Fig 2: Characterization of ADAR1 knockdown profile in HaCaT and SiHa cell line. (A) Relative mRNA of ADAR1 measured by quantitative PCR. ADAR1 expression was significantly downregulated in siADAR1, whereas expression level did not change in siNT. (B) Protein expression of innate immune factors involved in sensing of nucleic acids and IFN-I response in siRNA-treated cells. Downregulation of ADAR1p150 correlates with upregulation of RIG-I and MDA5, increased phosphorylation of STAT1 and transcriptional factor IRF7 in HaCaT and SiHa cell line. A representative Western blot is shown. The figure shows the cropped gels/blots obtained by each protein evaluation. Full-length blots of each tested protein are included in supplementary material. (C) mRNA expression of DDX58 in siRNA-treated cells measured by qPCR. DDX58 expression was significantly upregulated in siADAR1, whereas expression level did not change in siNT. (D) Relative mRNA expression of IFIH1 in siRNA-treated HaCaT and SiHa cells measured by quantitative PCR and normalized to GAPDH expression. IFIH1 gene expression was upregulated in siADAR1, whereas expression level in siNT did not change in both cell lines. (E) Relative mRNA expression of IFNB1 in siRNA-treated cells measured by qPCR and normalized to GAPDH expression. IFNB1 gene expression was upregulated in siADAR1, whereas expression level in siNT did not change in any cell line. (F) Relative mRNA expression of CXCL10 in siRNA-treated cells measured by qPCR and normalized to GAPDH expression. CXCL10 gene expression was upregulated in siADAR1, whereas expression level in siNT did not change in both cell lines. (G) Relative mRNA expression of IRF7 in siRNA-treated cells measured by qPCR and normalized to GAPDH expression. IRF7 gene expression was upregulated in siADAR1, whereas expression level in siNT did not change in both cell lines. Left side graphs represent HaCaT and right side SiHa cell line. Data represents mean ± SD of at least 5 independent experiments and is normalized to Mock-transfected SiHa or HaCaT cells. *p < 0.05; **p < 0.005; ***p < 0.0005.

Fig 3: ADAR1 knockdown blocks HIV-1 transcription in primary macrophages. (a) HIV-1 replication in ADAR1 and IFIH1 knockdown M-CSF macrophages, infected with a VSV-pseudotyped, GFP-expressing HIV-1. Infection was measured 72 h later by flow cytometry. Data represent percentage replication relative to mock-transfected macrophages (left panel). A representative flow cytometry dot plot showing infected macrophages is also depicted (right panel). Mean ± SD of at least 5 different donors performed in triplicate is shown. (b) HIV-1 replication in ADAR1 and IFIH1 knockdown M-CSF macrophages, infected with a full replicative HIV-1 BaL strain. Differentiated macrophages were infected for 7 days and CAp24 production was measured in culture supernatant by ELISA. Mean ± SD of 3 different donors performed in triplicate is shown. (c) Proviral DNA formation (left panel), viral integration (middle panel) and viral transcription (right panel) in ADAR1 and IFIH1 knockdown macrophages. siRNA-treated and subsequently differentiated macrophages were infected with HIV-1 BaL for 16 h (for proviral DNA formation), 20 h (for viral DNA integration) or 40 h (for viral transcription) before DNA or RNA extraction. All determinations were normalized to mock-treated infected macrophages and AZT (3 µM) or raltegravir (RAL; 2 µM), were included as controls. Mean ± SD of at least 3 different donors is shown. In all panels, isolated monocytes were transfected with the corresponding siRNA and differentiated to macrophages with M-CSF for 4 days, prior to infection with HIV-1- *p < 0.05; **p < 0.005; ***p < 0.0005.

Fig 4: Putative ADAR1-mediated A-to-I editing sites in HPV16 and NEIL1 transcripts. (A) Schematic representation of designed primers and putative A-to-I editing sites of HPV16 integrated sequence in SiHa cell line. InosinePredict was used to estimate site and number of ADAR1 A-to-I editing sites. (B) Representative DNA sequencing chromatogram of HPV16 E2 putative editing site with no A-to-I editing detected in nucleotide 3160 in SiHa-derived transcripts. HPV16 SiHa transcripts were amplified from RQ-PCR using primers represented in (A), sequenced and aligned siNT and siADAR1 sequences to detect A-to-I editing events. siNT and siADAR1 chromatograms are from the same experiment. (C) Representative DNA sequencing chromatogram of NEIL1 transcripts showing A-to-I editing at known K242 codon in siNT, but not in siADAR1 SiHa transcripts. A-to-I editing function is confirmed in siNT SiHa transcripts. siNT and siADAR1 chromatograms are from the same experiment. (D) Relative ADAR1 editing efficiency in SiHa NEIL1 transcript. Reduced edited peak in siADAR1 was relativized to edited peak in siNT. Edited adenosines to inosines are detected as Gs by direct sequencing. Data represents mean ± SD of at least 3 independent experiments. A representative chromatograms is shown. **p < 0.005.

Fig 5: Representation of signaling cascade regulated by ADAR1 in MDM. ADAR1 modulates the recognition of non-self dsRNA in the cytoplasm; consequently, sensing of non-self dsRNA induces expression of RNA sensors, RIG-I and MDA5, through downstream effectors, MAVS and TBK1, and transcription factors IRF3 and IRF7. Leading to induction of an innate immune response characterized by increase expression of type I IFN and ISG and subsequent over production of cytokines and chemokines able to block HIV-1.