Characterization of Parp14-Trim25 directed mitochondrial ISGylation and its antagonism by viral deISGylases

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Interferon stimulated gene 15 (ISG15) is a ubiquitin like post-translational modifier, and expressed in abundance upon immune challenges such as infection, and other cellular stresses such as mitochondrial disruption, ageing and DNA damage. ISG15 can modify substrates in a process referred to as ISGylation. Many viruses in turn have co-evolved the ability to hydrolyse ISG15 modifications via the reverse reaction referred to as deISGylation, allowing them to subvert host cell function and innate immune responses.Much of the innate immune signalling takes place at the mitochondrial membranes, which function as platforms or signalling hubs in collaboration with the endoplasmic reticulum membrane. These processes consist of various signalling branches with specificity for different pathogen classes. The role of ISGylation in antiviral signalling at the mitochondria and subsequent virus restriction are poorly understood processes.Our efforts to expand the knowledge on the role of ISGylation, recently led to the identification of a new antiviral branch defined by the Poly ADP-ribosyltransferase 14 (Parp14) enzyme together with an E3 ligase Trim25. We showed that this enzyme drives ISGylation of as yet unidentified substrates at the mitochondria. Many of these substrates in turn are deISGylated by a viral protease (SARS-CoV-2 PLpro). How Parp14-Trim25 functions to regulate ISGylation at the mitochondria, and its functional implications are not known. Similarly, the full repertoire of substrates of Parp14-Trim25 and how its ADP-ribosyltransferase enzymatic activity impacts ISGylation remain important issues to address. While not much is known about Parp14-Trim25, deletion of Parp14 leads to increased susceptibility to virus infection and aberrant innate immune signalling. Importantly, viruses encoding potent deISGylating activities are also known to contain ADP-ribosylhydrolase activities in macrodomains and have effective immune suppression abilities. Whether these hydrolytic activities function in concert to evade host immunity is currently unknown.This proposal combines genetic, biochemical and proteomics approaches in cultured and hIPS-derived macrophages to systematically address these open questions. Our work will provide comprehensive insight into the novel Parp14-regulated antiviral branch and shed light on its physiological role in a relevant cellular system. The findings will inform on the mechanisms of antiviral immunity and other stress conditions, and those disrupted by many RNA viruses to ultimately offer hints toward novel avenues for therapeutic intervention.