Effect of PNPLA3 148M/M on (log₁₀) FPR in 247 CHC patients according to different thresholds of age at infection.

<p>Effect of PNPLA3 148M/M on (log₁₀) FPR in 247 CHC patients according to different thresholds of age at infection.</p

Demographic and clinical features of the 132 HCV1 patients, stratified by presence of HCV defective particles or IL28B genotype.

<p>Continuous variables are expressed as median ± interquartile range (IQR). P values are calculated by Wilcoxon test and Fisher's test for continuous and categorised variables respectively; NS: not significant.</p><p>Demographic and clinical features of the 132 HCV1 patients, stratified by presence of HCV defective particles or IL28B genotype.</p

Multivariable logistic regression for Grading, ETR and Relapse.

<p>OR: odds ratio—CI: 95% confidence interval</p><p>^a: odds ratio and confidence intervals have been calculated considering an interval of 10 years.</p><p>^b: odds ratio and confidence intervals have been calculated considering an interval of 10⁵ units of viremia</p><p>^c: odds ratio and confidence intervals have been calculated considering an interval of 20 units of GGT</p><p>Multivariable logistic regression for Grading, ETR and Relapse.</p

Viral genome architecture of the defective forms identified in the serum of chronic hepatitis C patients (genotype 1 HCV).

<p>(A) Pictures of agarose-gel showing amplicons obtained after the second round of nested PCR for all the patients in which defective forms were identified (lanes 1–25) and a subset of patients negative for the defective form (lanes 26–36). Lanes M: molecular size markers. (B) Schematic representation of the architecture of the 25 defective variants identified in the sera of subjects chronically infected with genotype 1 HCV. (C) Picture of agarose gel showing amplicons obtained after nested PCR performed on synthetic HCV RNA mixtures assessing full-length/defective ratios from 1 to 1000 (lanes 3–12). FL: full-length RNA only (lane 1); D: deleted RNA only (lane 2). Lane M: molecular size markers.</p

<i>Sbds</i>^{<i>R126T/R126T</i>} and <i>Sbds</i>^<i>MOCK</i> cells show an increase in cell death in response to UV stress.

<p><b>(A-B)</b> UV exposure damages more SBDS mutant cells than wt or <i>Sbds</i>^{<i>RESCUE</i>}. (A) Experiment on <i>Sbds</i>^{<i>R126T/R126T</i>} versus wt. There is an increase in cell death in <i>Sbds</i>^{<i>R126T/R126T</i>} versus wild type cells. In basal conditions no differences are observed among the genotypes (first two bar triplets, left). Upon UV damage cell death affects more <i>Sbds</i>^{<i>R126T/R126T</i>} (second two bar triplets, right). Cells were stressed with UV irradiation (9999 μJ/cm², three times), recovered for 4 hours and stained for annexin with 7-AAD. <b>(B)</b> Observed ratios in all genotypes. Ratio ≥ 1 indicates higher sensitivity, the rescue with SBDS only partly restores the ratios. <b>(C-D)</b> COMET assay. Representative images of indicated cells treated as indicated in manufacturer protocol (Comet assay kit, Trevigen) in (C). Scale bars indicate 20 μm. (D) Absolute quantitation of the tail moment unveils a minor but significative difference.</p

Quantitative detection of total and defective HCV RNA in a subset of patients who carried defective HCV forms.

<p>Quantitative detection of total and defective HCV RNA in a subset of patients who carried defective HCV forms.</p

Virogical responses to PEG-IFNα/RBV stratified for HCV defective forms or IL28B genotype.

<p>RVR, EVR, ETR and SVR rates in the overall population as well as relapse rates in ETR-positive subjects, according to the presence of HCV deletions (A-B) or IL28B genotype (C-D), are reported. The presence of HCV defective particles does not have a significant effect on RVR, EVR, ETR or SVR rates, while it correlates with a higher probability of relapse in ETR-positive subjects (A-B). IL28B CT/TT genotypes are significantly associated with lower RVR, EVR, ETR and SVR rates and correlate with a higher probability of relapse (C-D).</p

SBDS deficiency reduces the maximal translational capability up to 70% due to a defect in 60S maturation that is partly associated with a change in eIF6–free 60S subunits.

