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Paul Lehner

Viral silencing and immune evasion pathways

The goal of our work is to identify novel genes and map intracellular pathways involved in virus:host interactions. Infected cells need to sense and respond appropriately to viral infection. In turn, viruses manipulate host cell signaling pathways to enable viral replication and evade immune recognition.

Why is viral evasion interesting and important?

Viruses are assiduous cell biologists. Deciphering 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 through the specific targeting of newly identified pathways.

Viral Silencing

All life forms defend their genome against DNA invasion. Mammalian cells counteract this threat to cellular integrity through innate immune system activation (eg release of interferon). An additional cellular response is to silence the incoming DNA, prior to transcription. Indeed DNA silencing through chromatinisation likely represents the default pathway. Perversely, viruses not only introduce and replicate their nucleic acid, but recruit host machinery to enable replication. Persistent viral survival depends on their ability to evade immune recognition, as well recognized with interferon antagonism, but less well realized in the context of DNA silencing.

Viruses adopt different strategies to enhance, prevent or de-repress silencing and are adept at manipulating their chromatin environment. Uniquely, retroviruses reverse transcribe their RNA into dsDNA and integrate into host chromosomal DNA ie they ‘become genes’. By aligning regulation of their viral genome with the host, retroviral integration allows unintegrated, poorly expressed retroviruses to escape extrachromosomal silencing. While most integrations are transcriptionally active, for HIV, the most clinically important lentivirus, it is the silenced, integrated HIV proviral pool which defies eradication and mandates lifelong HIV treatment.

Understanding different mechanisms of DNA silencing and how viruses usurp and overcome them provides unique insight into chromatin regulation and potential opportunities for manipulating these pathways.

 

Genome-wide forward genetic screens identifies novel pathways to heterochromatin formation, critical for repression of newly integrated viruses

We use insertional mutagenesis or CRISPR-Cas9 genome-wide forward genetic screens to interrogate intracellular pathways. This approach has identified critical cellular components used to control viruses and host proteins appropriated by viruses.

 To understand how newly integrated retroviruses are silenced we performed a forward genetic screen and identified a novel human epigenetic repressor complex we named HUSH (Human Silencing Hub) (Tchasovnikarova et al Science 2015. HUSH is composed of the three proteins, TASOR, MPP8, and periphilin which are recruited to genomic loci rich in H3K9me3, the canonical mark of repressive heterochromatin. Loss of HUSH results in derepression of integrated retroviruses. The effector functions of HUSH are: (i) To recruit the SETDB1 histone methyltransferase to deposit repressive H3K9me3 and (ii) To recruit the MORC2 chromatin remodeller to compact the chromatin. Thus HUSH represents a novel route to the establishment and assembly of repressive heterochromatin (Tchasovnikarova et al Nature Genetics 2017 49(7):1035-1044). Preventing viruses such as HIV from being silenced could provide a crucial step in their eradication.

          Lehner HUSH complex                                      

Schematic of the HUSH epigenetic repressor complex: HUSH components TASOR, MPP8 and periphilin are recruited, via MPP8, to H3 histones (H3K9me3). Subsequent recruitment of two effector proteins, SETDB1 and MORC2 remodel the locus through methylation and compaction leading to heterochromatin assembly and transcriptional repression

 

Viruses manipulate the host ubiquitin:proteasome pathway to degrade unwanted cellular receptors. Our genome wide CRISPR forward genetic screens have identified critical components of these pathways, in particular the ubiquitin E3 ligases pirated by viruses to degraded these cellular proteins. These screens provide fundamental insights into both viral evasion and normal cellular regulatory pathways eg sterol regulation.

