Nuclear Magnetic Resonance Structure of the N-Terminal Domain of Nonstructural Protein 3 from the Severe Acute Respiratory Syndrome Coronavirus

Abstract

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four ␤-strands and two ␣-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.Severe acute respiratory syndrome (SARS) is a viral infectious disease that has attracted worldwide attention since an outbreak in 2003 (26). It has been postulated that the SARS coronavirus (SARS-CoV) was introduced to the human population from animal CoVs (26). CoVs comprise a large group of enveloped, positive-sense, single-stranded RNA viruses that have been classified in the Nidovirales order. There are three groups of CoVs, based on serological cross-reactivity and phylogenetic relatedness. The SARS-CoV is distantly related to the group 2 viruses and has been classified in group 2b (38).The SARS-CoV represents one of the largest currently known RNA genomes. It is composed of at least 14 functional open reading frames that encode three classes of proteins, i.e., structural proteins (the S, M, E, N, 3a, 7a, and 7b proteins), nonstructural proteins (nsp1 to nsp16), and the accessory proteins (3b, 6, 8, 9b, and 14) (38). With regard to the nonstructural proteins, the translation of the SARS-CoV genome produces two large replicase polyproteins (pp1a and pp1ab), which are processed by two proteases to yield 16 mature nonstructural proteins that mediate RNA replication and processing. Since the SARS outbreak in 2003, knowledge of the structure, activity and function of some of these proteins has increased considerably (30,32,35,41,45); however, the biological roles of many of the SARS-CoV proteins remain unknown. In this paper we describe the nuclear magnetic resonance (NMR) structure determination and a preliminary functional characterization of nsp3a, the N-terminal domain of the largest of the nonstructural proteins, nsp3.SARS-CoV nsp3 is a 213-kDa polypeptide involved in RNA replication and has been proposed to consist of seven domains, nsp3a to nsp3g, which have been identified based on phylogenetic conservation and predicted amino acid secondary structure (38). The biological role of nsp3 is only partially understood, and so far structures have been determined of only the two domains nsp3b, which has been described as an ADP ribose-1Љ-phosphatase (34), and nsp3d, which is a papain-like protease (PLpro) involved in the proteolytic processing of pp1a and pp1ab. nsp3d contains thr...

Changes in nonstructural protein 3 are associated with attenuation in avian coronavirus infectious bronchitis virus

“…It is approximately 1,600 amino acid residues in length and consists of an acidic domain, an ADP-ribose 1 phosphatase, the PL2 protease (a deubiquitinating protease), Y and transmembrane domains. The acidic domain is of unknown function, however; there is some evidence that it possesses nucleic acid binding activity because it is consistently co-purified with singlestranded RNA [33]. Previous studies with other organisms indicate that electrostatic interactions from this type of domain play a key role in ligand binding [34].…”

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

“…5), but secondary structure prediction (JPred; (Drozdetskiy et al, 2015)) suggests that the Ub1 and PL1 pro domains adopt a conserved fold in all coronaviruses (data not shown). The NMR structure of the residues 1e112 of SARS-CoV nsp3 exhibits a globular ubiquitin-like fold with two additional helices which make the overall structure of the Ub1 domain somewhat more elongated than other ubiquitin-like proteins (Serrano et al, 2007b). In contrast, the following HVR was shown to be structurally disordered for SARS-CoV (Serrano et al, 2007b) and is dispensable for replication in MHV (Hurst et al, 2013).…”

“…Based on phylogenetic analysis of nidovirus nsp3 homologues, results from previously published studies (Gorbalenya et al, 2006;Ratia et al, 2006;Saikatendu et al, 2005;Serrano et al, 2007b;Thiel et al, 2003;Ziebuhr et al, 2001) and de novo domain prediction software (Jaroszewski et al, 2005), we estimate that the full repertoire of sequenced coronavirus nsp3 genes encodes 18 domains, with individual viruses having 10e16 domains each. Several of these domains are duplicated, including two ubiquitin-like domains Ub1 and Ub2, two PL pro , and three macrodomains (Mac1, Mac2 and Mac3).…”

From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses

“…Nsp1SARS-CoV (2HSX; 2GDT); TGEV (3ZBD)Almeida et al (2007) and Jansson (2013) Nsp3 UB1 SARS-CoV (2GRI) ; MHV (2M0I)Serrano et al (2007) andKeane and Giedroc (2013) Nsp3 PL1 pro TGEV (3MP2) Wojdyla et al (2010) Nsp3 X-domain SARS-CoV (2ACF; 2FAV); HCoV 229E (3EWQ; 3EJG); IBV (3EWO; 3EJF; 3EKE); FCoV (3ETI; 3EW5) Saikatendu et al (2005), Egloff et al (2006), Xu et al (2009a), Piotrowski et al (2009) and Wojdyla et al (2009) Nsp3 SUD SARS-CoV (2W2G; 2WCT; 2KQV; 2KQW; 2JZF; 2RNK) Tan et al (2009), Johnson et al (2010a) and Chatterjee et al . (2002, 2003), Yang et al (2003), Tan et al (2005), Zhao et al (2008) and Xue et al (2008) Nsp7 SARS-CoV (1YSY; 2KYS) Peti et al (2005), Johnson et al (2010b) Nsp7 + 8 complex SARS-CoV (2AHM); FCoV (3UB0) Zhai et al (2005) and Xiao et al (2012) Nsp9 SARS-CoV (1UW7; 1QZ8); HCoV 229E (2J97) Sutton et al (2004), Egloff et al (2004) and Ponnusamy et al (1XAK; 1YO4) Nelson et al (2005) and Hänel et al (2006) orf9b SARS-CoV (2CME) Meier et al (2006) Spike RBD alone and in complex with receptor SARS-CoV (2GHV; 2AJF); HCoV NL63 (3KBH); PRCV (4F5C); MHV (3R4D); MERS-CoV (4L3N; 4KR0; 4KQZ; 4L72) Hwang et al (2006), Li et al, 2005a, Wu et al (2009), Reguera et al (2012), Peng et al (2011), Chen et al (2013a), Lu et al (2013) and Wang et al (2013) Spike fusion core SARS-CoV (1WYY; 2BEQ; 2BEZ; 1ZV7; 1ZVB; 1ZV8; 1ZVA; 2FXP; 1WNC); MHV (1WDF; 1WDG); HCoV NL63 (2IEQ) Duquerroy et al (2005), Supekar et al (2004), Deng et al (2006), Hakansson-McReynolds et al (2006), Xu et al, 2004b and Zheng et al (2006) Nucleocapsid-NTD IBV (2C86; 2GEC; 2BXX); HCoV OC43 (4J3K); SARS-CoV (2OFZ; 2OG3; 1SSK); MHV (3HD4) Jayaram et al (2006), Fan et al (2005), Chen et al (2013b), Saikatendu et al (2007), Huang et al (2004) and Grossoehme et al (2009) Nucleocapsid-CTD IBV (2CA1; 2GE7; 2GE8); SARS-CoV (2CJR; 2JW8; 2GIB) Jayaram et al (2006), Chen et al (2007), Takeda et al (2008), Yu et al (2006) S2m SARS-CoV (1XJR) Robertson et al (2004) Structures elucidated by nuclear magnetic resonance (NMR) techniques are indicated by a Protein Data Bank (PDB) code in italics.…”