Comparison of surface charge and lipophilicity distributions between human and rat CD81 ectodomains.

<p>The structures of the resolved human CD81 (gray) and the homology-modeled rat CD81 (pink) shown in the ribbon were superposed. The major differences between the two CD81 structures are at the flexible loops from 173 to 186 a.a. (green: human CD81; red: rat CD81). (B) The surface charge distributions of the two CD81s show that the rat CD81 is more positively charged than human CD81 at the flexible loop region marked with a dashed line (blue: positive charge; red: negative charge). (C) The lipophilicity maps do not show much difference between the human and rat CD81s in the loop region (blue: hydrophilic; green: lipophilic).</p

Flow cytometry of E2 peptides binding to human and rat cells and inhibitions of anti-CD81 antibody/cell binding by E2 peptides.

<p>(A) and (B) show the fluorescence intensity of Huh-7 cells treated with fluorescent p_E2-site1 (E2-s1) and p_E2-site2 (E2-s2), respectively, at different concentrations; (C) and (D) are the fluorescence intensity measurements of the rat PC12 cells treated with fluorescent p_E2-site1 or p_E2-site2, respectively; and (E) and (F) are the inhibitions of fluorescent anti-CD81 antibodies targeting Huh 7 cells by HCV E2 peptides. In this experiment, untreated cells were used as negative controls, and the cells treated with fluorescent-labelled anti-CD81 antibodies were used as positive controls and set as 100% binding. p_m_E2-site1 (m-E2-s1) and p_m_E2-site2 (m-E2-s2) in (A), (B), (C) and (D) are mutant peptides of p_E2-site1 and p_E2-site2 indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177383#pone.0177383.s004" target="_blank">S1 Table</a>.</p

MM/PBSA binding free energy calculations for human and rat CD81s to HCV E2 protein.

<p>(A) For different HCV E2 sites (E2-site1, E2-site2, and E2-both sites) binding to human and rat CD81s, the binding free energies of human CD81 to HCV E2 were lower than those of rat CD81. HCV E2-site2 bound to human CD81 with the lowest binding free energy (H-E2-S2). (B) The detailed analysis of the components of binding free energies showed that the major difference for HCV E2-site2 binding to human and rat CD81s lies in the electrostatic interactions (H-E2-S2 and R-E2-S2). VDW dominates the binding of HCV E2-site1 to human CD81 (H-E2-S1). The figure represents the following. For H-E2-S1: the E2-site1 binding to human CD81; for H-E2-S2: the E2-site2 binding to human CD81; for H-E2-both: E2-both sites binding to human CD81; for R-E2-S1: the E2-site1 binding to rat CD81; and for R-E2-S2: the E2-site2 binding to rat CD81.</p

Surface charge and lipophilicity distributions for HCV E2 binding to human CD81.

<p>The complex structure is presented as a ribbon (orange: HCV E2; gray: human CD81). (A) and (B) are the surface lipophilicity distributions on HCV E2-site1 and human CD81 at the binding interface. In the figures, blue represents the hydrophilic part and green the hydrophobic part. The hydrophobic residues around the binding interface are labelled and presented as sticks. (C) and (D) are the surface charge distributions on HCV E2-site2 and human CD81 at the binding interface mapped according to the Poisson-Boltzmann equation. Blue and red correspond to positive and negative electrostatic potential, respectively. Charged residues around the binding interface are labelled and presented as sticks.</p

Molecular docking of human and rat CD81s to HCV E2 protein.

<p>Preferable sites of HCV E2 binding with human and rat CD81s are shown in A to C. (A) The HCV E2-site1 loop could bind to human and rat CD81s with similar RDOCK scores (human: −18.3 kcal/mol; rat: −16.2 kcal/mol). (B) The HCV E2-site2 loop was able to dock to human and rat CD81s, but the RDOCK score for human CD81 was more than twice as low as rat CD81 (human: −14.7 kcal/mol; rat: −6.2 kcal/mol). (C) HCV E2 bound to human CD81 with both E2-site1 and E2-site2 loops (RDOCK score: -19.1 kcal/mol). Green: E2-site1; blue: E2-site2; pink: the binding loops of human and rat CD81s.</p

Putative model of the HCV E2/CD81 binding process.

<p>The initial HCV E2/CD81 binding process can be divided into two steps. Step 1: E2 initially recognizes and approaches CD81 with the E2-site2 region. Step 2: The orientation of E2 changes to a more preferable binding pose with the E2-site1 region auxiliary binding to execute the processes that follow. The E2-site1 region, E2-site2 region, and CD81 binding loop are presented in green, blue, and pink, respectively.</p

Sequence alignment of human, chimpanzee, and rat CD81s.

<p>The sequence alignments of full-length human and chimpanzee CD81s display 100% identity, whereas that of human and rat CD81s show over 93% identity (97% similarity). The sequence identity of the LEL between humans and rats is 84% (93% similarity) and that of the transmembrane domain is 99%, indicating that the major differences between human and rat CD81s are in the LEL. The transmembrane domain at a.a. 1–112 and 202–236 is indicated with a green rod, whereas the LEL region at a.a. 113–201 is indicated with a red rod. The largest differences between the CD81s located within 160–190 a.a are within the black–framed box.</p

Suppression of vaccinia virus replication slightly by exogenously expressed Dicer protein.

<p>(A, B) HeLa cells were transfected with empty vector (2 ug, lanes 1 and 2), with the plasmid expressing T7-Dicer protein (2 ug, lanes 3 and 4) or with the plasmid expressing T7-Dicer with mutations in a.a. 1816 and 1817 (2 ug, lanes 5 and 6). Twenty-four hrs after transfection, the cells were mock-infected (lanes 1, 3 and 5) or infected with VV (M.O.I. = 10) (lanes 2, 4 and 6). Twenty-four hrs after infection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer, A type inclusion protein and β-actin (A) while the secreted virus particles from the supernatants (lanes 2, 4 and 6) were analyzed by the plaque assay (B). (p<0.01, **). (C) HeLa cells were transfected with scramble siRNA as a control or siRNA against Dicer. Twenty-four hrs after transfection, cell lysates were analyzed by SDS-PAGE and Western blotting with antibodies against the Dicer (upper panel) and β-actin proteins (bottom panel) as the loading control. (D) HeLa cells were transfected with scramble or Dicer siRNA. Twenty-four hrs after transfection, the cells were infected with VV (M.O.I. = 10). Twenty-four hrs after infection, the secreted viral particles from the supernatants were analyzed by plaque assay. NS: not significant.</p

Cytotoxicity of materials used for cargo delivery by PR9 as determined using the MTT assay.

<p>Cells were treated with QD, PR9, chloroquine, PR9/QD, QD/chloroquine, PR9/QD/chloroquine, QInP alone and PR9/QInP complexes for 24 h. Significant differences at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from three independent experiments.</p

Mechanism of cellular internalization of PR9/QD complexes.

<p>(A) Cellular uptake of PR9/QD complexes without or with inhibitors. Cells were treated with PBS as a negative control, QDs alone or PR9/QD complexes in the absence or presence of endocytic inhibitors. Low temperature (4°C), 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)-amiloride (EIPA), cytochalasin D (CytD), filipin and nocodazole inhibit different endocytic pathways. Flow cytometry was used to quantitate transduction efficiency. (B) Effect of the lysosomotropic agent chloroquine on cellular uptake of CPP/QD complexes. Significant differences at <i>P</i><0.05 (*) and <i>P</i><0.01 (**) are indicated. Data are presented as mean ± SD from seven (A) and eleven (B) independent experiments.</p