The sensing of nonself nucleic acids is powerful strategy of the host innate immunity against invading viruses. In cells infected, e.g. with DNA viruses, the unusual presence of DNA in cytosol can be indicative of non-self or „dangerous“DNA. Several DNA sensors have been recently identified, from which cGAS (cGAMP-AMP synthase) is best characterized. It binds dsDNA and catalyses the formation of cGAMP a dinucleotide that activates the endoplasmic reticulum resident protein STING. This protein serves as a platform for the recruitment of proteins leading to downstream activation of innate immune factors and interferon production. Another DNA sensor, IFI16 (in mouse cells, the analogous protein - p204), was shown to be capable to sense herpesvirus DNA in the cell nucleus and subsequently activate STING.
We have studied the mechanism of induction of interferon in cells infected by murine polyomavirus (MPyV). We found that the virus is hidden from recognition by DNA sensors during travelling to the cell nucleus but induces interferon production when its DNA genome replicates in the nucleus. Signalling after sensing proceeded through phosphorylation of the STING protein. We observed MPyV genomes associated with p204 and cGAS sensors in the nucleus and, also, some viral DNA leaked to the cytosol co-localizing with cGAS. Interestingly, only binding of genomes to p204 in the nucleus and to cGAS in cytosol resulted in IFN production (manuscript in the preparation). The meaning of cGAS binding to the viral minichromosomes remains unsolved. Also, until now, there is not a consensus in the molecular details of the STING activation by IFI16/p204.
The proposed PhD project will directly follow the above research. It will focus on understanding the mechanism of sensing MPyV and human BKPyV genomes by DNA sensor p204/IFI16 and uncovering the role of cGAS association with polyomavirus minichromosomes in the cell nucleus. Confocal, super resolution and immunoelectron microscopy will be used to follow i) dynamics and spatial and functional organisation of IFI16/p204 and cGAS interactions, ii) IFI16/p204 and STING dynamics of trafficking after their activation and iii) the possible interaction of cGAS with viral nucleosomes as well as with cellular SMC5/6 complex and other proteins associated with viral genomes during replication and DNA damage response. Western blot, co-immunoprecipitation, pulldown assays and mass spectroscopy will be used for further identification of cGAS and p204/ IFI16 interacting partners and for studies of posttranslational modifications of IFI16, cGAS and STING. The extension of research to human BKPyV will be facilitated by exploiting newly available cell lines (derived from lung or bladder vascular endothelial cells )recently proposed as reservoir of BKPyV instead of primary human kidney cells.
Ryabchenko B., Soldatova I, Schroller V., Forstová J and Huerfano S. Immune sensing of Mouse polyomavirus by the DNA sensors p204 and cGAS. 2020, Manuscript in preparation.
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