Severe acute respiratory syndrome (SARS) coronavirus infection and growth are dependent on initiating signaling and enzyme actions upon viral entry into the host cell. Proteins packaged during virus assembly may subsequently form the first line of attack and host manipulation upon infection. A complete characterization of virion components is therefore important to understanding the dynamics of early stages of infection. Mass spectrometry and kinase profiling techniques identified nearly 200 incorporated host and viral proteins. We used published interaction data to identify hubs of connectivity with potential significance for virion formation. Surprisingly, the hub with the most potential connections was not the viral M protein but the nonstructural protein 3 (nsp3), which is one of the novel virion components identified by mass spectrometry. Based on new experimental data and a bioinformatics analysis across the Coronaviridae, we propose a higher-resolution functional domain architecture for nsp3 that determines the interaction capacity of this protein. Using recombinant protein domains expressed in Escherichia coli, we identified two additional RNA-binding domains of nsp3. One of these domains is located within the previously described SARS-unique domain, and there is a nucleic acid chaperone-like domain located immediately downstream of the papain-like proteinase domain. We also identified a novel cysteine-coordinated metal ion-binding domain. Analyses of interdomain interactions and provisional functional annotation of the remaining, so-far-uncharacterized domains are presented. Overall, the ensemble of data surveyed here paint a more complete picture of nsp3 as a conserved component of the viral protein processing machinery, which is intimately associated with viral RNA in its role as a virion component.The severe acute respiratory syndrome coronavirus (SARSCoV) is an enveloped virus with a 29.7-kb positive-strand RNA genome (35). Replication of this genome and transcription are mediated by a large membrane-anchored RNA processing complex. Components of this complex are derived from the 16 nonstructural proteins (nsp1 to nsp16) that are processed from the open reading frame 1a (ORF1a) and ORF1b. The polyprotein 1a (pp1a) is translated from ORF1a, while the polyprotein 1ab (pp1ab) is formed by a Ϫ1 ribosomal frameshift upstream of the ORF1a stop codon, causing read-through into ORF1b. SARS-CoV encodes two proteinases, a "main proteinase" (nsp5) and a papain-like proteinase (PL2 pro domain of nsp3). These two proteins proteolytically cleave pp1a and pp1ab into the 16 mature nsp's (61). Specifically, SARS-CoV PL2 pro cleaves pp1a at the three sites 177 LNGG 2 AVT 183 , 815 LKGG 2 API 821 , and 2737 LKGG 2 KIV 2743 to release nsp1, nsp2, and nsp3, respectively.In current coronavirus terminology, the term "nonstructural protein" usually refers to peptides processed from pp1a and pp1ab, while "structural protein" refers to the N, M, S, and E proteins, which interact to coordinate the structure of the virion lipi...
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...
The J-UNIO (JCSG protocol using the software UNIO) procedure for automated protein structure determination by NMR in solution is introduced. In the present implementation, J-UNIO makes use of APSY-NMR spectroscopy, 3D heteronuclear-resolved [1H,1H]-NOESY experiments, and the software UNIO. Applications with proteins from the JCSG target list with sizes up to 150 residues showed that the procedure is highly robust and efficient. In all instances the correct polypeptide fold was obtained in the first round of automated data analysis and structure calculation. After interactive validation of the data obtained from the automated routine, the quality of the final structures was comparable to results from interactive structure determination. Special advantages are that the NMR data have been recorded with 6 to 10 days of instrument time per protein, that there is only a single step of chemical shift adjustments to relate the backbone signals in the APSY-NMR spectra with the corresponding backbone signals in the NOESY spectra, and that the NOE-based amino acid side chain chemical shift assignments are automatically focused on those residues that are heavily weighted in the structure calculation. The individual working steps of J-UNIO are illustrated with the structure determination of the protein YP_926445.1 from Shewanella amazonensis, and the results obtained with 17 JCSG targets are critically evaluated.
The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N-and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand -sheet holding two ␣-helices of three and four turns that are oriented antiparallel to the -strands. Two antiparallel two-strand -sheets and two 3 10 -helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.