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.
Highlights
The highly contagious severe acute respiratory syndrome (SARS) affected individuals in 30 countries in 2002 and 2003 [1]
An HA tagged at the M amino-terminus (HA-M) had no major effect on M release and N packaging; in contrast, a FLAG tagged at the M carboxyl terminus (M-FLAG) significantly affected N incorporation (Fig. 2B, lane 11)
This observation does not alter our conclusion that N release or virus-like particles (VLPs) production depends on the presence of secretion-competent M proteins
Summary
The highly contagious severe acute respiratory syndrome (SARS) affected individuals in 30 countries in 2002 and 2003 [1]. SARS-CoV encodes four structural proteins: spike (S), membrane (M), envelope (E) and nucleocapsid (N) [4,9]. Translated on free polysomes, N is associated with newly synthesized viral genomic RNA to form helical nucleocapsids [19]. The M membrane glycoprotein is cotranslationally inserted into the endoplasmic reticulum (ER) and transported to Golgi complexes [20,21]. M interacts with nucleocapsids on the cell membranes of ER or Golgi complexes [22,23,24,25,26]. S and E proteins are translated on membrane-bound polysomes, inserted into the ER, and transported to Golgi complexes, where E and M interact and trigger virion budding with enclosed nucleocapsids [14,19]. Smooth-walled vesicles that are exocytotically released from cells [4]
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