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

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

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

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

Generation of <i>P. falciparum</i> parasites lacking expression of P52.

<p>(A) Illustration of the DNA construct (m144) used for the targeted gene disruption of <i>p52</i> and the <i>p52</i>-genomic locus before and after integration. Shown are the p52 gene and target sequence (amplified using 1624 & 1625), the paralog of p52, p36, and the <i>T. gondii dhfr/ts</i> selection cassette. In addition, primer pairs and restriction sites for diagnostic PCR and Southern analysis are shown (see B and C). hrp – histidine rich protein. (B) Southern analysis of <i>BstN</i>I/<i>SnaB</i>I digested genomic DNA of Wt and <i>Δp52</i> demonstrates correct disruption of <i>p52</i>. DNA was hybridized with a <i>p52</i> specific probe detecting a 3.3 kb fragment in Wt, a 2.2 kb fragment for intact plasmid and the expected fragments of 1.3 kb and a 4.2 kb band (see A) in the two <i>Δp52</i> clones (<i>Δp52-1 and Δp52 -2</i>). (C) PCR analysis of genomic DNA of Wt and <i>Δp52</i> clones and the plasmid DNA (construct) demonstrates correct disruption of <i>p52</i>. Genomic DNA from Wt and <i>Δp52</i> asexual parasites and sporozoites was used as template for the PCR reactions. The Wt specific PCR was performed using primers 1638 and 1676 amplifying a 2.1 kb fragment. PCR primer pairs 1638 and L430, specific for integration of the DNA construct (see A) amplify a 2.0 kb fragment. Primer pairs 190 and 191 amplifying a 1.8 kb fragment from <i>T. gondii dhfr/ts</i> were used as a control.</p

Invasion capacity of Wt and Δ<i>p52</i> sporozoites in primary human hepatocytes <i>in vitro</i>.

<p>(A) Intra (In) and extracellular (Ex) sporozoites 3 hrs after incubation of sporozoites with primary human hepatocytes in culture. Sporozoites were first stained with anti-PfCSP antibodies (red). Then cells were permeabilised and sporozoites were stained with anti-PfCSP antibodies (green). Consequently, extracellular sporozoites will stain red AND green and intracellular sporozoites will stain only green. Nuclei of the hepatocytes (white arrow heads) were stained with DAPI (B) The percentage of intracellular/invaded sporozoites (Wt and Δ<i>p52</i> mutant lines) in primary human hepatocyte 3 hours after sporozoite incubation, as determined in the double anti-CSP staining immuno-fluorescence assay (see A). (C) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 3 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites. (D) The number of schizonts detected by IFA using anti-HSP70 antibodies and the nuclear dye DAPI formed 5 days after incubation with either Wt or Δ<i>p52</i> mutant sporozoites.</p