HR,Skodt HR, Dougan G, Nathan JA & Lehner PJ. TMEM129 is a Derlin-1 associated ERAD E3 ligase essential for virus-induced degradation of MHC-I. Proc Natl Acad Sci Jul 16. pii: 201409099 (2014).
Boname, JM, Bloor, S, Wandel, MP, Nathan, JA, Antrobus, R, Dingwell, KS, Thurston, TL, Smith, DL, Smith JC, RandowF, Lehner P J. Cleavage by Signal Peptide Peptidase is required for the degradation of selected tail-anchored proteins. J. Cell Biol. 205, 847-62 (2014).
Weekes, M. P., Tomasec, P., Huttling, E. L., Fielding, C. A., Nusinow, D., Stanton, R. J., Wang, E. C. Y., Aicheler, R., Murrell, I., Wilkinson, G. W.G, Lehner, P. J. and Gygi, S. P. Quantitative temporal viromics: a new approach to investigate
host-pathogen interaction. Cell (http://dx.doi.org/10.1016/j.cell.2014.04.028) (2014).
Timms, R. T., Duncan, L. M., Tchasovnikarova, I. A., Antrobus, R., Smith, D. L. et al. Haploid Genetic Screens Identify an Essential Role for PLP2 in the Downregulation of Novel Plasma Membrane Targets by Viral E3 Ubiquitin Ligases. PLoS Pathogens 9, e1003772 (2013).
Weekes, M. P., Tan, S. Y. L., Poole, E., Talbot, S., Antrobus, R., Smith, D. L., Montag, C., Gygi. S. P., Sinclair, J. H. and Lehner. P. J. Latency-associated degradation of the MRP1 drug transporter offers a therapeutic target for latent human cytomegalovirus (HCMV) infection. Science 340, 199–202 (2013).
Burr, M. L., Van den Boomen, D. J. H., Bye, H., Antrobus, P. R, Wiertz, E. J. and Lehner, P. J. MHC class I molecules are preferentially ubiquitinated on ER luminal residues during HRD1-mediated dislocation. Proc. Natl Acad. Sci. USA 110, 14290–14295 (2013).
Cano, F., Bye, H., Duncan, L. M., Buchet-Poyau, K., Billaud, M., Wills, M. R. and Lehner, P. J. The RNA-binding E3 ubiquitin ligase MEX-3C links ubiquitination with MHC-I mRNA degradation. EMBO J. 31, 3596–606 (2012).
Duncan, L. M., Timms, R. T., Zavodszky, E., Cano, F., Dougan, G., Randow, F. and Lehner, P. J. Fluorescence-Based Phenotypic Selection Allows Forward Genetic Screens in Haploid Human Cells. PLoS ONE 7, e39651 (2012).
Weekes, M. P., Antrobus, R., Talbot, S., Hör, S., Simecek, N., Smith, D. L., Bloor, S., Randow, F. and Lehner, P. J. Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6. J. Proteome Res. 11, 1475–1484 (2012).
Piper, R. C. and Lehner, P. J. Endosomal transport via ubiquitination. Trends Cell Biol. 21, 647 - 655 (2011).
Cano, F., Miranda-Saavedra, D. and Lehner, P. J. RNA-binding E3 ubiquitin ligases: novel players in nucleic acid regulation. Biochem Soc Trans. 38, 1621–1626 (2010).
Boname, J. M., Thomas, M., Stagg, H. R., Xu, P., Peng, J. and Lehner, P. J. Efficient Internalization of MHC I Requires Lysine-11 and Lysine-63 Mixed Linkage Polyubiquitin Chains. Traffic 11, 210–220 (2010).
Stagg, H. R., Thomas, M., Van den Boomen, D., Wiertz, E. H., Drabkin, H. A., Gemmill, R. M. and Lehner, P. J. The TRC8 E3 Ligase ubiquitinates MHC class I molecules before dislocation from the ER J. Cell Biol. 186, 685–692 (2009).
Randow, F. and Lehner, P. J. Viral avoidance and exploitation of the ubiquitin system. Nature Cell Biol. 11, 527–534 (2009).