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.

Graphical abstract

Highlights

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  A SARS-CoV replicon encoding Renilla luciferase as reporter protein is constructed.

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  The role of three macrodomains for the replication/transcription complex is analyzed.

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  In contrast to macrodomains X and SUD-N, SUD-M is found indispensable for replication.

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  Site-directed mutagenesis identifies charged SUD-M residues required for replication.

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  These residues have previously been shown to be involved in G-quadruplex binding.