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.

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles

“…Together this suggests that the cluster of three macrodomains in SARS-CoV nsp3 arose through gene duplication and that additional macrodomains may contribute to the function of nsp3 as an accessory to the viral replication process (Neuman et al, 2008). Interestingly, in the context of a SARS-CoV infectious cDNA clone, Mac1 and Mac2 were dispensable but Mac3 was essential for RNA replication (Kusov et al, 2015).…”

Nsp3 of coronaviruses: Structures and functions of a large multi-domain protein

“…In addition, these conserved domains are also present in several positivesense ssRNA (+ssRNA) viruses of the families Hepeviridae, Togaviridae, and Coronaviridae, such as hepatitis E virus (HEV), alphavirus, rubella virus, and all coronaviruses (Koonin et al, 1992;Snijder et al, 2003). Our group has shown that the X domain (Mac1) is dispensable for RNA replication in the context of a SARS-CoV replicon (Kusov et al, 2015). Recently, evidence accumulated showing that the X domain plays a role in counteracting the host innate immune response (Eriksson et al, 2008;Kuri et al, 2011;Fehr et al, , 2016.…”

“…A region corresponding to parts of SUD was found to exist in other coronaviruses, mostly of clades B, C, and D of the genus Betacoronavirus (Neuman, 2016). For example, domains similar to SUD-M and SUD-C (but not SUD-N) are also encoded by the MERS-CoV genome (Kusov et al, 2015;Ma-Lauer et al, 2016). Thus, it is no longer appropriate to call this domain "SARS-unique".…”

ADP‐ribosylation: new facets of an ancient modification

“…In addition to the MacroD‐type of macrodomains, a highly diverged macrodomain SUD‐M was found as a part of the nsP3 in SARS coronaviruses. This unique macrodomain binds nucleic acids, preferentially RNA, and is crucial for viral genome replication/transcription . Numerous other examples show that viral macrodomains affect virus replication and interferon‐response in humans .…”