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Virology. 2015 Oct;484:313-22. doi: 10.1016/j.virol.2015.06.016. Epub 2015 Jul 3.

A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

Author information

1
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.
2
Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Lübeck, Germany. Electronic address: tanjinzhi@gmail.com.
3
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma, Madrid, Spain. Electronic address: ealvarezmba@gmail.com.
4
Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB-CSIC), Campus Universidad Autónoma, Madrid, Spain. Electronic address: L.enjuanes@cnb.csic.es.
5
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.

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.

KEYWORDS:

G-quadruplex; MERS-CoV; Macrodomain; Reverse genetics; SARS-CoV replicon; SARS-unique domain; X-domain

PMID:
26149721
PMCID:
PMC4567502
DOI:
10.1016/j.virol.2015.06.016
[Indexed for MEDLINE]
Free PMC Article

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