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Intracellular behavior of rabies virus matrix protein (M) is determined by the viral glycoprotein (G)
K Nakahara1, H Ohnuma, S Sugita
1Department of Molecular Microbiology, Graduate School of Pharmaceutical Sciences, Kyoto University, Japan.
Abstract:
To investigate the nature and intracellular behavior of the matrix (M) protein of an avirulent strain (HEP-Flury) of rabies virus, we cloned and sequenced the cDNA of the protein. Using expression vectors pZIP-NeoSV(X)1 and pCDM8, the cDNA was transfected to animal cells (BHK-21 and COS-7) with or without coexpression of viral glycoprotein (G). When M protein alone was expressed in the cells, it displayed homogeneous distribution in the whole cell including the nucleus. In contrast, coexpression with G protein resulted in the abolishment of nuclear distribution of M antigen, and both of the antigens displayed a colocalized distribution in the cell, especially at the cellular membrane as seen in the virus-infected cells, while the distribution of G antigen was not affected by coexpressed M antigen. Immunoprecipitation studies revealed that M protein was coprecipitated with G protein by anti-G antibody, and vice versa, although cross-linking with dithiobis(succinimidyl propionate) was necessary for coprecipitation because of their easier dissociation in the presence of sodium deoxycholate. These results suggest that M protein intimately associates with G protein, which may affect or regulate the behavior (e.g., intracellular localization) of M protein. Studies with deletion mutants of M protein indicate that an internal region around the amino acids from 115 to 151 is essential for the M protein to preserve its binding ability to G protein.
Insights
Rabies virus matrix (M) protein associates with glycoprotein (G) protein, altering M protein
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- The matrix (M) protein of rabies virus plays a crucial role in viral assembly and pathogenesis.
- Understanding the intracellular behavior and interactions of viral proteins is essential for developing antiviral strategies.
Purpose of the Study:
- To investigate the intracellular localization and interactions of the matrix (M) protein from an avirulent rabies virus strain (HEP-Flury).
- To determine the influence of the viral glycoprotein (G) on the intracellular distribution of the M protein.
Main Methods:
- Cloning and sequencing of the M protein cDNA.
- Transfection of animal cells (BHK-21, COS-7) with M protein cDNA, with or without coexpression of G protein, using expression vectors.
- Immunofluorescence microscopy to visualize protein distribution.
- Immunoprecipitation studies to assess protein-protein interactions, including cross-linking experiments.
- Analysis of deletion mutants to identify regions involved in protein binding.
Main Results:
- Expressed M protein alone showed homogeneous distribution, including nuclear localization.
- Coexpression of M and G proteins resulted in M protein localization at the cellular membrane, with no effect on G protein distribution.
- Immunoprecipitation confirmed an association between M and G proteins, requiring cross-linking due to dissociation in detergents.
- A specific region (amino acids 115-151) of the M protein was identified as essential for binding to the G protein.
Conclusions:
- Rabies virus M protein intimately associates with the G protein.
- The G protein influences the intracellular localization of the M protein, potentially regulating its behavior.
- The identified M protein region (aa 115-151) is critical for G protein binding and likely for M protein's role in viral assembly.