Fig 1: Catalytic activity of TRAMP-like complexes is important for HAV translation, independent of regulating viral poly(A) tail length or RNA stability(A) Time course of luciferase activity expressed by electroporated HAV-FLuc subgenomic replicon RNA in WT, DKO and ZCCHC14 KO cells. Datasets represent mean ± SD (n = 2 biological replicates) for each time point.(B) Time course of luciferase activity expressed by electroporated HAV-FLuc replication-incompetent replicon RNA in WT, DKO cells, and ZCCHC14 KO. Datasets represent mean ± SD (n = 2 biological replicates) for each time point.(C) Northern blot of HAV RNA from WT or PAPD5/7 DKO cells, which were electroporated with replication-deficient HAV replicon and harvested at indicated time points post-electroporation. Membrane was stained using an in vitro-transcribed DIG-labeled anti-sense RNA probe targeting nucleotides 7118–7465 of the HAV genome. 18S ribosomal RNA was visualized using SYBR Safe dye on agarose gel prior to transfer onto membrane.(D) Poly(A) tail length assay. Total RNA from infected WT or KO cells was G/I tailed at 3' ends, reverse transcribed and PCR amplified by either genome-specific (GS) forward and GS reverse primers, or GS forward and G/I reverse primer. PCR products were analyzed by polyacrylamide gel electrophoresis. For HBV, total RNA was collected from infected WT or ZCCHC14 KO HepG2-NTCP cells 14 dpi. For HAV, total RNA was harvested from infected WT, DKO, or ZCCHC14 KO Huh7.5.1 cells 3 dpi. Arrow indicates a longer HBV poly(A) tail in WT cells.(E) Length distribution of Sanger-sequenced HAV poly(A) tails. An RNA linker was ligated to 3' ends of rRNA-depleted RNA from infected WT or DKO cells followed by reverse transcription and PCR amplification of the HAV poly(A) tail region. PCR products were TOPO cloned and Sanger sequenced.(F) Analysis of nucleotide composition of sequenced HAV poly(A) tails from (E).(G) Quantification of HAV RNA in WT, PAPD5 KO, and PAPD5 KO cells complemented with either WT or catalytically inactive (cat) PAPD5 cDNA 3 dpi by qRT-PCR. Viral RNA was normalized to 18S RNA, and results are displayed relative to infection in WT cells.Datasets represent mean ± SEM (n = 3 biological replicates). p values were determined by unpaired two-sided t tests using GraphPad Prism 8. ns, non-significant; ***p < 0.001.