Novel β-Barrel Fold in the Nuclear Magnetic Resonance Structure of the Replicase Nonstructural Protein 1 from the Severe Acute Respiratory Syndrome Coronavirus

Abstract

The nonstructural protein 1 (nsp1) of the severe acute respiratory syndrome coronavirus has 179 residues and is the N-terminal cleavage product of the viral replicase polyprotein that mediates RNA replication and processing. The specific function of nsp1 is not known. Here we report the nuclear magnetic resonance structure of the nsp1 segment from residue 13 to 128, which represents a novel ␣/␤-fold formed by a mixed parallel/antiparallel six-stranded ␤-barrel, an ␣-helix covering one opening of the barrel, and a 3 10 -helix alongside the barrel. We further characterized the full-length 179-residue protein and show that the polypeptide segments of residues 1 to 12 and 129 to 179 are flexibly disordered. The structure is analyzed in a search for possible correlations with the recently reported activity of nsp1 in the degradation of mRNA.After the major outbreak of severe acute respiratory syndrome (SARS) in the beginning of 2003, the SARS coronavirus (SARS-CoV) became a major topic of coronavirus research. The coronavirus genome is composed of a single plus-strand RNA of about 30 kb, which is the largest nonsegmented genome among known RNA viruses. About two-thirds of the coronavirus genome is devoted to encoding the replicase that mediates viral RNA synthesis (64). The replicase gene comprises two large open reading frames (ORFs) located at the 5Ј end of the genome. The first one, ORF1a, encodes a polyprotein of 450 to 500 kDa (polyprotein 1a), and the second one, ORF1b, is translated together with ORF1a after a Ϫ1 ribosomal frameshift, leading to the expression of the "polyprotein lab," which has a size of 750 to 800 kDa. The replicase polyprotein is processed by ORF1a-encoded viral proteinases, which leads to about 16 nonstructural proteins (nsp), which are numbered consecutively from the N terminus to the C terminus of the polyprotein (14,53).The large number of mature proteins produced from the polyprotein indicates a high level of complexity of the viral replication process. Some of the enzymatic activities that were detected or predicted in SARS-CoV to date include the main protease (nsp5), a papain-like proteinase (nsp3d; PLpro), an RNA-dependent RNA polymerase (nsp12), an RNA helicase (nsp13), an endoribonuclease (nsp15), an ADP-ribose-1Љ-phosphatase (nsp3b), a deubiquitinase (nsp3d), a 3Ј35Ј exoribonuclease (nsp14), and a ribose-2Ј-O-methyltransferase (nsp16) (3,5,18,21,22,44,59,70). For the two proteases, the endoribonuclease, and the ADP-ribose-1Љ-phosphatase, three-dimensional structures have been solved, which together with biochemical data have revealed some aspects of the enzyme mechanisms (27,54,56,58,63,67; reviewed in references 4, 35, and 65). The physiological functions of several of the other nonstructural proteins remain to be determined, but high-resolution structure determinations have allowed the identification of possible functional sites and provided the basis for further biochemical studies (15,30,52,61,62,68). Other replicase proteins still remain to be characterized.Nsp1 is the N-te...

Nsp1 proteins of group I and SARS coronaviruses share structural and functional similarities

“…Structures for the nsp1 proteins of HCoV-229E and HCoV-NL63 were computed with Modeller software (Marti-Renom et al, 2000) using the solution structure of NSP1 13-128 from the SARS-CoV (PDB code 2HSX) as a template structure (Almeida et al, 2007). Sequence alignment of the nsp1 proteins from HCoV-229E, HCoV-NL63 and SARS-CoV was performed with the program ClustalW and refined manually.…”

“…The nsp1 proteins of SARS-CoV and MHV promote host mRNA degradation and suppress host gene expression (Kamitani et al, 2006;Zust et al, 2007;Narayanan et al, 2008). Nuclear magnetic resonance (NMR) analysis revealed that SARS-CoV nsp1 has a novel b-barrel structure mixed with ahelixes (Almeida et al, 2007). More recently, SARS-CoV nsp1 was found to block host translational machinery function by binding to the ribosome small subunit (Kamitani et al, 2009).…”

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.…”

Atlas of coronavirus replicase structure

“…Nsp1 sequences are divergent between subgroups of betacoronavirus, and no sequence similarity between SARS-CoV nsp1 and betacoronavirus subgroup A nsp1 proteins could be identified using standard searching tools such as PSI-BLAST. Almeida et al (2006Almeida et al ( , 2007 determined the NMR structure of the nsp1 segment from residue 13 to 128 and also showed that the polypeptide segments of residues 1-12 and 129-179 are flexibly disordered (PDB ID 2GDT; 2HSX) . Residues 13-128 of nsp1 represents a novel ␣/␤-fold formed by a mixed parallel/antiparallel 6-stranded ␤-barrel, an ␣-helix covering one opening of the barrel, and a 3 10 -helix alongside the barrel (Fig.…”