Mutational effects on M protein release and VLP assembly. (A) Results from analysis of SARS-CoV M cysteine residues in VLP assembly.

<p>(A) 293T cells were cotransfected with SARS-CoV N and indicated wt or mutant M expression vector. C63/85S, C63/158S and C63/85/158S designate combined double or triple alanine substitutions in cysteine residues 63, 85 and 158. At 24 to 36 h post-transfection, supernatants and cells were collected and prepared for protein analysis. Medium pellet samples corresponding to 50% of total and cell lysate samples corresponding to 5% of total were fractionated by 10% SDS-PAGE and electroblotted onto nitrocellulose filters. SARS-CoV M was probed with rabbit antiserum, and N was detected with a mouse anti-N monoclonal antibody. M proteins in medium or cell samples were quantified by scanning mutant and wt M band densities from immunoblots. Ratios of M levels in medium to those in cells were determined for each mutant and normalized to wt medium/cell ratios in parallel experiments. Error bars indicate standard deviations. *, <i>p<</i>0.05; **, <i>p<</i>0.01. (B) A FLAG tagged at the M carboxyl terminus prevents VLP assembly. 293T cells were transfect with SARS-CoV N alone or together with the indicated wt or mutant M expression vector. At 24 to 36 h post-transfection, supernatants and cells were collected and prepared for protein analysis as described above. (C–D) Results from cross-linking analyses of SARS-CoV M and HIV-1 Gag proteins. Extracellular particles isolated from 293T culture supernatants expressing SARS-CoV M, M plus N, or HIV-1 Gag were mock-treated or treated with the cysteine-specific cross-linking chemical BMH (panel C, lane 4 and panel D, lanes 5 and 7) as described in Materials and Methods. Samples were subjected to Western immunoblotting following 1 h incubation at room temperature. Arrowhead indicates Pr55gag dimer position.