Viral antagonism of extracellular cGAMP responses

The presence of viral DNA outside of the nucleus is detected by the pattern recognition receptor cGAS during infection. cGAS then generates the cyclic dinucleotide 2’3’-cGAMP, which activates the central hub of DNA sensing – STING. This results in the production of proinflammatory cytokines, primarily type I interferons, driving antiviral responses.
Classically, 2’3’-cGAMP was thought to solely act within the cytosol, as its negative charge makes it membrane impermeable. Recently, five membrane spanning channels (ABCC1, SLC19A1, SLC46A2, P2X7 and LRRC8A:C/E) have been shown to import and export 2’3’-cGAMP allowing movement between cells. This finding implicates extracellular 2’3’-cGAMP in a range of functions: defence against viral invasion; T cell proliferation; natural killer cell recruitment; vaccine efficacy; cancer clearance; metastasis and immunotherapy. 2’3’-cGAMP may thus function like a cytokine to message between cells.
I theorised if exogenous 2’3’-cGAMP functions within the innate immune response then viruses would act to evade this mechanism. I screened viruses for the capacity to deplete cGAMP channels during infection. I found that viruses with dsDNA genomes deplete multiple cGAMP channels during infection, whereas no viruses with RNA genomes reduced cGAMP channel abundance. Remarkably, Herpes Simplex Virus 1 (HSV-1) degraded three of the five known cGAMP channels - P2X7, SLC46A2 and LRRC8A:C. Therefore, I chose HSV-1 as a model virus to study the effects of extracellular 2’3’-cGAMP.
By immunoprecipitating LRRC8C from HSV-1 infected cells and preforming mass spectrometry-based proteomics I identified UL56, as a viral interaction partner of LRRC8C. Overexpression of UL56 confirmed that it caused LRRC8A:C degradation. Unexpectedly I found UL56 also caused degradation of the P2X7 and SLC46A2 channels. Using the reverse approach, I found that HSV-1 lacking the UL56 gene (HSV-1-ΔUL56) was unable to degrade cGAMP channels. This proves that UL56 is necessary and sufficient to degrade all three of the cGAMP channels. Mechanistically UL56 contains PPXY motifs that act as a platform for recruitment of NEDD-4 family ubiquitin ligases. I showed that mutation of the PPXY motifs to AAXA prevented degradation of cGAMP channels by UL56, both when UL56-AAXA was over- expressed or upon infection with AXAA mutated virus (HSV-1-AAXA). Consistent with degradation mediated by NEDD-4 ligases, chemical inhibition of the proteasome appeared to rescue LRRC8A:C abundance.
To show UL56 inhibits 2’3’-cGAMP import, I developed in vitro models of 2’3’-cGAMP uptake for LRRC8A:C, SLC46A2 and P2X7. This was done either by activating channels endogenously expressed by 293 cells (LRRC8A:C) or by stably overexpressing the channels (P2X7 and SLC46A2) to allow increased 2’3’-cGAMP uptake. In this system, import of 2’3’-cGAMP can be blocked with channel-specific inhibitors or by overexpression of UL56. This showed functionally that UL56 prevents 2’3’-cGAMP from being imported into cells and so inhibits IFN-β production. In summary I show for the first time that a virus antagonises cGAMP channels, likely to avoid the effects of exogenous 2’3’-cGAMP, thus adding to the recent and exciting body of literature describing the role of 2’3’-cGAMP as a signalling molecule between cells.

Keywords: cGAMP
STING
HSV-1
Advisor: Rehwinkel, J