Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome

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.