# Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

## TL;DR

The paper introduces a cell-based assay to study how viral macrodomains reverse a key immune-related modification, offering a tool for drug discovery.

## Contribution

A novel cell-based screening system for macrodomain hydrolase activity using PARP15 isoform 1 is developed.

## Key findings

- PARP15 isoform 1 forms nuclear foci dependent on its ADP-ribosyltransferase activity.
- Active macrodomains dissolve these foci, allowing direct monitoring of hydrolase activity in living cells.
- The assay enables screening of macrodomain inhibitors with physiological relevance.

## Abstract

Mono-ADP-ribosylation, a modification of both proteins and nucleic acids, is implicated in innate immunity. Intracellularly, this modification is catalyzed by PARP enzymes, some induced in response to interferons. Mono-ADP-ribosylation is reversed by hydrolases including proteins with macrodomains, which are conserved across all kingdoms of life. Macrodomains encoded by certain positive-sense single-stranded RNA viruses, such as Chikungunya virus and SARS-CoV-2, antagonize host MARylation to enhance viral replication and suppress the immune response. While macrodomain hydrolase activity is essential for CHIKV replication, in SARS-CoV-2 it predominantly contributes to immune evasion, underscoring viral macrodomains as potential antiviral drug targets. Efforts to develop macrodomain inhibitors include computational modeling, crystallography-based methods, and in vitro assays. However, tools to study macrodomain activity directly in cells remain rare. Here, we established a cell-based assay using PARP15 isoform 1, which we found forms nuclear foci dependent on its ADP-ribosyltransferase activity. Enzymatically active macrodomains dissolve these foci, enabling hydrolase activity monitoring in living cells. Using stable cell lines, this system allows the screening of macrodomain inhibitors while simultaneously addressing cell permeability, toxicity, and physiological relevance. Adaptable to various macrodomains, our platform offers a versatile tool to study macrodomain function in living cells, analyzing mutants, and advancing drug discovery efforts.

In this study, the authors present a cellular assay to probe macrodomain ADP-ribosylhydrolase activity. This relies on PARP15, forming nuclear foci upon automodification, which are dissolved by loss of MARylation. The system enables functional studies and screening of macrodomain and PARP15 inhibitors in physiologically relevant conditions, capturing cell permeability and cytotoxicity.