The PRWWz assembly defect is HIV-1 protease activity-dependent.

<p>(A) 293T cells were transfected with wt or mutant plasmids. At 4 h post-transfection, cells were replated on four dish plates and either left untreated (lanes 1 and 5) or treated with the HIV-1 protease inhibitor (PI) Saquinavier at concentrations of 0.01 (lanes 2 and 6), 0.1 (lanes 3 and 7), or 1.0 µM (lanes 4 and 8). At 48–72 h post-transfection, cells and culture supernatant were collected, prepared, and subjected to Western immunoblot analysis. (B–G) Transmission electron microscopy images of concentrated culture supernatant from 293T cells expressing the wt or PRWWz. Culture supernatants from PI-treated or untreated transfectants were collected, filtered, and pelleted through 20% sucrose cushions. Virus-containing pellets resuspended in PBS buffer were subjected to Western immunoblotting (panel B) and transmission electron microscopy. The high-power view (×60,000 magnification) in the inset shows cone-shaped cores (arrowheads) that are characteristic of mature wt virus particles (Panel C). Bars, 200 nm.</p

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

Effects of leucine zipper domain insertion on Gag-PR multimerization and assembly.

<p>(A) Schematic representations of HIV-1 Gag and Gag-LZ chimeras. HIV-1 Gag domains, <i>pol</i>-encoded PR, and the transframe peptide p6<i>^pol</i> are indicated; <i>fs</i> denotes a frameshift mutation that forces <i>gag</i> and <i>pol</i> into the same reading frame. The fused leucine zipper (LZ) domains wt (Wz) or mutant (Kz) at the PR C-terminus are indicated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032845#pone-0032845-g001" target="_blank">Figure 1</a> legend; x denotes substitution mutations in CA, NC (NC15A), and PR that blocked either Gag assembly or PR activity. (B–E) Assembly and multimerization of HIV-1 Gag mutants. 293T cells were transfected with designated constructs. At 48–72 h post-transfection, culture supernatants and cells were collected and subjected to Western immunoblotting (panels B and C). (D) Gag-associated proteins from medium or cell samples were quantified by scanning immunoblot band densities (C). Ratios of Gag in media to Gag in cells were determined for each construct, and compared with wt release level by dividing the release ratio for each chimera by the wt ratio. (E) Velocity sedimentation analysis of cytoplasmic Gag precursor and Gag-LZ chimeras. Homogenized and extracted cytoplasmic lysates were centrifuged through consecutive 25%, 35%, and 45% sucrose gradients at 130,000×<i>g</i> for 1 hour. Fractions were collected from the top of each gradient. Aliquots of each fraction were subjected to SDS-PAGE (10%) and probed with a monoclonal antibody directed against HIV-1 CA.</p

Effects of leucine zipper insertion on virus assembly and processing.

<p>(A–B) 293T cells were transfected with designated constructs Panel A: At 48–72 h, cells and culture supernatant were collected. Panel B: At 4 h post-transfection, equal amounts of cells were placed on two dish plates. Cells and culture supernatants were collected at 24 and 48 h post-transfection and subjected to Western immunoblotting. HIV-1 Gag proteins were probed with an anti-p24CA monoclonal antibody. Pr55<i>^gag</i>, p41<i>^gag</i>, and p24<i>^gag</i> positions are indicated. Cellular Pr55<i>^gag</i> and p24<i>^gag</i> levels were quantified by scanning Pr55<i>^gag</i> and p24<i>^gag</i> band densities from immunoblots. Ratios of p24<i>^gag</i> to p55<i>^gag</i> were determined for the wt and each mutant (panel A, bottom), or normalized to those of wt in parallel experiments (panel B). Values were derived from three independent experiments. Bars indicate standard deviation. **, <i>p</i><0.01. (C) 293T cells were transfected with 10 µg of D25, PRWz, or PRWWz plasmid alone (lanes 1–3); 10 µg D25 plus 10 µg PRWz (lane 4) or PRWWz (lane 5); or 10 µg D25 plus 2 µg PRWz (lane 6) or PRWWz (lane 7). For each transfection, plasmid DNA amounts were maintained at 20 µg by adding pBlueScript SK. At 48–72 h post-transfection, culture supernatant and cells were collected and subjected to Western immunoblotting.</p

Effects of LZ domain insertion on Gag cleavage.

<p>(A) Schematic representations of HIV-1 mutants. Indicated are domains for HIV-1 Gag and <i>pol</i>-encoded p6<i>^pol</i>, PR, and RT. Wild-type (Wz) and mutant (Kz) leucine zipper domains and “x” substitution mutations blocking Gag assembly are indicated as described above. (B–C) 293T cells were transfected with designated constructs. At 4 h post-transfection, equal amounts of cells were placed on two dish plates. Panel B: Cells were collected 24 and 48 h post-transfection and subjected to Western immunoblotting. Cellular Pr55<i>^gag</i> and p24<i>^gag</i> levels were quantified by scanning immunoblot band densities. Ratios of p24<i>^gag</i> to p55<i>^gag</i> were determined for each mutant and normalized to those of wt in parallel experiments. Bars indicate standard deviation. **, <i>p</i><0.01. Panel C: Supernatants and cells were collected 48–72 h post-transfection following treatment with or without 5 µM of HIV-1 protease inhibitor Saquinavier. Samples were prepared and subjected to Western immunoblotting.</p

The enhancement effect of LZ on Gag cleavage is independent of myristylation.

<p>293T cells were transfected with designated constructs containing a normal myristylation or myristylation-minus (myr-) mutation (lanes 5–8). At 4 h post-transfection, equal amounts of cells were placed on two dish plates. Cells and culture supernatants were collected at 24 and 48 h post-transfection and analyzed by Western immunoblotting.</p

Schematic representations of HIV-1 Gag and Gag-PR-leucine zipper expression constructs.

<p>WT and D25 both expressed Pr55<i>^gag</i> and Pr160<i>^gag-pol</i>. Indicated are the HIV Gag protein domains MA (matrix), CA (capsid), NC (nucleocapsid), p6, <i>pol</i>-encoded p6<i>^pol</i>, PR, and RT. The “X” in D25 denotes a PR-inactivated mutation. Gag/PR contains a stop codon insertion at the PR-RT junction, with a codon sequence of 5′-TTT <u>CCC ATT AGC CCT</u> TAG-3′ (RT codons underlined). Striped (Wz) and dotted (Kz) boxes denote wild-type (wt) and mutant (Kz) leucine zipper (LZ) domains, respectively, with each placed individually or in tandem repeat at the end of Gag/PR. Note that the chimeric constructs contain a linker of four Gly residues between the end of PR and beginning of LZ, and a separate linker of three Gly residues between the linked LZ domains.</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