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

Purnima Kumar, Vithiagaran Gunalan, Boping Liu, Vincent T.K. Chow, Julian Druce, Chris Birch, Mike Catton, Burtram C. Fielding, Yee Joo Tan, Sunil K. Lal

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32 Citations (Scopus)

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 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.

Original languageEnglish
Pages (from-to)293-303
Number of pages11
JournalVirology
Volume366
Issue number2
DOIs
Publication statusPublished - 30 Sep 2007
Externally publishedYes

Keywords

  • Accessory protein
  • Immunoprecipitation
  • Non structural protein
  • Protein-protein interaction
  • SARS coronavirus
  • Yeast two-hybrid system

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