The severe acute respiratory syndrome coronavirus (SARS-CoV) genome is predicted 
to encode 14 functional open reading frames, leading to the expression of up to 
30 structural and non-structural protein products. The functions of a large 
number of viral ORFs are poorly understood or unknown. In order to gain more 
insight into functions and modes of action and interaction of the different 
proteins, we cloned the viral ORFeome and performed a genome-wide analysis for 
intraviral protein interactions and for intracellular localization. 900 pairwise 
interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 
positive non-redundant interactions, including six self interactions, were 
identified. About 38% of interactions were subsequently confirmed by CoIP in 
mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with 
other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, 
nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major 
player within the replication complex machinery. It was shown by others that 
nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show 
that also accessory protein ORF9b does not play a pivotal role for viral 
replication, as it can be deleted from the virus displaying normal plaque sizes 
and growth characteristics in Vero cells. However, it can be expected to be 
important for the virus-host interplay and for pathogenicity, due to its large 
number of interactions, by enhancing the global stability of the SARS proteome 
network, or play some unrealized role in regulating protein-protein 
interactions. The interactions identified provide valuable material for future 
studies.