Viral evolution in the presence of increasing HMAb HC33.1 concentrations.

<p>(<b>A</b>) Dual antibody immunofluorescence staining of Huh7.5 cells infected with JFH1 2a HCVcc during multiple passages in increasing concentration of HC33.1. IFA is shown for P0, P18, P27 and P31. HCV E2 glycoprotein was stained with HC33.1 under which viral escape variants were selected (green, upper set of panels), or with CBH-5, a neutralizing domain B HMAb that does not share the same epitope with HC33.1 on E2 (green, lower set of panels). Total virus-infected cells were stained with anti-NS3 antibody labeled with Alexa-594 (red). The cells were counterstained with Hoechst nuclear stain H33342 (blue). The captured images were superimposed (merge). (<b>B and C</b>) Sequence analysis of escape variants in increasing concentrations of HC33.1. Circle graph represents the change in composition of variants from selected passages, as indicated on the top, P0–P34. Specific variants are color coded as indicated in the legend. The table presents the corresponding HC33.1 concentration and the relative intensity in IFA binding by HC33.1. The arrow line divides the viral evolution into four phases (Phase I–IV), based on the appearance in each phase of a variant(s) bearing a specific mutation that became dominant. Viral RNA in the corresponding cell culture supernatants were analyzed by single colony sequencing following RT-PCR amplifications.</p

Analysis of escape variants on their sensitivity to HC33.1-mediated neutralization and binding, and their effect on in vitro viral fitness.

<p>(<b>A</b>) HC33.1 dose-dependent neutralization against viral pool in culture supernatants collected from P0, P18, P27 and P34 with their respective dominant variants bearing the following mutations: N417S, N417T/N434D/K610R, N417T/S419N/N434D/K610R and S395/L413I/N417T was performed by FFU-reduction assay. (<b>B</b>) Dose-dependent neutralization against recombinant HCVcc variants bearing specific mutations, as identified in each phase of viral escape selection was performed by SEAP reporter assay. The IC₅₀ value against each variant is tabulated in the legend. (<b>A, B</b>) Each assay was performed in triplicates and data are shown as percent neutralization, the mean of two experiments ±SD. (<b>C</b>) HC33.1 binding to specific variants, as identified in the legend, by ELISA. Recombinant JFH1 E1E2 wt or the indicated variant E1E2 lysate was captured by GNA in microtiter wells. The wells were then incubated with HC33.1 at the indicated concentrations (0–150 µg/ml). Binding was detected after anti-human IgG-labeled horseradish peroxidase. The <i>y</i>-axis shows the mean optical density values for triplicate wells, the mean of two experiments ±SD. (<b>D</b>). Effect of specific mutations on in vitro viral fitness was determined by measuring virus yield of wt or variant HCVcc bearing the indicated mutations in the focus forming unit assay at an MOI of 0.1. Each assay was performed in triplicates and data are shown as FFU/ml, the mean of two experiments ±SD.</p

Dose-dependent escape variants and their stability with and without continuing immune pressure.

<p>(<b>A</b>) The composition of the viral pool at P18 containing the dominant variant_{N417T+N434D+K610R} during five additional passages at a constant concentration of 4.5 µg/ml HC33.1. (<b>B</b>) The composition of the viral pool at P27 containing the dominant variant_{N417T+S419N+N434D+K610R} during 15 additional passages after HC33.1 was removed. (<b>C</b>) The composition of the viral pool at P18 containing the dominant variant_{N417T+N434D+K610R} during 15 additional passages after HC33.1 was removed. (<b>D</b>) The composition of the viral pool at P34 containing the dominant variant_{S395P+L413I+N417T} during 8 additional passages after HC33.1 was removed. (<b>A–D</b>) The identified specific variants are color coded as indicated in the legend.</p

Human and mouse antibodies against amino acid 412–423 have different neutralization profiles.

