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

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