The SARS-Unique Domain (SUD) of SARS Coronavirus Contains Two Macrodomains That Bind G-Quadruplexes

Abstract

Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389–652 (“SUDcore”) of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 Å resolution, respectively) revealed that SUDcore forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUDcore as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5–6 nucleotides, but more extended G-stretches are found in the 3′-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.

The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: Implications for the antiviral activity of a G-quadruplex ligand

“…In both cases, G-quadruplex ligands were able to stabilize the G-quadruplex conformations and to exert antiviral activity (Perrone et al, 2014). In the SARS coronavirus, G-quadruplexes targeting by a key viral protein was proposed to control host's cell response to viral infection (Tan et al, 2009).…”

The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

“…G4s have been reported in the SARS coronavirus7, the human papilloma, hepatitis C, Zika and Ebola virus genomes891011. Among herpesviruses, RNA G4s have been implicated in the regulation of DNA replication and translation of the Epstein–Barr virus1213.…”

The VIZIER project: Overview; expectations; and achievements

“…Interestingly, some difficult problems were solved by dedicated scientists highly specialized on software for crystal structure determination, as well as crystal structure determination of challenging targets. This was the case for the West Nile RdRp and the VPg-3D RdRp Coxsackie virus complex (Gruez et al, 2008), for which a large proportion of amino acids were ill-defined in the structural model, the arenavirus L1 protein domain whose phases could not be easily found, and the coronavirus SUD domain (Tan et al, 2009). The availability of dedicated software whose improved beta versions were made available prior to dissemination in the structural biology community surely helped to solve or improve structural models generated in VIZIER.…”

“…It could be thus questionable to focus on these domains and their production and structure determination, even though these domains belong to the "low hanging fruit" category. Actually, the structural and functional study of 5 more macro-domains showed significant structural differences in substrate affinity and selectivity amongst coronaviruses (Tan et al, 2009;Piotrowski et al, 2009). The invested efforts for coronavirus macro-domain design and production were helpful alphavirus macro-domain definition and crystallization .…”