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Title: The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein

Abstract

Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is themore » first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.« less

Authors:
 [1];  [2]; ;  [3]; ; ;  [4]; ;  [2];  [5]
  1. Virology Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box: 10504, Aruna Asaf Ali Road, New Delhi 110067 (India)
  2. Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore)
  3. Microbiology Department, National University of Singapore, Kent Ridge, Singapore 117597 (Singapore)
  4. Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria (Australia)
  5. Virology Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box: 10504, Aruna Asaf Ali Road, New Delhi 110067 (India), E-mail: sunillal@icgeb.res.in
Publication Date:
OSTI Identifier:
21077969
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 366; Journal Issue: 2; Other Information: DOI: 10.1016/j.virol.2007.04.029; PII: S0042-6822(07)00314-5; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ANIMAL GROWTH; GENES; IN VITRO; MEMBRANES; PROTEINS; TRANSCRIPTION; VIRUSES; YEASTS

Citation Formats

Kumar, Purnima, Gunalan, Vithiagaran, Liu Boping, Chow, Vincent T.K., Druce, Julian, Birch, Chris, Catton, Mike, Fielding, Burtram C., Tan, Yee-Joo, and Lal, Sunil K.. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. United States: N. p., 2007. Web. doi:10.1016/j.virol.2007.04.029.
Kumar, Purnima, Gunalan, Vithiagaran, Liu Boping, Chow, Vincent T.K., Druce, Julian, Birch, Chris, Catton, Mike, Fielding, Burtram C., Tan, Yee-Joo, & Lal, Sunil K.. The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. United States. doi:10.1016/j.virol.2007.04.029.
Kumar, Purnima, Gunalan, Vithiagaran, Liu Boping, Chow, Vincent T.K., Druce, Julian, Birch, Chris, Catton, Mike, Fielding, Burtram C., Tan, Yee-Joo, and Lal, Sunil K.. Sun . "The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein". United States. doi:10.1016/j.virol.2007.04.029.
@article{osti_21077969,
title = {The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein},
author = {Kumar, Purnima and Gunalan, Vithiagaran and Liu Boping and Chow, Vincent T.K. and Druce, Julian and Birch, Chris and Catton, Mike and Fielding, Burtram C. and Tan, Yee-Joo and Lal, Sunil K.},
abstractNote = {Severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) caused a severe outbreak in several regions of the world in 2003. The SARS-CoV genome is predicted to contain 14 functional open reading frames (ORFs). The first ORF (1a and 1b) encodes a large polyprotein that is cleaved into nonstructural proteins (nsp). The other ORFs encode for four structural proteins (spike, membrane, nucleocapsid and envelope) as well as eight SARS-CoV-specific accessory proteins (3a, 3b, 6, 7a, 7b, 8a, 8b and 9b). In this report we have cloned the predicted nsp8 gene and the ORF6 gene of the SARS-CoV and studied their abilities to interact with each other. We expressed the two proteins as fusion proteins in the yeast two-hybrid system to demonstrate protein-protein interactions and tested the same using a yeast genetic cross. Further the strength of the interaction was measured by challenging growth of the positive interaction clones on increasing gradients of 2-amino trizole. The interaction was then verified by expressing both proteins separately in-vitro in a coupled-transcription translation system and by coimmunoprecipitation in mammalian cells. Finally, colocalization experiments were performed in SARS-CoV infected Vero E6 mammalian cells to confirm the nsp8-ORF6 interaction. To the best of our knowledge, this is the first report of the interaction between a SARS-CoV accessory protein and nsp8 and our findings suggest that ORF6 protein may play a role in virus replication.},
doi = {10.1016/j.virol.2007.04.029},
journal = {Virology},
number = 2,
volume = 366,
place = {United States},
year = {Sun Sep 30 00:00:00 EDT 2007},
month = {Sun Sep 30 00:00:00 EDT 2007}
}
  • No abstract prepared.
  • No abstract prepared.
  • ABSTRACT Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impactmore » on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting. IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that effectively targets the highly conserved 2'O-MTase activity of CoVs for attenuation. With clear understanding of the IFN/IFIT (IFN-induced proteins with tetratricopeptide repeats)-based mechanism, NSP16 mutants provide a suitable target for a live attenuated vaccine platform, as well as therapeutic development for both current and future emergent CoV strains. Importantly, other approaches targeting other conserved pan-CoV functions have not yet proven effective against MERS-CoV, illustrating the broad applicability of targeting viral 2'O-MTase function across CoVs.« less
  • The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbsmore » as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.« less
  • The spike (S) protein of SARS coronavirus (SARS-CoV) is responsible for viral binding with ACE2 molecules. Its receptor-binding motif (S-RBM) is located between residues 424 and 494, which folds into 2 anti-parallel {beta}-sheets, {beta}5 and {beta}6. We have previously demonstrated that fragment 450-650 of the S protein (S450-650) is predominantly recognized by convalescent sera of SARS patients. The N-terminal 60 residues (450-510) of the S450-650 fragment covers the entire {beta}6 strand of S-RBM. In the present study, we demonstrate that patient sera predominantly recognized 2 linear epitopes outside the {beta}6 fragment, while the mouse antisera, induced by immunization of BALB/cmore » mice with recombinant S450-650, mainly recognized the {beta}6 strand-containing region. Unlike patient sera, however, the mouse antisera were unable to inhibit the infectivity of S protein-expressing (SARS-CoV-S) pseudovirus. Fusion protein between green fluorescence protein (GFP) and S450-650 (S450-650-GFP) was able to stain Vero E6 cells and deletion of the {beta}6 fragment rendered the fusion product (S511-650-GFP) unable to do so. Similarly, recombinant S450-650, but not S511-650, was able to block the infection of Vero E6 cells by the SARS-CoV-S pseudovirus. Co-precipitation experiments confirmed that S450-650 was able to specifically bind with ACE2 molecules in lysate of Vero E6 cells. However, the ability of S450-510, either alone or in fusion with GFP, to bind with ACE2 was significantly poorer compared with S450-650. Our data suggest a possibility that, although the {beta}6 strand alone is able to bind with ACE2 with relatively high affinity, residues outside the S-RBM could also assist the receptor binding of SARS-CoV-S protein.« less