Related Experiment Videos
Characterization of the coronavirus M protein and nucleocapsid interaction in infected cells
K Narayanan1, A Maeda, J Maeda
1Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019, USA.
Abstract:
Coronavirus contains three envelope proteins, M, E and S, and a nucleocapsid, which consists of genomic RNA and N protein, within the viral envelope. We studied the macromolecular interactions involved in coronavirus assembly in cells infected with a murine coronavirus, mouse hepatitis virus (MHV). Coimmunoprecipitation analyses demonstrated an interaction between N protein and M protein in infected cells. Pulse-labeling experiments showed that newly synthesized, unglycosylated M protein interacted with N protein in a pre-Golgi compartment, which is part of the MHV budding site. Coimmunoprecipitation analyses further revealed that M protein interacted with only genomic-length MHV mRNA, mRNA 1, while N protein interacted with all MHV mRNAs. These data indicated that M protein interacted with the nucleocapsid, consisting of N protein and mRNA 1, in infected cells. The M protein-nucleocapsid interaction occurred in the absence of S and E proteins. Intracellular M protein-N protein interaction was maintained after removal of viral RNAs by RNase treatment. However, the M protein-N protein interaction did not occur in cells coexpressing M protein and N protein alone. These data indicated that while the M protein-N protein interaction, which is independent of viral RNA, occurred in the M protein-nucleocapsid complex, some MHV function(s) was necessary for the initiation of M protein-nucleocapsid interaction. The M protein-nucleocapsid interaction, which occurred near or at the MHV budding site, most probably represented the process of specific packaging of the MHV genome into MHV particles.
Insights
The M protein and N protein interact within the mouse hepatitis virus (MHV) budding site, crucial for packaging the viral genome. This interaction, independent of viral RNA, requires specific MHV functions for initiation.
Area of Science:
- Virology
- Molecular Biology
- Cellular Biology
Background:
- Coronaviruses assemble using structural proteins (M, E, S) and a nucleocapsid (genomic RNA and N protein).
- Understanding coronavirus assembly, particularly macromolecular interactions, is key to viral replication studies.
Purpose of the Study:
- To investigate the macromolecular interactions during mouse hepatitis virus (MHV) assembly.
- To elucidate the roles of M and N proteins in coronavirus particle formation and genome packaging.
Main Methods:
- Coimmunoprecipitation assays to detect protein-protein interactions.
- Pulse-labeling experiments to track newly synthesized proteins.
- RNase treatment to assess RNA independence of interactions.
Main Results:
- Demonstrated a direct interaction between M protein and N protein in MHV-infected cells.
- Identified M protein-N protein interaction occurring in a pre-Golgi compartment, a budding site.
- Showed M protein interacts with genomic RNA (mRNA 1), while N protein interacts with all MHV mRNAs, forming a nucleocapsid complex.
Conclusions:
- M protein interacts with the N protein-genomic RNA nucleocapsid, independent of S and E proteins.
- This M protein-nucleocapsid interaction requires specific MHV functions for initiation, occurring at the budding site.
- The interaction likely represents the specific packaging of the MHV genome into new viral particles.