Fig 1: HAV depends on functional UFM1 conjugation of the ribosomal protein RPL26 for optimal translation(A) Schematic of cellular UFMylation pathway. #, rank in gene enrichment analysis of CRISPR screen.(B) UFM1 immunoblot of WT, UBA5 KO, UFSP2 KO, UFM1 KO, and UFM1 KO cells complemented with WT, Gly83Ala, or ?C3 UFM1 as well as UBA5 KO cells complemented with WT or catalytically inactive (cat) UBA5. Top bands represent UFM1 conjugation products, and the bottom band represents free UFM1. P84 was used as loading control.(C) Quantification of HAV RNA in WT, UFSP2 KO, UBA5 KO, UFM1 KO, and UFM1 KO cells complemented with WT, Gly83Ala, or ?C3 UFM1. Viral RNA was normalized to 18S RNA, and results are displayed relative to infection in WT cells.(D) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replicon RNA in WT, UBA5 KO, and UFSP2 KO cells.(E) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replication-incompetent replicon RNA in WT, UBA5 KO, and UFSP2 KO cells.(F) Immunoblots of uninfected and HAV-infected WT, UBA5 KO, UFM1 KO, and UFSP2 KO cells. Membranes were stained with anti-UFM1, anti-HAVVP1, or anti-RPL26 antibodies. Vinculin served as loading control.(G) FLAG immunoblot of WT, UFM1 KO, and UFSP2 KO cells transduced with lentivirus expressing FLAG-tagged WT-RPL26 or 5KR-RPL26. Vinculin served as loading control.(H) Quantification of HAV RNA in WT, UFM1 KO, and UFSP2 KO cells expressing WT-RPL26 or 5KR-RPL26. Viral RNA was normalized to 18S RNA and results are displayed relative to infection in WT cells expressing WT-RPL26.For all qRT-PCR experiments, datasets represent mean ± SEM (n = 3 biological replicates). For luciferase experiments, datasets represent mean ± SD (n = 2 biological replicates)for each time point. p values were determined by unpaired two-sided t tests using GraphPad Prism 8. ns, non-significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.