Expression and membrane integration of SARS-CoV M protein.
SARS-CoV M gene fragment was cloned and expressed as a recombinant protein fused with a V5 tag at the C-terminus in Vero E6 cells. In addition to un-glycosylated and glycosylated proteins, one product with smaller size initiated in-frame from the third Met residues probably through ribosomal re-initiation was also detected. Translation initiated in-frame from the third Met is unusual since the sequence around the first Met of SARS-CoV M protein contains the optimal consensus Kozak sequence. The function of this smaller translated product awaits further investigation. Similar to other N-glycosylated proteins, glycosylation of SARS-CoV M protein was occurred co-translationally in the presence of microsomes. The SARS-CoV M protein is predicted as a triple-spanning membrane protein lack of a conventional signal peptide. The second and third trans-membrane regions (a.a. 46-68 and 78-100) are predicted to be the primary type helices, which will be able to penetrate into membrane by themselves, while the first trans-membrane region (a.a. 14-36) is predicted to be the secondary type helix, which is considered to be stabilized by the interaction with other trans-membrane segments. As expected, the second and third trans-membrane regions were able to insert a cytoplasmic protein into the endoplasmic reticulum membrane more efficiently than the first one. These results should be important for the study of SARS-CoV morphogenesis.
Ma
Hsin-Chieh
HC
Graduate Institute of Medical Sciences, Tzu Chi University, 701, Section 3, Chung Yang Road, Hualien, Taiwan.
Fang
Chiu-Ping
CP
Hsieh
Yi-Ching
YC
Chen
Shih-Chi
SC
Li
Hui-Chun
HC
Lo
Shih-Yen
SY
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002462:Cell Membrane / Q000821:virology; D002522:Chlorocebus aethiops; D000086862:Coronavirus M Proteins; D017931:DNA Primers; D006031:Glycosylation; D008561:Membrane Fusion; D014176:Protein Biosynthesis; D013347:Subcellular Fractions / Q000378:metabolism; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*
D000086862:Coronavirus M Proteins; D017931:DNA Primers; D014763:Viral Matrix Proteins