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Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.

ABSTRACT: The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The mature SARS-CoV protein is present in cells several hours post-infection and co-localizes to the viral replication complex, but its function in the viral life cycle remains unknown. Furthermore, nsp1 sequences are highly divergent across the CoV family, and it has been suggested that this is due to nsp1 possessing a function specific to viral interactions with its host cell or acting as a host specific virulence factor. In order to initiate structural and biophysical studies of SARS-CoV nsp1, a recombinant expression system and a purification protocol have been developed, yielding milligram quantities of highly purified SARS-CoV nsp1. The purified protein was characterized using circular dichroism, size exclusion chromatography, and multi-angle light scattering.

SUBMITTER: Brucz K

PROVIDER: S-EPMC1862784 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.

Brucz Kimberly K Miknis Zachary J ZJ Schultz L Wayne LW Umland Timothy C TC

Protein expression and purification 20061114 2

The coronavirus (CoV) responsible for severe acute respiratory syndrome (SARS), SARS-CoV, encodes two large polyproteins (pp1a and pp1ab) that are processed by two viral proteases to yield mature non-structural proteins (nsps). Many of these nsps have essential roles in viral replication, but several have no assigned function and possess amino acid sequences that are unique to the CoV family. One such protein is SARS-CoV nsp1, which is processed from the N-terminus of both pp1a and pp1ab. The ma ...[more]

PMID: 17187987

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Project description:The ongoing pandemic of coronavirus disease 2019 (COVID-19), which results from the rapid spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a significant global public health threat, with molecular mechanisms underlying its pathogenesis largely unknown. Small non-coding RNAs (sncRNAs) are known to play important roles in almost all biological processes. In the context of viral infections, sncRNAs have been shown to regulate the host responses, viral replication, and host-virus interaction. Compared with other subfamilies of sncRNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs), tRNA-derived RNA fragments (tRFs) are relatively new and emerge as a significant regulator of host-virus interactions. Using T4 PNK‐RNA‐seq, a modified next‐generation sequencing (NGS), we recently found that nasopharyngeal swabs (NPS) samples from SARS-CoV-2 positive and negative subjects show a significant difference in sncRNA profiles. There are about 166 SARS-CoV- 2-impacted sncRNAs. Among them, tRFs are the most significantly affected and almost all impacted tRFs are derived from the 5’-end of tRNAs (tRF5). Using a modified qRT-PCR, which was recently developed to specifically quantify tRF5s by isolating the tRF signals from its corresponding parent tRNA signals, we validated that tRF5s derived from tRNA GluCTC (tRF5-GluCTC), LysCTT (tRF5-LysCTT), ValCAC (tRF5-ValCAC), CysGCA (tRF5-CysGCA) and GlnCTG (tRF5-GlnCTG) are enhanced in NPS samples of SARS-CoV2 patients and SARS-CoV2-infected airway epithelial cells. In addition to host-derived ncRNAs, we also identified several sncRNAs derived from the virus (svRNAs), among which a svRNA derived from CoV2 genomic site 346 to 382 (sv-CoV2-346) has the highest expression. The induction of both tRFs and sv-CoV2-346 has not been reported previously, as the lack of the 3’-OH ends of these sncRNAs prevents them to be detected by routine NGS. In summary, our studies demonstrated the involvement of tRFs in COVID-19 and revealed new CoV2 svRNAs.

Project description:The spike (S) glycoprotein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates the receptor interaction and immune recognition and is considered a major target for vaccine design. However, its antigenic and immunogenic properties remain to be elucidated. In this study, we immunized mice with full-length S protein (FL-S) or its extracellular domain (EC-S) expressed by recombinant baculoviruses in insect cells. We found that the immunized mice developed high titers of anti-S antibodies with potent neutralizing activities against SARS pseudoviruses constructed with the S proteins of Tor2, GD03T13, and SZ3, the representative strains of 2002 to 2003 and 2003 to 2004 human SARS-CoV and palm civet SARS-CoV, respectively. These data suggest that the recombinant baculovirus-expressed S protein vaccines possess excellent immunogenicity, thereby inducing highly potent neutralizing responses against human and animal SARS-CoV variants. The antigenic structure of the S protein was characterized by a panel of 38 monoclonal antibodies (MAbs) isolated from the immunized mice. The epitopes of most anti-S MAbs (32 of 38) were localized within the S1 domain, and those of the remaining 6 MAbs were mapped to the S2 domain. Among the anti-S1 MAbs, 17 MAbs targeted the N-terminal region (amino acids [aa] 12 to 327), 9 MAbs recognized the receptor-binding domain (RBD; aa 318 to 510), and 6 MAbs reacted with the C-terminal region of S1 domain that contains the major immunodominant site (aa 528 to 635). Strikingly, all of the RBD-specific MAbs had potent neutralizing activity, 6 of which efficiently blocked the receptor binding, confirming that the RBD contains the main neutralizing epitopes and that blockage of the receptor association is the major mechanism of SARS-CoV neutralization. Five MAbs specific for the S1 N-terminal region exhibited moderate neutralizing activity, but none of the MAbs reacting with the S2 domain and the major immunodominant site in S1 showed neutralizing activity. All of the neutralizing MAbs recognize conformational epitopes. These data provide important information for understanding the antigenicity and immunogenicity of S protein and for designing SARS vaccines. This panel of anti-S MAbs can be used as tools for studying the structure and function of the SARS-CoV S protein.

Dataset Information

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.

Publications

Expression, purification and characterization of recombinant severe acute respiratory syndrome coronavirus non-structural protein 1.

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