Fig. 5: Nuclear localization signals as a common pattern of HVR insertions.

a The insertion containing HVRs was analysed for the presence of NLS sequences or predicted nuclear localization by using the tools NLS Mapper (score normalized to 0 to 1). NucPred (Score 0 to 1), NLSTradamus (score 0 or 1) and Prosite (score 0 or 1). b Amino acid sequence of the RPS17 insertion from the Kernow-C1-p6 strain is shown. Alanine scanning mutagenesis was used to disrupt the NLS sequence. The generated constructs are shown in the alignment, Green areas above amino acids indicate codons. c, e The replicon system were used to analyse the impact of NLS mutants on the replication capacity of the Kernow-C1-p6 (p6) strain over time. Kernow-C1-p1 (p1) and p6 (grey triangles) were included as references while constructs of interest are depicted in green. Plotted are mean (+/- SD) relative light units (RLU) of n = 3 biologically independent experiments for construct j, n = 4 for other constructs, normalized to the four-hour value. d, f Respective 96-hour replication values are depicted as a column diagram. g The full-length system was used to validate the impact of the NLS mutants on the virus production. A representative picture of a whole 96-well infected with non-enveloped HEVcc stained for the ORF2 protein (black) is shown above each column. Plotted are the means with standard deviation (+/- SD) of n = 8 biologically independent experiments for p1 and p6, n = 3 for other constructs. h Huh7 cells were transfected with plasmids encoding a triple eYFP alone (control) or in tandem with the HVRs of indicated constructs. The cells were fixed after 16-20 hours and mean fluorescence intensity (MFI) for eYFP was assessed for each compartment. See Supplementary. Movies 4–13. i The eYFP MFI was measured for each compartment for n = 11 cells per construct, individual outliers was removed applying ROUT (Q = 1) method implemented in GraphPad Prism. Depicted is the range of individual data points as violin plots with straight lines as median and dashed lines as quartiles. Source data are provided in Source Data file.