Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome

et al (2007) Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome. PLoS ONE 2(5): e459. doi:10.1371/journal.pone.0000459 Analysis of Intraviral Protein-Protein Interactions of the SARS Coronavirus ORFeome Albrecht von Brunn - 0 1 2 Carola Teepe 0 1 2 Jeremy C. Simpson 0 1 2 Rainer Pepperkok 0 1 2 Caroline C. Friedel 0 1 2 Ralf Zimmer 0 1 2 Rhonda Roberts 0 1 2 Ralph Baric 0 1 2 Ju rgen Haas 0 1 2 0 Funding: This work was supported by the LMU Mu nchen (FoeFoLe Predoctoral Program to C.T.) and in part by a grant provided by the Bayerisches Staatsministerum f u r Wissenschaft, Forschung und Kunst. The work was also supported in part by a grant from the National Institute of Health AI059136 and AI059443 to RSB 1 Academic Editor: Dong-Yan Jin, University of Hong Kong , China 2 1 Genzentrum, Max-von-Pettenkofer-Institut , Lehrstuhl Virologie , Ludwig-Maximilians-Universita t (LMU) , M u nchen, Germany , 2 European Molecular Biology Laboratory (EMBL) Heidelberg , Heidelberg, Germany, 3 Institut fu r Informatik , Ludwig-Maximilians-Universita t (LMU) , M u nchen, Germany , 4 University of North Carolina at Chapel Hill , Chapel Hill, North Carolina , United States of America 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. - INTRODUCTION The observation of atypical pneumonias in the Chinese province Guangdong in November 2002 led to the identification of the severe acute respiratory syndrome (SARS). Within a few months, the disease spread to a large number of countries and caused more than 8,000 cases and almost 800 deaths. The causative pathogen identified was shown to be a new human coronavirus designated the SARS-CoV [1]. Tight intervention strategies limited the further spread of the pathogen. Sequence analysis of the first isolates of the newly identified SARS-CoV revealed characteristic features typical of the known three coronavirus groups [24]. According to several phylogenetic analyses the virus is grouped either a novel group IV or an early split-off of group II coronaviruses [5,6]. The genome of the SARS-CoV consists of a positive-stranded RNA of approximately 29,700 nt in length. The replicase genes span the first two-thirds of the genome containing the two overlapping ORF1a and ORF1b, which are connected by a ribosomal frameshift. The two polyproteins expressed are predicted to encode and to be cleaved by a papain-like proteinase 2 (PL-Pro = part of nsp3) and a 3C-like proteinase (3CLPro = nsp5) to 16 mature replicase proteins [1,6]. They are well conserved between SARS-CoV and other coronaviruses. It is suggested that they are required for the synthesis of the full-length genome and subgenomic RNA synthesis as well as for virus replication [6,7]. Functions of the processed proteins include a single-stranded RNA-binding protein (nsp9) an RNA-dependendent RNA polymerase (RdRp = nsp12) as well as a non-canonical RdRp (nsp8) synthesizing short primers for nsp12, a superfamily 1like helicase (HEL1 = nsp13), and a uridylate-specific endoribonuclease (NendoU = nsp15) [715]. Nsp3, nsp14, nsp16 are thought to have ADP-ribose 19-phosphatase, 39-.59 exonuclease and 29-O-ribose methyltransferase activities, respectively [6]. But many of the functions of the nsps are still unknown. At their 59terminus the subgenomic mRNAs share a common leader sequence encoded at the 59- end of the genome, which is joined to the respective gene sequences at specific transcription regulatory sequences. Their common 39- ends extend to the end of the genome. The last third of the genome encodes the S, E, M and N structural genes with the group-specific genes interspaced among them. Former are encoded by mRNAs 2, 4, 5, and 9, latter by transcripts 3, 6, 7, 8, and 9, respectively. These genes, ORF3a/b, ORF6, ORF7a/b, ORF8a/b, and ORF9b are not found in other coronaviruses and their functions with respect to replication and pathogenesis are not well understood. There is evidence that some of the accessory ORFs can be deleted individually or in combination with almost no impact on in vitro growth, RNA synthesis, or on in vivo virus replication in a murine model [16]. Also, the nsp2 replicase protein of murine hepatitis virus (MHV) and SARS-CoV is dispensable for virus replication in cell culture. Its deletion results in attenuation of viral growth and RNA synthesis [17]. There are reports that a number of MHV and SARS-CoV replicase proteins colocalize and eventually interact in cytoplasmic membrane bound complexes, in which viral RNA synthesis occurs [18,19]. Direct interactions of nsp7 and nsp8 in a hexadecameric supercomplex could be demonstrated by crystallography [20]. Interactions of the structural N and M proteins were demonstrated by a mammalian two-hybrid system [21]. For the elucidation of molecular mechanisms during the course of viral growth and propagation there is a need to systematically examine possible interactions of all viral proteins. We therefore cloned the SARS-CoV ORFeome by recombinatorial cloning (GATEWAY technology) and performed a genome-wide analysis for viral protein interactions by yeast-two-hybrid (Y2H) matrix screen. RESULTS Generation of a SARS-CoV ORFeome We have designed a set of nested PCR primers to amplify all viral non-structural, structural and accessory ORFs at the predicted prote (...truncated)