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 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.
authors.affiliation: Virology Group, International Centre for Genetic Engineering and Biotechnology, P. O. Box: 10504, Aruna Asaf Ali Road, New Delhi 110067, India.
authors.forename: Purnima
authors.initials: P
authors.lastname: Kumar
authors.forename: Vithiagaran
authors.initials: V
authors.lastname: Gunalan
authors.forename: Boping
authors.initials: B
authors.lastname: Liu
authors.forename: Vincent T K
authors.initials: VT
authors.lastname: Chow
authors.forename: Julian
authors.initials: J
authors.lastname: Druce
authors.forename: Chris
authors.initials: C
authors.lastname: Birch
authors.forename: Mike
authors.initials: M
authors.lastname: Catton
authors.forename: Burtram C
authors.initials: BC
authors.lastname: Fielding
authors.forename: Yee-Joo
authors.initials: YJ
authors.lastname: Tan
authors.forename: Sunil K
authors.initials: SK
authors.lastname: Lal
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000737:chemistry; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D045473:Severe acute respiratory syndrome-related coronavirus / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D017361:Viral Nonstructural Proteins; C087633:nonstructural protein, coronavirus