Fig 2: Murine CypA has a diminished ability to facilitate HCV replication.(A) An amino acid sequence alignment of CypA from diverse species. Similar amino acids are indicated in boxed, bold capital letters while differences are lowercase. Species are arranged from top to bottom in increasing evolutionary distance from human. For pigtailed macaque, all experiments utilized a TRIM5-CypA fusion – only the residues of the CypA portion of the fusion are depicted here. (B) Huh7.5 cells expressing an shRNA against endogenous human CypA (Huh7.5-shRNA CypA) were transduced to express different CypA orthologs and then infected with a HCV reporter genome expressing secreted Gaussia luciferase (Jc1-Gluc, MOI = 0.1). At five dpi, the luciferase activity of the supernatants was assessed as a proxy for viral replication. CypA rescue efficiency is shown normalized to Huh7.5-shRNA CypA transduced with human CypA, which is 100% identical at the amino acid level to chimpanzee, bonobo, gorilla, olive baboon and rhesus macaque CypA. Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Dunnett’s multiple comparisons test with all means compared to that of the +human CypA line. Chimp/Ch, chimpanzee; Bo, bonobo; Go, gorilla; Orang, orangutan; Rhesus mac/RhM, rhesus macaque; Pt mac, pigtailed macaque; Olive bab/Bab, olive baboon; Sq monkey, squirrel monkey. ****, p<0.0001; ns, not significant.

Fig 3: Characterizing the amino acid basis for the differing efficiencies of murine and human CypA in HCV replication.(A) Schematic depicting the humanized murine CypA and murinized human CypA constructs tested. (B) Modeled structure of human CypA (PDB 1CWA) with the six residues differing between murine and human CypA altered to those of murine CypA and shown labeled. The residues that directly interact with cyclosporine A (CsA) (Arg55, Phe60, Met61, Gln63, Gly72, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122 and His126) (Ke et al., 1994) are depicted in gray as stick models. The six residues that comprise the active site (His54, Arg55, Phe60, Gln111, Phe113, and His126) (Zydowsky et al., 1992), five of which also interact with CsA, are shown in black as stick models. Huh7.5-shRNA CypA cells were transduced with the singly murinized human (C), the singly humanized murine (D) or the triply murinized/humanized (E) CypA mutants, infected with Jc1-Gluc at MOI = 0.1 and supernatants assessed for Gaussia luciferase activity as a proxy for HCV replication at five dpi. The rescue efficiency of each mutant was normalized to Huh7.5-shRNA CypA cells transduced with human CypA. Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Sidak’s multiple comparison test, with all means compared to that of the +human CypA line (C) or the +mouse CypA line (D). For (E), Tukey's multiple comparison test was used to compare all the means to one another. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant.

Fig 4: AIF(370–394) delivery abrogates glutamate-mediated cell death. (a and b) HT-22 cells were transfected with three doses of AIF(370–394) or AIF(343–360) peptides (10 µM, 25 µM, 50 µM) or with an unrelated peptide (NC), used as negative control, at the highest concentration tested (50 µM). Cell viability after glutamate treatment (2 mM for 12 h) was assessed by the MTT assay. As shown, only the presence of AIF(370–394), which binds to CypA, protected cells from the glutamate-induced death in a concentration-dependent manner. Results are presented as percentage of controls considered to be 100% and represent the mean±S.D. of at least four independent experiments performed in quadruplicate. (c) To confirm that apoptosis occurred in glutamate-treated cells and that peptide protection influenced this process, peptide-transfected HT-22 cells were treated with 2 mM glutamate (12 h) or untreated (control) and labeled with Annexin-V-FITC/PI. Finally, they were analyzed by flow cytometry. As shown, the number of apoptotic cells was strongly diminished in the presence of increasing concentrations of AIF(370–394). Percentages reported in the bar graph refer to double-positive stained cells (n=4). (d) HT-22 cells were treated with 2 mM glutamate after transfection with AIF(370–394) or with an unrelated peptide used as negative control (NC). Data show that up to 15 h from the initial glutamate-induced damage, AIF(370–394) afforded protection in a time- and dose-dependent way

Fig 5: CypA regulates glutamate-mediated HT-22 cell death. (a) Western blot analysis of CypA (18 kDa) content in the nuclear and cytosolic fraction of HT-22 cells either untreated or treated with 2 mM glutamate for 6, 8 and 12 h. As shown, CypA levels persistently increased in the nucleus with a peak at 6 h. Lamin A/C (80 kDa) and tubulin (55 kDa) were used as nuclear and cytosolic markers, respectively. (b) Densitometric analysis of the bands obtained 0, 6, 8, and 12 h after glutamate treatment. Bars represent the mean±S.D. of three independent experiments (*P<0.05). (c) Confocal immunofluorescence detection of AIF and CypA in apoptotic HT-22 cells compared with untreated cells showing that the two proteins timely colocalize following the apoptotic insult (see merged image). Scale bar: 5 µm. (d) HT-22 cells were treated with glutamate (Glut, 12–14 h) and stained with DAPI to visualize nuclei. Data are reported as average±S.D. (n=4), ***P<0.001. (e) Co-immunoprecipitation of AIF and CypA in glutamate-treated (2 mM for 12–14 h) and untreated cells. After immunoprecipitation of AIF and immunodetection of AIF or CypA, we found that CypA was captured only in apoptotic cells (upper panel). The panel on the right shows a western blot analysis of the total amount of AIF and CypA, using the tubulin protein as a loading marker