Genetic and proteomic analysis of viral evasion
The goal of our work is to identify novel genes and map intracellular pathways involved in virus:host cell interactions. Infected cells need to sense and respond appropriately to intracellular viral infections. In turn, viruses manipulate host cell signalling pathways to enable viral replication and evade immune recognition.
To facilitate this work we have developed a gene discovery programme that uses functional proteomic approaches to identify receptors manipulated by viruses and genome-wide genetic screens to identify key components of intracellular signalling pathways.
Why is the study of viral evasion mechanisms of interest?
Viruses are assiduous cell biologists. We study critical host receptors and pathways used to both attenuate viruses and those which are targeted by viruses. Understanding the molecular mechanisms viruses use to manipulate cellular processes provides unique insights into fundamental cell biological pathways, informs us about viral evasion and has the capacity to offer novel therapeutic approaches, for instance through the specific targeting of these newly identified pathways.
Functional proteomic approaches to study viral evasion pathways:
Cell surface receptors are modulated by all viruses. 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. A major goal has been to develop techniques to establish a temporal unbiased view of cellular receptors or intracellular proteins whose expression is altered upon viral infection. We developed ‘Plasma Membrane Profiling’ (PMP), a metabolic (SILAC) proteomics approach or, more recently, a chemical (TMT)-based proteomics approach which allows us to determine how expression of >1000 cell surface receptors, or ~8000 total cellular proteins changes following viral infection. This technology has identified immune evasion as well as metabolic receptors whose expression is dramatically altered upon HCMV (Weekes et al Cell 157:1460-72, 2014) or HIV (Matheson et al. Cell Host Microbe 18, 409–423 2015) infection. Using genetic, biochemical and cell biological approaches we then determine how newly identified cell surface proteins are compromised by viruses. For example, 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 and CRISPR-Cas9 genome-wide screens in haploid human cells assigns function to new genes:
We complement these functional proteomic technologies with genetic screens in human cells, using either insertional mutagenesis-based screens in haploid cells, or CRISPR-Cas9 genome-wide library screens. This approach has identified critical cellular components used to control viruses as well as host proteins appropriated by viruses as part of their immune evasion strategy. For example, we identified a novel human epigenetic repressor complex we named HUSH (Human Silencing Hub) which is critical for silencing newly inserted retroviruses. HUSH is composed of the three proteins, TASOR, MPP8, and periphilin which are recruited to genomic loci rich in H3K9me3, the canonical mark of heterochromatin. Loss of HUSH results in decreased H3K9me3 at endogenous genomic loci, altered transcription, as well as derepression of integrated retroviruses. Preventing viruses such as HIV from being silenced could provide a crucial step in their eradication.
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.
Timms RT, Menzies, SA, Tchasovnikarova IA, Christensen, LC, Williamson, JC, Antrobus R, Dougan G, Ellgaard, L, Lehner PJ. Genetic dissection of mammalian ERAD through comparative haploid and CRISPR forward genetic screens. Nature Comm. (2016) in press
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).
Matheson NJ, Sumner J, Wals K, Rapiteanu R, Weekes MP, Vigan R, Weinelt J, Schindler M, Antrobus R, Costa ASH, Frezza C, Clish CB, Neil SJD & Lehner PJ. Cell Surface Proteomic Map of HIV Infection Reveals Antagonism of Amino Acid Metabolism by Vpu and Nef. Cell Host Microbe 18, 409–423 (2015).
Cano F, Rapiteanu R, Winkler GS & Lehner PJ. A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C. Nature Comm. 6, 8670 (2015).
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).
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).