<p>(<b>A</b>) Epitope alignment. Epitopes of three HMAbs: HC33.1; HC33.4 and HC33.8 are compared with murine MAb AP33. Recombinant E1E2 mutant proteins were expressed in 293T cells and cell lysates were analyzed by ELISA. Individual protein expression was normalized by binding of CBH-17, an anti-HCV E2 HMAb to a linear epitope <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Keck1" target="_blank">[25]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Hadlock1" target="_blank">[47]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004297#ppat.1004297-Keck8" target="_blank">[51]</a>. Red indicates 0–20%, orange 21–40%, brown 41–60%, white 61–100% and green >100% binding, when the residue was replaced by alanine, relative to binding to wt. Dose-dependent neutralization of (<b>B</b>) wt JFH-1 HCVcc, (<b>C</b>) HCVcc variant bearing N417S mutation and (<b>D</b>) bearing N417T mutation were performed by SEAP reporter assay. (<b>B, C, D</b>) Either wt HCVcc or variant HCVcc was incubated with HC33.1 or AP33, at concentrations ranging from 0.1 to 50 µg/ml, prior to infecting the Huh7J-20 cells. Virus infectivity levels were determined by measurement of the SEAP activity released into the medium. (<b>F</b>) Antibody concentration (µg/ml) required to reach 50% neutralization (IC₅₀) for each antibody is summarized. (<b>E</b>) The effect of N417S or N417T mutation on in vitro viral fitness as measured by focus forming assay at an MOI of 0.1. (<b>B–E</b>) Each assay was performed in triplicates and data are shown as percent neutralization, the mean of two experiments ±SD.</p

Assessment of lipid uptake in WT and <i>sg/sg</i> BMMs.

<p>(a) TLC was performed on lipid extracted from WT and <i>sg/sg</i> BMMs (n = 3 littermate pairs). Lanes 2–4 and 5–7 correspond to untreated WT and <i>sg/sg</i> samples respectively while lanes 8–10 and 11–13 correspond to 4 h 400 μM oleic acid-treated WT and <i>sg/sg</i> samples respectively. Lanes 1 and 14 are ladder standards. Quantification was performed by normalizing the relative density of triglyceride bands in each lane to WT untreated controls (arbitrarily set as 1.0) and is expressed as the mean ± S.E.M. relative fold-difference (n = 3 littermate pairs). Statistical analyses were performed using two-way ANOVAs with Bonferroni post test applied where *P<0.05. (b) Representative images of WT and <i>sg/sg</i> BMMs with endocytosed fluorescent-acLDL were captured using the Olympus upright wide-field epifluorescence microscope with actin (phalloidin; white) and nucleus (DAPI; blue) labeling, and fluorescent-acLDL (red). Quantification of fluorescent-acLDL uptake is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) normalized to number of cells analyzed (n = ~200 cells per mouse) from n = 4 biological replicates (littermate pairs). Relative fold-difference is calculated with the mean of WT values set as 1.0 and is available on the right Y-axis. Statistical analysis was performed using an unpaired Student’s t-test where **P<0.01. Scale bars = 20 μm. (c) Electron microscopy of LDs. Representative images of WT and <i>sg/sg</i> BMMs treated with 400 μM oleic acid overnight show clusters of LDs accumulated in the cytoplasm. And interspersed with endoplasmic reticulum. N = nucleus, scale bars = 1 μm. Quantification of LD size is expressed as a histogram of area/LD frequency distribution.</p

Assessment of lipid storage in WT and <i>sg/sg</i> BMMs.

<p>(a) Representative images of BMMs from WT and <i>sg/sg</i> littermates fixed and stained for LDs using Oil Red O (red). Images were taken using the Personal Deltavision Deconvolution microscope with wheat germ agglutinin (white) and nucleus (DAPI; blue) labeling. Quantification of LDs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (n = ~100 cells/mouse) from n = 4 littermate pairs. (b) Representative images of WT and <i>sg/sg</i> BMMs immunolabeled for ADRP (red). Images were taken using the Personal Deltavision Deconvolution microscope (maximum projection of 30-step 0.1 μm slices) with actin (white) and nucleus (DAPI; blue) labeling. Quantification of LDs was performed on 700–1300 LDs acquired from 10 fields of view per biological replicate (n = 3 littermate pairs) and is expressed as the mean ± S.E.M. top-down cross sectional area, perimeter, and Feret’s diameter (distance between two parallel tangential planes) per LD. Statistical analyses for were performed using unpaired two-tailed Student’s t-tests where *P<0.05; **P<0.01. Scale bars = 10 μm.</p

Increased <i>Vldlr</i> mRNA expression correlates with increased lipid uptake in <i>sg/sg</i> BMMs.

