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 signalling 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.
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 identify 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.
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 the 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).
Tchasovnikarova IA, Timms RT, Matheson NJ, Wals K, Antrobus R, Göttgens B, Dougan G, Dawson MA, Lehner PJ.GENE SILENCING. Epigenetic silencing by the HUSH complex mediates position-effect variegation in human cells. Science 2015. 348, 1481-1485. doi: 10.1126/science.aaa7227.
Tchasovnikarova IA, Timms RT, Douse CH, 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. 49, 1035-1044. doi: 10.1038/ng.3878
Burr ML, Sparbier CE, Chan YC, Williamson JC, Woods K, Beavis PA, Lam EYN, Henderson MA, Bell CC, Stolzenburg S, Gilan O, Bloor S, Noori T, Morgens DW, Bassik MC, Neeson PJ, Behren A, Darcy PK, Dawson SJ, Voskoboinik I, Trapani JA, Cebon J, Lehner PJ, Dawson MA. CMTM6 maintains the expression of PD-L1 and regulates anti-tumour immunity. Nature 2017. 549, 101-105. doi: 10.1038/nature23643.
Robbez-Masson L, Tie CHC, Conde L, Tunbak H, Husovsky C, Tchasovnikarova IA, Timms RT, Herrero J, Lehner PJ, Rowe HM. The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes. Genome Research 2018. 28, 836-845. doi: 10.1101/gr.228171.117.