Genome-Wide Analysis of Protein-Protein Interactions and Involvement of Viral Proteins in SARS-CoV Replication

Abstract

Analyses of viral protein-protein interactions are an important step to understand viral protein functions and their underlying molecular mechanisms. In this study, we adopted a mammalian two-hybrid system to screen the genome-wide intraviral protein-protein interactions of SARS coronavirus (SARS-CoV) and therefrom revealed a number of novel interactions which could be partly confirmed by in vitro biochemical assays. Three pairs of the interactions identified were detected in both directions: non-structural protein (nsp) 10 and nsp14, nsp10 and nsp16, and nsp7 and nsp8. The interactions between the multifunctional nsp10 and nsp14 or nsp16, which are the unique proteins found in the members of Nidovirales with large RNA genomes including coronaviruses and toroviruses, may have important implication for the mechanisms of replication/transcription complex assembly and functions of these viruses. Using a SARS-CoV replicon expressing a luciferase reporter under the control of a transcription regulating sequence, it has been shown that several viral proteins (N, X and SUD domains of nsp3, and nsp12) provided in trans stimulated the replicon reporter activity, indicating that these proteins may regulate coronavirus replication and transcription. Collectively, our findings provide a basis and platform for further characterization of the functions and mechanisms of coronavirus proteins.

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…Apparently, in the context of a viral infection, when other viral proteins are present, MHV nsp2 and HCV NS5A are immobilized at the replicative structures presumably due to protein-RNA or protein-protein interactions. In agreement herewith, large-scale protein-protein interaction studies demonstrated that nsp2 of MHV and SARS-CoV is engaged in a multitude of interactions with itself, nsp3, nsp4, nsp6, nsp7, nsp8, nsp11, nsp15 and nsp16 [82,114,115,116]. These observations are remarkable in view of the dispensability of nsp2 during CoV infection in vitro [59].…”

“…CoV replicative structures/RTCs are macromolecular assemblies, the components of which are engaged in a plethora of protein-protein, protein-RNA, and protein-lipid interactions [114,115,116,131]. Currently, the exact composition of the replicative structures/RTCs is not known, let alone the full arsenal of interactions occurring within these structures.…”

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

“…Impact of nsp10 IDAs on the Interaction with nsp16 in Mammalian Cells-Interaction of nsp10 with nsp16 has already been detected in mammalian cells using the two-hybrid system and confirmed using pull-down assays (26). However, mammalian two-hybrid systems detect interaction within the cell nucleus, whereas these viral proteins are localized in the cytoplasm during infection.…”

“…with nsp16-We used RY2H with nsp10 and nsp16 to isolate interaction-defective alleles (IDAs) and thereby delineate their surface of interaction (25,26). IDAs are alleles that contain mutations affecting their ability to interact with their wild type binding partners, leading to the identification of specific amino acid residues involved in the interaction between nsp10 and nsp16 (39).…”

SARS-CoV nucleocapsid protein antagonizes IFN-β response by targeting initial step of IFN-β induction pathway, and its C-terminal region is critical for the antagonism

“…pCMV-tag2b-N was constructed as described [15]. pCMV-Myc-N was constructed by substituting the Flag-tag coding sequence of pCMV-tag2b-N with Myc-tag coding sequence.…”