Effect of carbon monoxide treatment on neuronal apoptosis.

<p>(<b>A</b>) Representative micrographs of neurons treated or not with 20 µM of glutamate and 10 µM of CO. Apoptotic hallmarks were analyzed by fluorescent microscopy. <i>Upper panel</i>, for the photos taken with the filter for phase contrast, <i>middle panel</i>, for Hoechst (white arrows for nuclei with condensed chromatin) and <i>lower panel</i>, for propidium iodide (white arrows for cells which membrane integrity was lost). (<b>B</b>) Primary cultures of neuronal cells were pre-treated with 10 µM CO, followed by 24 h of glutamate (10–30 µM) treatment. Cell viability was assessed by counting cells containing normal nuclei and plasmatic membrane integrity. For each coverslip, at least 1500 cells were counted. All values are mean ± SD (error bars), n = 5; *<i>p</i><0.05 compared to control. (<b>C</b>) The effect of 10 µM CO treatment on Bcl-2 expression was assessed by its mRNA quantification.</p

BEVS-driven FL-gB expression screening.

<p>(<b>A</b>) bBst2x was used to infect Sf9 (left panel) or High Five (right panel) cells at 0.3 (circles), 1 (diamonds), 2.5 (triangles) or 4 (crosses) ×10⁶ cell/mL (CCI)) with mutliplicities of infection (m.o.i.) of 0.1, 1, 3, 5 or 10 pfu/cell. Cells were harvested at 72 hours after infection and detergent-soluble protein extracts were analysed by densitometric analysis of anti-gB TM immunoblots. Overall relative quantification was made by comparing samples from different experimental series in the same immunoblots (hereafter n = 3 with error bars representing 95% confidence interval, C.I.) and setting as 100% the mean of most intense signals. (<b>B</b>) Comparison of FL-gB expression pattern and kinetics in Sf9 and High Five cell lines, bBst2x-infected with m.o.i. 5 at CCI 0.3 (Sf9) or 2.5 (High Five, Hi5). Cells were sampled at the indicated hours <i>post</i>-infection (h.p.i.) and detergent-soluble cell extracts probed in immunoblot (IB) with anti-gB TM. Positions of the molecular weight markers are indicated and expressed in kDa. (<b>C</b>) Theroretical FL-gB relative volumetric yields computed from (A) for m.o.i. 5 series, according to CCI and cell viability at 72 h.p.i.</p

BEVS-driven FL-gB expression screening.

<p>(<b>A</b>) bBst2x was used to infect Sf9 (left panel) or High Five (right panel) cells at 0.3 (circles), 1 (diamonds), 2.5 (triangles) or 4 (crosses) ×10⁶ cell/mL (CCI)) with mutliplicities of infection (m.o.i.) of 0.1, 1, 3, 5 or 10 pfu/cell. Cells were harvested at 72 hours after infection and detergent-soluble protein extracts were analysed by densitometric analysis of anti-gB TM immunoblots. Overall relative quantification was made by comparing samples from different experimental series in the same immunoblots (hereafter n = 3 with error bars representing 95% confidence interval, C.I.) and setting as 100% the mean of most intense signals. (<b>B</b>) Comparison of FL-gB expression pattern and kinetics in Sf9 and High Five cell lines, bBst2x-infected with m.o.i. 5 at CCI 0.3 (Sf9) or 2.5 (High Five, Hi5). Cells were sampled at the indicated hours <i>post</i>-infection (h.p.i.) and detergent-soluble cell extracts probed in immunoblot (IB) with anti-gB TM. Positions of the molecular weight markers are indicated and expressed in kDa. (<b>C</b>) Theroretical FL-gB relative volumetric yields computed from (A) for m.o.i. 5 series, according to CCI and cell viability at 72 h.p.i.</p

Analysis of purified FL-gB.

<p>(<b>A</b>) Purified reduced (left panel) or non-reduced (right panel) FL-gB was loaded on SDS-PAGE; amounts of FL-gB loaded in the reducing SDS-PAGE are indicated; for non-reducing electrophoresis, 2.5 µg of FL-gB were heated or not in the absence of reducing agents. The positions of the uncleaved FL-gB, TM and SU chains are indicated as migrating before or after the reduction of the gB protomer disulfide bridges. (<b>B</b>) 5 µg of purified FL-gB were analysed by reducing SDS-PAGE after incubating the protein for 4 hours at 30°C with or without the indicated units (U) of recombinant furin and loaded on reducing SDS-PAGE. (<b>C</b>) FL-gB glycosylation was analysed by incubating 2.5 µg of either denatured or native FL-gB with PNGase F for 3 hours or endoglycosidase F isoforms for 1 hour, respectively; the control sample was left undigested. Denaturation was by heating FL-gB at 99°C for 20 min in 0.2% SDS and 1% 2-mercaptoethanol.</p

The concentration of assembly intermediates for three initial protein concentration scenarios:

<p>1) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (stoichiometric ratios – white bars); 2) <b>[vp2]₀</b> = 1.2 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 0.78 M (limitation of vp7 – black bars); 3) <b>[vp2]₀</b> = 12 M, <b>[vp6]₀</b> = 7.8 M and <b>[vp7]₀</b> = 7.8 M (excess of vp2 – grey bars). The Gibbs free energy of vp2, vp6 and vp7 subunit association was assumed to be equal (<b>ΔG⁰_vp2,3-mer</b> = <b>ΔG⁰_vp6,13-mer</b> = <b>ΔG⁰_vp7,13-mer</b> = −4.08 kcal.mol⁻¹).</p

The effect of ΔG⁰_vp7,13-mer on RLP assembly efficiency when the initial protein concentration of vp2, vp6 and vp7 is 1.2 M, 7.8 M and 7.8 M, respectively, and the Gibbs free energy of vp2 and vp6 subunit association (ΔG⁰_vp2,3-mer and ΔG⁰_vp6,13-mer, respectively) is equal to −4.08 kcal.mol⁻¹.

<p><b>ΔG⁰_vp7,13-mer</b> varies between −4.08×[0.05 to 1.4] kcal.mol⁻¹.</p

gB peptides identified by MS/MS.

1<p>start and end amino acids as by conceptual translation of HCMV UL55 ORF.</p>2<p>Confidence Interval.</p

Purified FL-gB is in the post-fusion conformation.

<p>(<b>A</b>) Negatively-stained FL-gB particles have been imaged by EM and averaged as described in M&M. 2D class obtained is shown. Structural features of gB protein are highlighted. (<b>B</b>) Cartoon view of HSV-1 gB_ecto trimer (PDB 4HSI) is showed for reference. Dashed lines mark gaps of missing residues in the model.</p

SLP assembly efficiency as function of the initial vp2 concentration, [vp2]₀, for different Gibbs free energy of vp2 subunit association values, ΔG⁰_vp2,3-mer.

<p>The full line represents <b>ΔG⁰_vp2,3-mer</b> = −4.08 kcal.mol⁻¹ as estimated by Zlotnick 1994, the dash lines represent <b>ΔG⁰_vp2,3-mer</b>>−4.08 kcal.mol⁻¹ and the dot lines represent <b>ΔG⁰_vp2,3-mer</b><−4.08 kcal.mol⁻¹. <b>K_d,app</b> represents the pseudo-critical concentration that satisfies the constraint <b>[1]</b> (concentration of unassociated vp2 subunits at equilibrium - <b>[1]</b> = <b>[svp2]_e</b>) = <b>[20]</b> (complete SLP) = <b>K_d,app</b> at equilibrium for a specific <b>ΔG⁰_vp2,3-mer</b>.</p