Characterization of protein-protein interactions between the nucleocapsid protein and membrane protein of the SARS coronavirus

Abstract

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.

Fig. 1

Mammalian two-hybrid assay on SARS-CoV nucleocapsid protein and membrane protein. Constructs labeled in the figure were co-transfected into Huh7 cells. Forty-eight hour post-transfection, cells were harvested and subjected to the SEAP assay. Chemiluminescence values were used to measure the interaction activities.

Fig. 2

Mutation analysis of the SARS-CoV nucleocapsid (N) protein. Schematic description of the DpM-N related mutants (a), and SEAP chemiluminescence values of the mammalian two hybrid assay on protein–protein interaction between SARS-CoV membrane (M) protein and the N protein truncation mutants (b).

Fig. 3

Sequential deletion analysis of the SARS-CoV nucleocapsid (N) protein. Schematic description of the S-ΔpM-N related mutants (a), and SEAP chemiluminescence values of the mammalian two-hybrid assay on protein–protein interaction between SARS-CoV membrane (M) protein and the N protein sequential deletion mutants (b). The mutants were constructed using pM vector as labeled in (b). Each mutant was then co-transfected with the full-length N gene in pVP16 vector (pVP-N). The pM vector co-transfected with pVP-N was used as the negative control.