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Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity.

ABSTRACT: While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)-a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase-deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

SUBMITTER: Jayabalan AK

PROVIDER: S-EPMC8017970 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-related CoVs encode 3 tandem macrodomains within nonstructural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated antiviral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) Mac1 domains exhibit similar structural folds, and all 3 proteins bound to ADP-ribose with affinities in the low micromolar range. Importantly, using ADP-ribose-detecting binding reagents in both a gel-based assay and novel enzyme-linked immunosorbent assays (ELISAs), we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate than the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCE SARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused more than 1.2 million deaths worldwide. With no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic posttranslational process that is increasingly being recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here, we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose and describe its ADP-ribose binding and hydrolysis activities in direct comparison to those of SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

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Stress granule formation, disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity.

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