title: Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.
abstract: Severe acute respiratory syndrome coronavirus (SARS-CoV) membrane (M) proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs) when coexpressed with SARS-CoV nucleocapsid (N) protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219) is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.
authors.affiliation: Department of Medical Research and Education, Taipei Veterans General Hospital, Taipei, Taiwan.
authors.forename: Ying-Tzu
authors.initials: YT
authors.lastname: Tseng
authors.forename: Chia-Hui
authors.initials: CH
authors.lastname: Chang
authors.forename: Shiu-Mei
authors.initials: SM
authors.lastname: Wang
authors.forename: Kuo-Jung
authors.initials: KJ
authors.lastname: Huang
authors.forename: Chin-Tien
authors.initials: CT
authors.lastname: Wang
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000086862:Coronavirus M Proteins; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D045473:Severe acute respiratory syndrome-related coronavirus / Q000502:physiology*; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
chemical_list: D000086862:Coronavirus M Proteins; C000706237:M protein, SARS-CoV; D014763:Viral Matrix Proteins