<p>(a) <i>Vldlr</i> mRNA expression from Lipoprotein Signaling and Cholesterol Metabolism RT² Profiler PCR Array (Qiagen) expressed as the mean ± S.E.M. relative quantification (RQ) (fold-change) in <i>si</i>Ch25h cells (black bar) compared to control siRNA (set as 1.0; white bar). The Genorm software embedded in StatMiner determined <i>Actb</i>, <i>Gapdh</i>, and <i>Hsp90ab1</i> to be the three most stable and robust endogenous controls and all data were normalized to the median of their Ct values. The P-values were calculated using empirical Bayes statistics where *P<0.05. (b) Targeted qPCR analysis was performed using Taqman Gene Expression Assays for <i>Vldlr</i>. <i>Hprt1</i> was used as the endogenous control. Analysis was performed using RNA acquired from n = 4 littermate pairs of WT and <i>sg/sg</i> BMMs in triplicate experiments and represented as the mean ± S.E.M relative quantification (fold-change) with WT set as 1.0. Statistical analysis was performed using unpaired two-tailed Student’s t-test where *P<0.05; **P<0.01. (c) RAW264.7 cells were transiently transfected with either an empty HA-tagged pcDNA3.1 vector or a vector containing HA-VLDLR for 48 h. Cells were stained for LDs using Oil Red O and quantification of LDs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (~350 cells per experiment, n = 3 independent experiments). Statistical analysis was performed using an unpaired two-tailed Student’s t-test where *P<0.05. Representative images of RAW264.7 cells transiently transfected with either an empty HA-tagged pcDNA3.1 vector or a vector containing HA-VLDLR for 48 h were captured using the Olympus upright wide-field epifluorescence microscope are shown in the bottom panel. Red denotes LDs (Oil Red O staining) and blue denotes nuclei (DAPI). Scale bars = 100 μm.</p

<i>Ch25h</i> knockdown up-regulates LDs.

<p>(a) Western blot analysis of Ch25h protein in WT (lanes 1 to 4) and <i>sg/sg</i> (lanes 6 to 9) BMMs (n = 4 littermate pairs). Lane 5 was only loaded with sample buffer. Quantification was performed by normalizing the relative densities of Ch25h bands (32 kDa) to the loading control ERp72 (72 kDa) and is represented as the mean ± S.E.M. relative density from n = 4 littermate pairs. Statistical analysis was performed using an unpaired two-tailed Student’s t-test where **P<0.01. (b) Two separate siRNA oligomers (individually or pooled) were used to knockdown expression of <i>Ch25h</i> in WT BMMs. The degree of mRNA knockdown was assessed by qPCR in unactivated macrophages with <i>Hprt1</i> as the housekeeping gene and represented as the mean ± S.E.M relative quantification (fold-change) with WT cells treated with siRNA negative control set as 1.0. Statistical analysis was performed using a one-way ANOVA with Bonferroni’s post test applied, comparing Ch25h mRNA expression in all treatments relative to the siRNA negative control (n = 4 biological replicates) where *P<0.01; n.s. denotes non-significant. (c) Quantification of LDs in WT control and <i>si</i>Ch25h BMMs is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs (Oil Red O) normalized to number of cells analyzed (~500–700 cells per biological replicate (n = 4)). Statistical analysis was performed using an unpaired two-tailed Student’s t-test where ***P<0.001. (d) Representative images of LDs (stained using Oil Red O) in WT control and <i>si</i>Ch25h BMMs. Images were taken using the Olympus upright wide-field epifluorescence microscope. Blue denotes nucleus (DAPI) labeling and red denotes LD (Oil Red O) labeling. Scale bars = 50 μm. (e) Representative images of anti-ADRP (red) labeling in WT control and <i>si</i>Ch25h BMMs. Images were taken using the Personal Deltavision Deconvolution microscope and displayed as the maximum projection of 30-step z-stack (0.1 μm slices). White denotes actin (phalloidin) labeling and blue denotes nucleus (DAPI) labeling. Quantification of LDs was performed on 600–700 LDs acquired from 5 fields of view per biological replicate (n = 4 biological replicates) and is expressed as the mean ± S.E.M. cross sectional area, perimeter, and Feret’s diameter per LD. Statistical analyses were performed using unpaired two-tailed Student’s t-tests for each parameter where **P<0.01. Scale bars = 10 μm. (f) <i>sg/sg</i> BMMs treated with vehicle control (0.01% DMSO) or 25HC (0.025 μM to 0.1 μM) for 4 h. Representative images were taken using the Olympus upright wide-field epifluorescence microscope are shown on the left panel where blue denotes nucleus (DAPI) labeling and red denotes LD (Oil Red O) labeling. Quantification of LDs (low 25HC treatment) is expressed as the mean ± S.E.M. fluorescence intensity (integrated density) of stained LDs normalized to number of cells analyzed (n = ~100 cells per mouse per treatment). Statistical analysis was performed using a one-way ANOVA with Bonferroni’s post test (n = 3 biological replicates), comparing the amount of LDs in each treatment to the vehicle control <i>sg/sg</i> cells where **P<0.01; ***P<0.001; n.s. denotes non-significant. Scale bars = 50 μm.</p