Fig 2: IL28A expression was activated by ATG10S and ATG10 mutants with Cys44-Cys135 mutation, and mediated autolysosome formation and HCV degradation. (A) Protein levels of the immune factors (IL28A, IRF3, and IRF7) and autophagy flux-related proteins (LC3B, P62, and LAMP2) were analyzed by Western blotting. (B) Transcription levels of IL28A, IRF3, and IRF7 were tested by qPCR. (C) Co-Immunoprecipitation showed the interactions among LAMP2, IL28A, P62, and LC3B using anti-LAMP2 antibody, and the interactions among IL28A with ATG10 mutant proteins and LAMP2 using anti-Flag antibody (labeling the ATG10, ATG10S, and the ATG10 mutants). (D) Immunofluorescence analysis shows co-localization of IL28A with LAMP2 by using anti-IL28A and anti-LAMP2 antibodies and co-localization of ATG10 mutants with IL28A by using anti-Flag and anti-IL28A antibodies in the HCV subreplicon cells. (E) The interactions between autophagosomes and lysosomes were disappeared in IL28A-knockdown cells by immunoprecipitation with anti-LC3B antibody (upper two panels). The replication of HCV subgenomic replicon was restored in IL28A-knockdown cells detected by Western blotting and RT-PCR tests in which HCV CORE and NS5B proteins and core RNA were obviously elevated via IL28A downregulation (lower two panels). Scale bars, 15 µm. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Ctrl; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. the replicon model.

Fig 3: Mechanism of homoplasmy; Integration of IL29 and loss of aadA cassette in plastome of TN72 occurs by two homologous recombination events

Fig 4: Generation and confirmation of IL29 transformants. a The pSRSapI-IL29 plasmid contains IL29 under the control of the psaA exon-1 promoter/5'UTR and rbcL 3'UTR. This construct comprises psbH mutated in the TN72 strain of Chlamydomonas reinhardtii. b PCR confirmation of IL29 integration in pSRSapI expression vector through gene-specific primers. Transformants yield the 564 bp product