<p><b>(A-B) Polysome profiles.</b><i>Sbds</i>^{<i>R126T/R126T</i>} cells show an increase in free 60S and lower 80S peaks on sucrose gradient compared to wt (A). The phenotype is completely restored in <i>Sbds</i>^{<i>RESCUE</i>} cells (B). Note, the increase of free 60S is two-fold. A₂₅₄ nm was measured after 15–50% sucrose gradient sedimentation. Representative experiment of n≥5. (<b>C-D) Nucleoli analysis.</b> <i>Sbds</i>^{<i>R126T/R126T</i>} (C) and <i>Sbds</i>^<i>MOCK</i> cells (D) do not have differences in the number of nucleoli respect to their control cell lines (wild type and <i>Sbds</i>^{<i>RESCUE</i>}). Distribution of cells containing less or more of six nucleoli per nucleus in wild type, <i>Sbds</i>^{<i>R126T/R126T</i>}_, <i>Sbds</i>^{<i>RESCUE</i>} and <i>Sbds</i>^<i>MOCK</i> cells was counted with Volocity Sofwtare, by analyzing cells stained for the nucleolar marker nucleophosmin (NPM) (n≥200, n = number of nuclei analyzed per genotype). <b>(E-F) SBDS and NPM localization.</b> Confocal images wild type and <i>Sbds</i>^{<i>R126T/R126T</i>} cells indicate a co-localization of SBDS and NPM proteins within the nucleolus, and a cytoplasmic SBDS. There are no visible differences among all genotypes. Scale bar 25 μm (E) and 2 μm (F). <b>(G-H)</b> Measurement of eIF6 binding sites by iRIA technique shows that wt cells have 25% more free 60S as detected by eIF6 binding. (G) iRIA technique outline: <i>Sbds</i>^{<i>R126T/R126T</i>} and wild type cellular extracts were immobilized on a 96 well and biotinylated eIF6 is added. This assay is able to detect the binding between eIF6 and 60S. (H) <i>Sbds</i>^{<i>R126T/R126T</i>} fibroblasts have less binding sites for eIF6, respect to wild type cell line, i.e. 0.12 arbitrary units versus 0.16. Representative technical triplicate experiment of n≥4 biological replicates.</p

<i>SBDS</i> mutation results in increased lysomome trafficking and activity, and a decrease in ATP and lactate levels predicted by changes in the steady-state of the mRNAs identified by RNA-seq.

<p><b>(A)</b> Changes in the steady-state levels (total, <i>bona fide</i> for transcriptional) of genes with peptidase activity as detected both on polysomes and total. Heat maps representing absolute gene expression levels in <i>Sbds</i>^<i>MOCK</i> and <i>Sbds</i>^{<i>RESCUE</i>} samples for the subset of gene sets with peptidase activity by Gene Ontology analysis. (<b>B)</b> qPCR validation of selected genes associated to lysosome activity. Real time analysis of selected genes associated to lysosome trafficking and activity. Data shown for <i>n</i>≥3; mean±s.d., T-test, paired, two-tailed. <b>(C)</b> <i>Sbds</i>^<i>MOCK</i> cells have a decrease in intracellular pH value. Representative graph showing increased lysotracker fluorescence intensitity in <i>Sbds</i>^<i>MOCK</i> cells respect to <i>Sbds</i>^{<i>RESCUE</i>} cells. This result suggests more lysosome trafficking and activity in <i>Sbds</i>^{<i>R126T/R126T</i>} cells. Data shown for <i>n</i>≥3; mean±s.d., T-test, paired, two-tailed. <b>(D-E)</b>. Lamp1 immunostaining (representative cells, D) and quantitation (E). Scale bar indicates 8.3μm. Globally, A-E show increase in the proteolitic potential of <i>Sbds</i>^<i>MOCK</i> respect to <i>Sbds</i>^{<i>RESCUE</i>} cells. <b>(F-G)</b> SBDS levels positively regulate ATP accumulation (F) and lactate/pyruvate production, an index of glycolysis (G). All graphs represent mean ± s.d. Statistic applied was T-test, paired, two-tailed, n≥4.</p

Model for SBDS-induced changes.

<p><b>(A-B)</b> We speculate that SBDS mutation poises cells at a low energy level due to impaired global translation. Cells have a complex transcriptional rewiring and adapt by increasing the proteolytic flux. In this condition, they are less responsive to growth stimuli (example <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006552#pgen.1006552.g001" target="_blank">Fig 1</a>), but more sensitive to stressors respect to wt cells. Interventions to increase translational efficiency are predicted to increase their fitness. <b>(B)</b> Summar table indicating all phenotypes observed in this study in <i>Sbds</i> mutant cells.</p