Molecular Insights into Quorum Sensing in the Human Pathogen Pseudomonas aeruginosa from the Structure of the Virulence Regulator LasR Bound to Its Autoinducer

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Structural characterization of the Hepatitis C Virus NS3 protease from genotype 3a: The basis of the genotype 1b vs. 3a inhibitor potency shift

AbstractThe first structural characterization of the genotype 3a Hepatitis C Virus NS3 protease is reported, providing insight into the differential susceptibility of 1b and 3a proteases to certain inhibitors. Interaction of the 3a NS3 protease with a P2–P4 macrocyclic and a linear phenethylamide inhibitor was investigated. In addition, the effect of the NS4A cofactor binding on the conformation of the protease was analyzed. Complexation of NS3 with the phenethylamide inhibitor significantly stabilizes the protease but binding does not involve residues 168 and 123, two key amino acids underlying the different inhibition of genotype 1b vs. 3a proteases by P2–P4 macrocycles. Therefore, we studied the dynamic behavior of these two residues in the phenethylamide complex, serving as a model of the situation in the apo 3a protein, in order to explore the structural basis of the inhibition potency shift between the proteases of the genotypes 1b and 3a

Enhanced expression of full-length human cytomegalovirus fusion protein in non-swelling baculovirus-infected cells with a minimal fed-batch strategy.

Human cytomegalovirus congenital infection represents an unmet medical issue and attempts are ongoing to develop an effective vaccine. The virion fusion players of this enveloped virus are the natural targets to achieve this goal and to develop novel anti-viral therapies. The secreted ectodomain of the viral fusion factor glycoprotein B (gB) has been exploited so far as an alternative to the cumbersome expression of the wild type trans-membrane protein. In the soluble form, gB showed encouraging but limited potential as antigen candidate calling for further efforts. Here, the exhaustive evaluation of the Baculovirus/insect cell expression system has been coupled to an orthogonal screening for expression additives to produce full-length gB. In detail, rapamycin was found to prolong gB intracellular accumulation while inhibiting the infection-induced cell swelling. Not obvious to predict, this inhibition did not affect Baculovirus growth, revealing that the virus-induced cell size increase is a dispensable side phenotype. In parallel, a feeding strategy for the limiting nutrient cysteine has been set up which improved gB stability. This multi-modal scheme allowed the production of full-length, mutation-free gB in the milligram scale. The recombinant full-length gB obtained was embedded into a stable mono-dispersed particle substantially larger than the protein trimer itself, according to the reported association of this protein with detergent-resistant lipid domains

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

FL-gB particle is sensitive to ionic detergents.

<p>10 µg of FL-gB were incubated for 1 hour at 37°C in the presence of the indicated concentrations of SDS and then loaded onto a Blue Native-PAGE gel.</p

Primers used to construct bBst2x¹.

1<p>Fw and Rv stand for forward and reverse primers, respectively; restriction sites are in boldface; start and stop codons are in lowercase; overlapping sequences for 5′ extension PCR are matched as underlined and italics.</p

Cysteine supplementation further increases FL-gB expression in infected High Five.

<p>(<b>A</b>) 300x concentrated cysteine supplement was prepared in culture medium to obtain the indicated final Cys concentrations and added alone to High Five cell cultures 24 h.p.i. with bBst2x at CCI 2.5 with m.o.i. 5. Cys was added alone (left panel) or in combination with 50 nM rapamycin treatment (right panel). Cells were harvested at 72 h.p.i. and detergent-soluble FL-gB relative quantification performed as already described (controls were made by adding fresh medium to infected cell cultures previously supplemented or not with 0.1% DMSO at the time of infection). (<b>B</b>) FL-gB expression was compared in detergent-soluble (Sol) or -insoluble (Ins) protein extracts obtained from cells treated as in (A right panel). Equal loading was by resuspending the insoluble pellets in the same volume used to prepare the soluble fractions. Immunoblot (left panel) and densitometric analysis (right panel) are shown. (<b>C</b>) Effect on the dissolved oxygen (DO) control and cell viability in bioreactors of bBst2x-infected High Five - CCI 2.5, m.o.i. 5, rapamycin 50 nM - by either cysteine shot addition (Shot, dashed lines and triangles) or continuous feeding (Cont, solid lines and circles). Bioreactors were sampled for cell viability, while DO was recorded in real-time and expressed as the percentage of air saturation.</p

Effect of rapamycin on FL-gB expression.

<p>(<b>A</b> left panel) High Five cells were infected with bBst2x at CCI 2.5 with m.o.i. 5 in the presence of either 0.1% DMSO (cnt) or the indicated final concentrations of rapamycin, harvested at 72 h.p.i. and relative FL-gB expression analysed by immunoblot with ECL mean signals of control samples set as 1; (right panel) anti-gB TM immunoblot on detergent-soluble protein extracts from High Five cells infected as above in the presence of 0.1% DMSO (cnt) or with 50 nM rapamycin added at the indicated time points. (<b>B</b> left panel) High Five cells were infected as in (A) in the presence of 50 nM rapamycin (diamonds) or DMSO alone (stars) and cell cultures were sampled at the indicated h.p.i. to measure viral titers (solid lines) and cell volume (dashed lines); (right panel) High Five cell were infected as in (A right panel) and the recorded cell volume plotted as cell swelling inhibition (stars) along with relative FL-gB expression (diamonds) detected in the same culture samples. (<b>C</b>) Kinetic expression of FL-gB and <i>Ac</i>MNPV gp64 analysed by immunoblot of detergent-soluble protein extracts from High Five cell cultures infected and sampled as in (B left panel).</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