Nucleocytoplasmic shuttling mechanisms of the matrix (M) protein of bovine ephemeral fever virus

• Main Authors: Yi-Jyun Lin, 林怡均
• Other Authors: 劉宏仁
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/97152030262631618861

碩士 === 國立中興大學 === 分子生物學研究所 === 103 === Bovine ephemeral fever virus (BEFV) has five structure proteins, including the matrix (M) protein. In this study, we demonstrate for the first time that the M protein of BEFV is a nucleocytoplasmic shuttling protein. Western blotting assays of cell fractionations of nucleus, membrane, and cytosol proteins indicated that M protein is present at the nucleus, the nucleolus and cell membrane. To explore the mechanisms underlying BEFV M shuttling between the nucleus and the cytoplasm, apyrase, wheat germ agglutinin (WGA), N-ethylmaleimide (NEM), actinomycin D (AMD) and leptomycin B (LMB) were used to inhibit ATP, nucleoporin proteins, importin and import pathways, RNA polymerase, and CRM1, respectively. Our results reveal that M protein translocated into the nucleus is energy- and carrier-dependent and through the classical import pathway. By using bioformatic analysis of M protein, we found that M protein has putative NLS at its C-terminal. Deletion analysis of M protein further defined the NLS at its C-terminal (aa 170-223). In our reciprocal co-immunoprecipation assays, we found that M protein would interact with CRM-1 and an important protein of cell nuclear structure, Lamin A/C, suggesting that CRM-1 is a carrier protein for M protein nuclear export and that Lamin A/C also plays a critical role in mediating M protein nuclear export.