The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains several accessory open reading frames (ORFs) without known functions. One of them, ORF 3a, encodes a predicted protein 3a consisting of 274 amino acids and contains three putative transmembrane domains. In this study we systematically investigate 3a protein's interactions with other viral proteins and a human lymph node cDNA library, using the Matchmaker Gal4 Two-Hybrid System. It was found that no interactions exist between the 3a protein domain and the tested SARS-CoV proteins (3a, 3b, 9b, and nucleocapsid). On the other hand, an unknown protein named 'DKFZP434H132' from the lymph node was detected to interact specifically with the intracellular domain of the 3a protein. This protein-protein interaction was v...[ Read more ]

The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains several accessory open reading frames (ORFs) without known functions. One of them, ORF 3a, encodes a predicted protein 3a consisting of 274 amino acids and contains three putative transmembrane domains. In this study we systematically investigate 3a protein's interactions with other viral proteins and a human lymph node cDNA library, using the Matchmaker Gal4 Two-Hybrid System. It was found that no interactions exist between the 3a protein domain and the tested SARS-CoV proteins (3a, 3b, 9b, and nucleocapsid). On the other hand, an unknown protein named 'DKFZP434H132' from the lymph node was detected to interact specifically with the intracellular domain of the 3a protein. This protein-protein interaction was verified by re-transformation and stringent selection in the yeast two-hybrid system. Co-IP and in vitro binding studies were also performed to further confirm the interaction between 3a and DKFZP434H132 proteins. The results showed that the two proteins could interact in vitro with a dissociation constant in micromolar range. More experiments are required to confirm the interaction in mammalian cells and to identify the function of DKFZP434H132 protein.