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

Abstract

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.

Figure 1. Analysis of SARS-Co viral protein interactions by Y2H matrix screen and CoIP in mammalian cells.

Y2H matrix screen was performed by mating S. cerevisiae strains AH109 and Y187 containing prey and bait vectors with the respective SARS-CoV ORFs on selective media. All ORFs were tested against each other. Positive interactions in yeast (black and grey squares) were retested by CoIP in mammalian cells (293cells) using anti- HA (preys) and anti-c-myc (baits) antibodies. Interactions tested positive in 293 cells by CoIP are encircled in red.

Figure 2. CoIPs of non-structural proteins nsp2 and nsp8.

293 cells were infected with vaccinia virus vTF-7 and subsequently co-transfected with HA- and c-myc- tagged plasmids carrying the respective SARS-CoV ORFs. After 20 hours half of the cell lysate was immunoprecipitated with anti-HA, the other half with anti- c-myc antibody. Bound proteins were subjected twice to 12,5% SDS-PAGE and Western Blot transfer, and probed cross-wise with the two antibodies. Co-precipitated proteins are indicated in the right panels. HA and c-myc tags are expressed as N-terminal fusions with the corresponding SARS-CoV ORF in plasmids pGADT7 and pGBKT7, respectively. Stars indicate expression products by IP, arrows by CoIP.

Figure 3. Deletion of ORF9b from SARS-CoV.

DNA sequences of wild-type and of modified ATG start codons of ORF9b are given on the left. The primary sequence of the N protein was maintained. Viral growth curves are shown on the right.

Figure 4. SARS-CoV-host interaction network.

Viral interactions are based on the experimental Y2H findings. Human interactions were taken from the combined human interaction network described in Table 2. Interactions between SARS and human proteins were gathered from the literature and are listed in Supplemental Table 1. The figure shows the SARS interaction network (A) and proteins and interactions which are separated from the SARS network by no more than 2 (B) and 3 (C) interactions, respectively. SARS-CoV proteins are depicted in dark red, their direct targets (distance 1) in light red, neighbours of the direct targets (distance 2) in orange and neighbours of the latter (distance 3) in yellow.

Figure 5. Subcellular localization analysis of SARS-CoV ORFs.

Expression plasmids containing N- and C- terminally FLAG -tagged ORFs were transfected into Hela cells and analysed after 24 hours with an anti- FLAG antibody for expression and localization of their products.