Investigating HSV-1 pUL56 mediated antagonisation of DNA sensing mechanisms

Abstract

Herpes Simplex Virus 1 (HSV-1) is a large, enveloped DNA virus that infects around two-thirds of the world population. HSV-1 is known to successfully remodel the host cell microenvironment to allow for efficient replication and propagation of progeny virions. One of the ways in which HSV-1 achieves this, is through the viral protein pUL56, a type-II transmembrane protein within the tegument of HSV-1, that protrudes into the cytoplasm when expressed in infected cells. HSV-1 pUL56 funcAons as an E3 ubiquitin ligase adaptor protein, interacting with the WW domains of the NEDD4 family of HECT E3 ubiquitin ligases via three PPxY motifs present within pUL56. HSV-1 pUL56 has been shown to remodel the host cell proteome and plasma membrane proteome during infection, targeting a range of host cell proteins for ubiquitin-mediated degradation. This thesis aimed to follow up on potential pUL56 degradation targets that are involved in the production and secretion of cyclic AMP-GMP (cGAMP). Under normal physiological conditions, DNA should not be present in the cytosol, and as such, is a powerful pathogen associated molecular pattern (PAMP) that triggers activation of pattern recognition receptors (PRR) within the cell. One such PRR, is the cGAMP synthase (cGAS) enzyme, that produces a secondary messenger molecule, cGAMP, which in turn leads to the production of type-I interferons (IFN), resulting in an anti-viral response. As well as acting internally within the cell, cGAMP can also be secreted from the cell by cGAMP transport channels. This enables cGAMP to be delivered to neighbouring cells, thereby initiating a signalling cascade that results in the production of type-I IFN, priming immunity in bystander uninfected cells to restrict virus spread. One such cGAMP transport channel of interest is the volume regulated anion channels (VRAC). The research in this thesis has demonstrated that VRACs are removed from the cell surface and subsequently degraded in a pUL56-dependent manner during HSV-1 infecAon. Recombinant HSV-1 viruses that lack pUL56 or the PPxY motifs within pUL56 are unable to degrade VRACs during infection, and type-I IFN transcript production is increased during infection of viruses lacking pUL56 expression or expressing pUL56 lacking the PPxY motifs. Type-I IFN production caused by these recombinant viruses returned to wild-type levels during infection of VRAC knockout cells. Immunoprecipitation of both full length and truncated forms of the obligatory subunit of VRACs, LRRC8A, co-expressed with a panel of overexpressed pUL56 mutants were conducted to identify the minimum binding region between the two proteins. Conclusion of these experiments have mapped the binding region to be between residue of 105-157 of pUL56, likely to be dependent on the PPxY motif present in this region, and between residues 347-426 of LRRC8A. During these investigations, a report was published that suggested pUL56 directly antagonises cGAS, lowering the overall production of cGAMP within infected cells. Given that this presents a confounding variable when investigating the function of cGAMP secretion during infection, the potential interaction between pUL56 and cGAS was investigated. Overexpression studies showed the interaction between the two proteins was dependent on a previously uncharacterised arginine rich region of pUL56 and further that interaction was dependent on the presence of nucleic acids, presumably serving as a linker. However, interaction between pUL56 and endogenous cGAS during infection could not be detected in multiple experiments. Transcriptional analysis further showed little-to-no impact of pUL56 expression on the expression of type-I IFN or pro-inflammatory cytokines, suggesting limited biological significance of the interaction. These data therefore suggest that pUL56 does not antagonise cGAS directly during HSV-1 infection, but can effectively remove VRACs from the cell surface of infected cells constituting a novel immune- evasion function of pUL56 that could increase HSV-1 spread from an initial site of infection by dampening paracrine cGAMP signalling.