Mutation of a highly conserved isoleucine residue in loop 2 of several β-coronavirus macrodomains indicates that enhanced ADP-ribose binding is detrimental for replication.

Autor:	Kerr CM; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., Pfannenstiel JJ; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., Alhammad YM; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., O'Connor JJ; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., Ghimire R; Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma, USA., Shrestha R; Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma, USA., Khattabi R; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., Saenjamsai P; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., Parthasarathy S; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA., McDonald PR; High Throughput Screening Laboratory, University of Kansas, Lawrence, Kansas, USA., Gao P; Protein Production Group, University of Kansas, Lawrence, Kansas, USA., Johnson DK; Molecular Graphics and Modeling Laboratory and the Computational Chemical Biology Core, University of Kansas, Lawrence, Kansas, USA., More S; High Throughput Screening Laboratory, University of Kansas, Lawrence, Kansas, USA.; Oklahoma Center for Respiratory and Infectious Diseases, College of Veterinary Medicine, Stillwater, Oklahoma, USA., Roy A; High Throughput Screening Laboratory, University of Kansas, Lawrence, Kansas, USA., Channappanavar R; Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma, USA.; Oklahoma Center for Respiratory and Infectious Diseases, College of Veterinary Medicine, Stillwater, Oklahoma, USA., Fehr AR; Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas, USA.
Jazyk:	angličtina
Zdroj:	Journal of virology [J Virol] 2024 Nov 19; Vol. 98 (11), pp. e0131324. Date of Electronic Publication: 2024 Oct 10.
DOI:	10.1128/jvi.01313-24
Abstrakt:	All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in non-structural protein 3. Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the glycine-isoleucine-phenylalanine motif. While we previously demonstrated the importance of the glycine residue for CoV replication and pathogenesis, the impact of the isoleucine and phenylalanine residues remains unknown. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that correlated with attenuated replication and pathogenesis of F-A mutant MERS-CoV and SARS-CoV-2 viruses in cell culture and mice. In contrast, the I-A mutant proteins had normal enzyme activity and enhanced ADP-ribose binding. Despite only demonstrating increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 viruses were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication. Importance: The conserved coronavirus (CoV) macrodomain (Mac1) counters the activity of host ADP-ribosyltransferases and is critical for CoV replication and pathogenesis. As such, Mac1 is a potential therapeutic target for CoV-induced disease. However, we lack a basic knowledge of how several residues in its ADP-ribose binding pocket contribute to its biochemical and virological functions. We engineered mutations into two highly conserved residues in the ADP-ribose binding pocket of Mac1, both as recombinant proteins and viruses for Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Interestingly, a Mac1 isoleucine-to-alanine mutant protein had enhanced ADP-ribose binding which proved to be detrimental for virus replication, indicating that this isoleucine controls ADP-ribose binding and is beneficial for virus replication and pathogenesis. These results provide unique insight into how macrodomains control ADP-ribose binding and will be critical for the development of novel inhibitors targeting Mac1 that could be used to treat CoV-induced disease. Competing Interests: The authors declare no conflict of interest.
Databáze:	MEDLINE
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