The A and B helices of CD81 LEL confer CD9/CD81 chimeric molecules the ability to support infection by <i>P. yoelii</i> sporozoites.

<p>A: Amino acid sequence alignment of CD81, CD9, and chimeras. Only the sequence of the LEL is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices is indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined. CD81 LEL residues presumably in contact with the SEL are indicated with an asterisk. Open circles shows residues known to be involved in the interaction with HCV E2 glycoprotein. B and C: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras and infected two days later with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells was determined by immunofluorescence in triplicate wells. Results are expressed as mean±s.d. **, p<0.01 and *, p<0.05 as compared to CD9-transfected cells.</p

A CD81 mAb binds poorly to the non-functional mutant VVD (135–137)→AAA but does not block infection.

<p>A: Hepa 1–6 cells were transfected with the indicated construct in pEGFP-N3 and analyzed for the surface expression and recognition of the transgene by several CD81 mAb using flow-cytometry analysis. Data are expressed as mean fluorescence intensity. In this experiment, the antibodies were used at 20 µg/ml (JS64, M38, JS81) or at 1/100 ascitic fluid dilution (all other mAbs). B: HepG2-A16/CD81 cells were infected with <i>P. yoelii</i> sporozoites in the presence of the indicated mAbs at 25 µg/ml except when otherwise indicated. All mAbs are directed to CD81 except TS9 which is a CD9 mAb and does not inhibit <i>P. yoelii</i> infection.</p

3D structure of CD81 LEL.

<p>The drawing of CD81 LEL (PDB #1g8q) was generated in MolMol. Four helices (A, C, D, E) are drawn in red while the B helix, crucial for <i>P. yoelii</i> infection is displayed in blue. The black balls indicate the CCG ubiquitous motif. The crucial D137 as well as D138 and D139 are in purple while V135 and V136 are in royal blue. Residues V146, T149, F150, T153 and L154 putatively involved in contact with the SEL are indicated in dark blue. T163, F186 and D196 residues, in yellow, have been reported to play a role in the HCV E2 glycoprotein binding to CD81-LEL. Residues V135, V136, T163, F186 and D196 projected backward, behind the drawing plane. The two disulfides bridges are colored light coral. Hydrophilic residues K144, K148 and E152 located on the top of the B helix are in green. The SEL, in cyan, is in front of the drawing plane.</p

The VVD (135–137)→AAA and DDD (137–139)→AAA mutants unable to support infection by <i>P. yoelii</i> sporozoites interact with CD9P-1 and EWI-2.

<p>CHO cells were transiently transfected with WT or mutant CD81 plasmids (in pEGFP-N3), together with a CD9P-1 (top) or a EWI-2 (bottom) cDNA. After 48 h, the cells were lysed with digitonin and immunoprecipitations with antibodies against CD81, CD9P-1 and EWI-2 were performed. After electrophoresis and transfer, the membranes were incubated with biotin-labelled mAbs to CD81 (TS81), CD9P-1 (1F11) and EWI-2 (8A12). The mutants are designed as follows: VVD: VVD (135–137)→AAA; DDD: DDD (137–139)→AAA</p

21 residues of CD81 in a CD9 backbone are sufficient to render hepatocytic cells susceptible to <i>P. yoelii</i> sporozoites infection.

<p>A: Amino acid sequence alignment of CD9, CD81 and chimeras. Only the sequence of the large extracellular loop of the different chimeras is shown. The origin of the flanking domains (TM3 and TM4) is shown on both sides of the sequence. The position of CD81 helices are indicated on the top of the alignment. CD81 residues are shown in red capital letters and CD9 residues in blue small letters. The CCG consensus site and other conserved cysteines, as well as a functionally important site (VVDDD) are underlined B: HepG2-A16 cells were transiently transfected with plasmids expressing CD9, CD81, or CD81/CD9 chimeras and infected two days later with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells in triplicate wells was determined by immunofluorescence. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells. C: HepG2-A16 cells stably expressing CD81, CD9, CD81ccg9 or CD9[81B] were infected with <i>P. yoelii</i> sporozoites. After two days incubation, the number of EEF-infected cells was determined in triplicate wells by immunofluorescence. Results are expressed as mean±s.d. **, p<0.01 as compared to mock-transfected cells.</p

Impact of murine syncytins and myomaker overexpression on myoblast cell-cell fusion <i>ex vivo</i>.

<p>C2C12 myoblasts were co-transfected with an empty (cont), syncytin-A (synA),–B (synB) or myomaker expression vector, supplemented with a GFP expression vector. Differentiation was induced 2 days later (D0). Cells were fixed at D0 or after four days of differentiation (D4) in 4% PFA, stained with an anti-desmin antibody (muscle cell marker), and nuclei were counterstained with DAPI. (<i>A</i>) The fusion index at D0 was calculated as the percentage of nuclei in GFP-positive cells with at least 2 nuclei. (<i>B</i>) The fusion index at D4 of differentiation was calculated as the percentage of nuclei in desmin-positive cells with at least 2 nuclei. (<i>C</i>) Representative images of desmin- and DAPI-labelled cells transfected with the control, syncytin-A, syncytin-B or myomaker vectors at D4. Data are the mean ± SEM (three independent experiments; * p<0.05, ** p<0.01, Student’s t-test).</p

Myonuclei number in 12 week old SynB^-/- mice.

