Viral antagonism of extracellular cGAMP responses
Keywords: cGAMP
STING
HSV-1

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.