title: A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.
abstract: The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity.
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel Site, University of Lübeck, Germany. Electronic address: koussov@biochem.uni-luebeck.de.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kusov
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany. Electronic address: tanjinzhi@gmail.com.
authors.forename: Jinzhi
authors.initials: J
authors.lastname: Tan
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma, Madrid, Spain. Electronic address: ealvarezmba@gmail.com.
authors.forename: Enrique
authors.initials: E
authors.lastname: Alvarez
authors.affiliation: Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma, Madrid, Spain. Electronic address: L.enjuanes@cnb.csic.es.
authors.forename: Luis
authors.initials: L
authors.lastname: Enjuanes
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany; German Center for Infection Research (DZIF), Hamburg - Lübeck - Borstel Site, University of Lübeck, Germany. Electronic address: hilgenfeld@biochem.uni-luebeck.de.
authors.forename: Rolf
authors.initials: R
authors.lastname: Hilgenfeld
mesh_terms: D019943:Amino Acid Substitution; D017930:Genes, Reporter; D049410:Luciferases, Renilla / Q000032:analysis / Q000235:genetics; D017434:Protein Structure, Tertiary; D059386:Reverse Genetics; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000502:physiology*; D017384:Sequence Deletion; D013194:Staining and Labeling; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D017361:Viral Nonstructural Proteins; D049410:Luciferases, Renilla
keywords: G-quadruplex; MERS-CoV; Macrodomain; Reverse genetics; SARS-CoV replicon; SARS-unique domain; X-domain