<p>(<i>A</i>) Quantification of the myonuclei number per 100 fibers in SOL, EDL and TA muscles of WT and SynB^-/- male and female mice. The number of myonuclei was estimated by analyzing at least 300 myofibers for each condition. Data are the mean ± SEM (4–6 mice analyzed per sex and per genotype; * p<0.05, ** p<0.01, Mann and Whitney test). (<i>B</i>) Nuclei corresponding to myonuclei (white arrow) are located within the myofibers. Nuclei outside myofibers (white asterisk), including those from satellite cells, were not counted.</p

Role of <i>syncytins</i> in myoblasts from human, sheep and dog.

<p>Left panels: level of expression of the human <i>syncytin-1</i> and <i>-2</i> (<i>A</i>), the ruminant <i>syncytin-Rum1</i> (<i>B</i>) and the carnivore <i>syncytin-Car1</i> (<i>C</i>) genes (arbitrary units, A. U.) during proliferation and differentiation of primary myoblasts from human, sheep and dog, as determined by quantitative RT-PCR and normalized by the beta2-microglobulin, SDHA and PPIA housekeeping genes, respectively; the fold-change in gene expression was expressed relative to the values of proliferating myoblasts, arbitrarily settled to one. Middle and right panels: impact of siRNA-mediated knockdown of <i>syncytins</i> on <i>ex vivo</i> cell-cell fusion of myoblasts from human, sheep and dog, respectively. Cells were transfected with siRNA (control or specifically designed against the corresponding <i>syncytins</i>), and differentiation induced 2 days later. Following 2 days of differentiation, cells were fixed in 4% PFA, stained with an anti-desmin antibody (muscle cell marker), and nuclei were counterstained with DAPI. The fusion index (number of nuclei in cells with >2 nuclei per total number of nuclei) was determined (middle panels). Representative images of desmin-immunolabeled and DAPI-stained myoblasts transfected with either control or <i>syncytin</i>-specific siRNAs, respectively, are shown (right panels). Data are the mean ± SEM (at least three independent experiments; * p<0.05, ** p<0.01, Student’s t-test).</p

Phenotype of 12 week old SynB^-/- and WT mice.

<p>(<i>A</i>) Body weight. (<i>B</i>) Representative WT and SynB^-/- male (m) and female (f) mice. (<i>C</i>) Muscle mass of three skeletal muscles located in the hindlimb, TA (<i>Tibialis Anterior</i>), SOL (<i>Soleus</i>), EDL (<i>Extensor Digitorum Longus</i>). (<i>D</i>) Mass of heart and kidney, length of tibia and femur. Data are the mean ± SEM (number of mice per sex and per genotype: 13–17 in <i>A</i>, 5–11 in <i>C</i>, 4–7 in <i>D</i>; * p<0.05, ** p<0.01, Mann and Whitney test).</p

Murine syncytin expression and impact of their knockdown on myoblast cell-cell fusion <i>ex vivo</i>.

<p>Primary myoblast cells were transfected with control siRNA (si cont) or siRNAs specifically designed against <i>syncytin-B</i> (si synB),–<i>A</i> (si synA) or myomaker (si myomaker), and differentiation was induced 2 days later. Following two days of differentiation, cells were fixed in 4% PFA, stained with an anti-desmin antibody (muscle cell marker), and nuclei were counterstained with DAPI. (<i>A</i>) Level of expression of <i>syncytin-B</i> and <i>-A</i> (arbitrary units, A. U.) during myoblast fusion in murine primary myoblasts, either actively proliferating (P: proliferating non confluent cells, D0: proliferative confluent cells), or in differentiation and fusion (D2 to D4), as analyzed by quantitative RT-PCR. All quantifications were normalized by RPL0, and the fold-change in gene expression expressed relative to the values of proliferating myoblasts, arbitrarily settled to one. (<i>B</i>) Efficiency of siRNA knockdown measured by quantitative RT-PCR. (<i>C</i>) Representative images of desmin- and DAPI-labelled myoblast cells transfected with control, <i>syncytin-B</i> or <i>syncytin-A</i> siRNAs. Representative myotubes are indicated by white arrows whereas white triangles correspond to mononucleated myoblast cells. (<i>D</i>) Fusion index (number of nuclei in cells with ≥2 nuclei per total number of nuclei). (<i>E</i>) Nuclei distribution per myotube. Data are the mean ± SEM (four independent experiments; * p<0.05, ** p<0.01, Mann and Whitney test).</p