Fig 2: Effect of RNF121 knockout (KO) and overexpression (OVX) on AAV transduction.A, Western blot of whole cell lysates from Scr and RNF121 KO cell lines. B, Luciferase reporter expression of Scr and RNF121 KO Huh7 cells following transduction with AAV1 (10,000 vg/cell), AAV2 (5,000 vg/cell), AAV6 (10,000 vg/cell), and AAV9 (20,000 vg/cell). C, Luciferase reporter expression of Scr and RNF121 KO in HEK293 and U87 cell lines following transduction with AAV2. D, Luciferase reporter signal with increasing dose of AAV2 on Scr and RNF121 KO Huh7 cells. E, Luciferase reporter signal of Scr and RNF121 KO HEK293 cells following transfection with a control plasmid or plasmid containing RNF121 cDNA. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.

Fig 3: Effect of AAV genome architecture and promoter elements on transduction in RNF121 KO cells.A, GFP expression in Scr and RNF121 KO Huh7 cells transduced with self-complementary (scGFP; 2000 vg/cell) and single-stranded (ssGFP; 10,000 vg/cell) AAV2 vectrors. B, Flow cytometric quantitation of GFP positive cells in the AAV2-scGFP treated group. C, GFP expression in Scr and RNF121 KO Huh7 cells with AAV2 vectors packaging scGFP cassettes driven by Transthyretin (TTR) and human Alpha 1 Antitrypsin (hAAT) promoters. D, Luciferase expression in Scr and RNF121 KO HEK293 cells with AAV2 vectors packaging ssLuciferase cassettes driven by the CMV promoter. E, Transfection of Scr and RNF121 KO HEK293 cells with vector genomic DNA extracted from AAV2 Luciferase preparations, in the presence or absence of AAV8 capsids packaging a Factor IX cassette (100,000 vg/cell). A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.

Fig 4: Effect of RNF121 KO on helper adenovirus infection and early steps in the AAV infectious pathway.A, Flow cytometric analysis and quantification of Scr and RNF121 KO Huh7 cells transduced with recombinant human Ad5-GFP (0.1 vg/cell). B, Luciferase reporter expression of Scr and RNF121 KO Huh7 cells following AAV2 transduction, with or without wild type hAd5 coinfection. C, Binding and D, Uptake of AAV1 and AAV9 capsids by Scr and RNF121 KO Huh7 cells. E, Immunoblot of AAVR expression relative to actin in Scr and RNF121 KO cells. F, Reporter expression of AAV4 (AAVR-independent serotype) in Scr and RNF121 KO cells. G, Quantitation of AAV vector genomes in cytoplasmic and nuclear fractions of Scr and RNF121 KO Huh7 cells following transduction with AAV2. H, Uncoating of vector genomes, quantified by qPCR of DNAse resistant genomes relative to total vector genomes in nuclear extract from Scr and RNF121 KO Huh7 cells. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.

Fig 5: Transcriptomic and proteomic analysis of RNF121 KO cells.A, mRNAs enriched in RNF121 KO, with genes with pAdj<0.05 highlighted in blue. B, Ingenuity pathway analysis of AAV capsid binding partners in RNF121 KO cells showing pathways involved in RNA processes (green), translation (blue), viral infection (lime), DNA damage (purple), and miscellaneous (red). C, U2snRNP inhibitor PladB (20nM) treatment of Scr and RNF121 KO cells. D, DNA-PK inhibitor treatment of Scr and RNF121 KO cells (20uM). E, ATM/ATR Kinase inhibition of Scr and RNF121 KO cells (20uM). F, StringDB interactome of DNA damage and proteasomal machinery in RNF121 KO cells. G, Summary of findings highlighting VCP mediated transcriptional arrest in the absence of RNF121, an essential AAV host factor. A two-tailed unpaired t test was used unless otherwise indicated, with *, p<0.05; **, p<0.01; ***, p<0.005.