Effects of M mutations on SARS-CoV VLP assembly.

<p>Effects of M mutations on SARS-CoV VLP assembly.</p

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.</p

Schematic representations of SARS-CoV M mutations.

<p>Wild-type (wt) SARS-CoV M protein is shown with three predicted transmembrane domains (shaded boxes). Amino acid substitutions at M codon positions are indicated. An HA or FLAG epitope tagged at the amino or carboxyl terminus is designated as HA-M and M-FLAG, respectively. W57A/L and F95A/L indicate an Ala or Leu substitution at W57 and F95. Alanine substitutions of the di-leucine motif at codons 15–16 and 218–219 are designated as 15LL/AA and 218LL/AA, respectively. Underlined mutations denote that changing the aromatic residue to Ala or Leu markedly affected M secretion, but replacement with another aromatic residue did not. The ability for each construct to release or produce VLPs with coexpressed N is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064013#pone-0064013-t001" target="_blank">Table 1</a>.</p

Co-precipitation of M mutants with SARS-CoV N.

<p>(A–C) GST pull-down assay. 293T cells were cotransfected with GST-N (SARS-CoV N fused to the GST carboxyl terminus) and N, wt or indicated M mutant plasmid. Aliquots of cell lysates preceding and flowing GST pull-down were subjected to Western immunoblotting using anti-GST, anti-M and anti-N antibodies as probes. (D) Co-immunoprecipitation assay. 293T cells were cotransfected with SARS-CoV N with the indicated wt or mutant M plasmid. Aliquots of cell lysates were subjected to co-immunoprecipitation with an anti-N monoclonal antibody.</p

Effects of substitution mutations on M-M interaction.

<p>(A) Associations between mutant M proteins and FLAG-tagged or HA-tagged wt M. 293T cells were transfected with the indicated plasmid alone (upper panels) or with a FLAG-tagged (M-FLAG) or HA-tagged (HA-M) SARS-CoV M expression vector. At 24–36 h post-transfection, cells and medium were harvested and subjected to Western immunoblot analyses as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064013#pone-0064013-g002" target="_blank">Figure 2</a> caption. Arrowheads and asterisks denote M and FLAG-tagged M positions, respectively. (B) Co-immunoprecipitation of M with FLAG-tagged M. 293T cells were transfected with the SCoV M wt or indicated mutant plasmid alone or with its FLAG-tagged counterpart (lanes 2, 4, 6, 8 and 10). Cell lysates were subjected to Western immunoblotting 48 h post-transfection. Equal amounts of cell lysates were mixed with anti-FLAG affinity gel and incubated for 2 h at 4°C. Bead-bound complexes were pelleted, washed, and subjected to Western immunoblotting.</p

Effects of substitution mutations on SARS-CoV M secretion or VLP assembly.

<p>293T cells were transfected with the indicated SARS-CoV M construct plus SARS-CoV N expression vector. At 24 to 36 h post-transfection, cells and supernatants were collected, prepared, and subjected to Western immunoblot analyses. M proteins were quantified and mutant M medium/cell ratios were normalized to those of wt M in parallel experiments as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064013#pone-0064013-g002" target="_blank">Figure 2</a> caption. Blots are representative of three independent experiments. Error bars indicate standard deviations. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

SARS-CoV VLP analysis.

<p>293T cells were cotransfected with SARS-CoV N and wt or indicated M mutant plasmid. At 48 h post-transfection, cells and supernatants were collected. Cells (panels A to I) were fixed and prepared for electron microscopy analysis as described in Material and Methods. Supernatants (panels J to L) were filtered and pelleted through 20% sucrose cushions, resuspended in PBS buffer and stained. Cell and supernatant samples were observed with a transmission electron microscope. The high-power view in the inset shows VLPs near cell nuclei (arrowheads). Bars, 100 nm.</p

Genetic map and results of QTL analyses for resistance to tomato late blight.

<p>The naming of the RAD markers was in the format “00g00000000”. The first two digits indicate chromosome number, while the last eight digits indicate the physical position of the <i>PstI</i> cutting site on the tomato genome reference sequence build SL2.40. The naming of the VeraCode SNP markers was designated in the same way as the RAD markers, except for replacing “g” with “n”, and the last eight digits indicating the physical position of the SNP site. VeraCode SNP markers names are highlighted green. The red bar indicates the location of the QTL resistant to isolate Pi733, and the hollow bar indicates the location of the QTL resistant to isolate Pi39A.</p

Relationship between read number and missing data.

<p>Circles indicate each line of the F2 mapping population. The 96 filled circles represent samples used for genetic map construction, while the 24 open circles represent samples removed because of a high number of missing data in the population.</p

Disease severity rating (DSR) for isolates Pi39A and Pi733.

<p>(A) Distribution of DSR in the mapping population. (B) Relationship of the two DSR score sets generated from independent inoculations of Pi39A and Pi733.</p