Kumar, PurnimaGunalan, VithiagaranLiu, BopingChow, Vincent T.K.Druce, JulianBirch, ChrisCatton, MikeFielding, Burtram C.Tan, Yee-JooLal, Sunil K.2014-01-062014-01-062007Kumar, P., et al. (2007). The nonstructural protein 8 (nsp8) of the SARS coronavirus interacts with its ORF6 accessory protein. Virology, 366(2): 293-3030042-6822http://hdl.handle.net/10566/914http://dx.doi.org/10.1016/j.virol.2007.04.029Severe 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 replicationenThis is the post-print version of the article published available online at http://dx.doi.org/10.1016/j.virol.2007.04.029SARS coronavirusProtein–protein interactionYeast two-hybrid systemNon structural proteinAccessory proteinImmunoprecipitationArticle