Association of SARS-CoV Membrane Protein with Nucleocapsid Protein

• Main Authors: Chia-Hui Chang, 張佳惠
• Other Authors: Chin -Tien Wang
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/26554433316831398482

碩士 === 國立陽明大學 === 公共衛生研究所 === 98 === Severe acute respiratory syndrome coronavirus (SARS-CoV) encodes four structural proteins i.e. membrane (M), nucleocapsid (N), envelope (E) and spike (S). Coronavirus M is necessary for virus-like particles (VLPs) formation. A number of studies have shown that co-expression of M and N can generate substantial amoutns of VLPs and that the M carboxyl-terminal region is important for M-N interaction. Our previous study, however, demonsated that the M can self-assemble and release from cells. We aim to map the domain functionally involved in M-M self-association and M-N interaction. A panel of M mutatns was constructed by site-directed mutagenesis, and each of the resutant M mutants was transiently expressed or co-expressed with N. The release efficiency of VLPs was determined by Western blot. The M-M association and M-N interaction domain was further determined by pull-down assay. The oligomerization and localization of secretion-defective M mutants were analyzed. The data showed that HA tagged at the amino-terminus of M does not significantly affect M self-assembly or M plus N VLP formation. However, substitutions or FLAG tagged at the very carboxyl-terminus of M markedly impaired M-N interaction. Blocking glycosylation of M (N4Q) has no major impacts on M self-assembly and VLP release. Alanine substitution in either W19 or W91 significantly reduced VLP production. The release efficiency of VLPs was also markedly reduced when Y94 and F95 were replaced with alanine or leucine residues. However, the secretion-defective M can release into medium when co-expressed with wild-type M. In addition, GST pull-down assays suggest that these secretion-defective M mutants can interact with N protein. The localization of glycosylation-deficient M and secretion-defective M was similar to wild-type M in that they localized in the plasma membrane and perinuclear areas. Velocity sedimentation analysis suggests that multimerization of most secretion-defective was impaired to a certain extent. These results suggest that although the substitution mutations in M have no detrimental effects on M-M or M-N interaction but they significantly affect M self-assembly and release.