Proteomic approaches to study viral evasion, identifies new viral therapeutic targets and a novel function for HIV-Vif

Cell surface receptors and intracellular proteins are modulated by all viruses. Our work on viral evasion of MHC-I antigen presentation has driven our interest in the role of ubiquitin in immunoreceptor regulation. We developed techniques to gain a temporal, unbiased, systematic overview of cellular receptors and intracellular proteins whose expression is altered upon viral infection. Plasma Membrane Profiling, using TMT-based proteomics, allows us to determine how expression of >1000 cell surface receptors, or ~8000 total cellular proteins changes upon viral infection or in tumour formation, and is applicable to primary human cells. This technology has identified multiple receptors whose expression is altered upon viral infection eg HCMV (Weekes et al Cell 157:1460-72, 2014) or HIV (Matheson et al. Cell Host Microbe 18, 409–423 2015) infection. We identified a novel, evolutionarily conserved function for HIV-Vif– degradation of the highly abundant cellular PP2A phosphatase. Thus HIV-Vif remodels the HIV-infected cellular phosphoproteome (Greenwood et al ELife 2016 5:e18296).

Related approaches identified cells that are latently infected with human cytomegalovirus, providing a potential strategy for their removal prior to transplantation (Weekes et al. Science 340, 199-202; 2013).

 

 

Lehner lab

Key papers

Tchasovnikarova IA, Timms RT,Douse DH, Roberts RC, Dougan G, Kingston RE Modis Y, Lehner PJ . Hyper-activation of HUSH complex function by Charcot-Marie-Tooth disease mutation in MORC2. Nature Genetics (2017) Jul;49(7):1035-1044. PMID:28581500

Burr ML,Sparbier CE, Chan-YC,Williamson JC, Woods K, Beavis P,Lam EYN, Henderson MA, Bell CC, Stolzenburg S, Gilan O, Noori T, Morgens D, Bassik MC, Neeson PJ, Behren A, Darcy PK, Dawson S-J, Voskoboinik I, Trapani JA, Cebon J, Lehner PJ, Dawson MA. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. † Joint corresponding authors Nature (2017) 549(7670):101-105. PMID:28743740

GreenwoodEJD, Matheson NJ, Wals K, van den Boomen DJ,  Antrobus R, Williamson JC, Lehner PJ. Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants. eLife (2016) 5:e18296 PMID:27690223

Timms RT, Tchasovnikarova IA, Dougan G, Antrobus R, Lehner PJ. ATF7IP-mediated stabilization of the histone methyltransferase SETDB1 is essential for heterochromatin formation by the HUSH complex. Cell Reports (2016) 17(3):653-659 PMID:27732843

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) 7:11786. PMID:27283361

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 (2015). doi: 10.1126/science.aaa7227 PMID: 26022416

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 (2015) 18, 409–423.

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. (2015) PLoS Pathogens DOI:10. 1371/journal.ppat.1004811

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 (2014) Jul 16. pii: 201409099.

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. (2014) 205, 847-62.

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 (2014). (http://dx.doi.org/10.1016/j.cell.2014.04.028)

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 (2013) 340, 199–202.

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. (2013) USA 110, 14290–14295.

 

Paul Lehner

Professor Paul Lehner

Professor of Immunology and Medicine, Wellcome Trust Principal Research Fellow

Honorary Consultant, Department of Medicine

Department: Medicine

contact: pjl30@cam.ac.uk

Plain English

 

Viruses play ‘cat and mouse’ with the immune system. All cells are equipped to recognize viruses and silence their DNA to prevent viral genes entering and exerting unwanted effects. Viruses need to evade immune recognition and overcome cellular silencing. Our group studies how cells repress viruses, and in turn how the viruses avoid this silencing, evade immune recognition and manipulate the cell for their own ends. We use state-of-the-art technologies including genome-wide CRISPR/Cas9 screens to assign cellular genes to signalling pathways and systematic proteomic approaches to determine which cellular proteins are modified during viral infection. This has led to the identification of novel HIV silencing pathways and therapeutic approaches for derepressing HIV. We have also identified cells that are latently infected with human cytomegalovirus, providing a potential strategy for their removal prior to transplantation.

Group members

Stuart Bloor · Dick van den Boomen · Marian Burr · Thomas Crozier · Lidia Duncan · Liane Dupont · Ildar Gabaev · Dan Greaves · Ed Greenwood · Natalie Rebeyev · Marta Seczynska · Agata Sinkiewicz · Norbert Volkmar · James Williamson

Funding

Wellcome Trust

Addenbrooke's Charitable Trust

Medical Research Council

GSK Discovery Partnership with Academia