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. 2020 Sep 24;11:4845. doi: 10.1038/s41467-020-18542-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Sequence alignment of overlapping NLS-RXL elements in M97 and U69 kinases. Stretches of basic residues characteristic for nuclear localization signals (NLS) are highlighted in blue, conserved leucine and phenylalanine residues participating only in the RXL/Cy motif in pink. A mutation strategy was designed to separate NLS function from cyclin binding. RXL-AXA mutations affect both RXL and NLS sequences, LXF-AXA and LF-AA mutations only the RXL/Cy motif. b HEK-293T cells were transfected with HA-tagged wild-type (WT) or mutant forms of U69 and M97 kinases. Cyclin A co-IP was carried out at 2 days post transfection and analyzed by immunoblotting for the presence of viral kinases and Cyclin A. The immunoblots are representative of three independent experiments with similar results. c Sequence fragments encompassing the NLS-RXL region of U69/M97 mutant and wild-type kinases were cloned between and in-frame with the green fluorescent protein (GFP) and β-galactosidase genes in pHM830. d, e 293T cells were transfected with the M97/U69-NLS reporter constructs, as indicated. The subcellular localization of GFP was analyzed at 24 h post transfection using confocal live-cell imaging microscopy. Nuclei were counterstained with Hoechst-33342. Scale bars: 10 μm. e The indicated number of cells were categorized based on the subcellular localization of the GFP reporter relative to the Hoechst stain.