Research On Nuclear Localization Mechanism Of PFV Transactivator Bell

Bell, the transactivator of prototype foamy virus (PFV), plays pivotal role in the life cycle of PFV. Bell bears a nuclear localization signal (NLS), but so far the amino acid sequences are inconclusive. Early studies have shown that residues211to225and/or209to226are necessary and sufficient for Bell nuclear localization. Later studies show that another two basic amino acids R199H200also regulate Bell nuclear localization, which suggests that Bell carries a bipartite NLS consisting of residues199to200and residues211to223.We take this as a starting point to analyze the differences between different research results. In order to define the amino acid sequences which are required for nuclear localization of Bell, we employed an enhanced green fluorescent protein-glutathione S transferase (EGFP-GST) system that has been widely utilized in studying subcellular localization of a number of proteins including retrovirus transactivators. The results of mutagenesis studies have shown that residues R221R222R223are essential for Bell nuclear localization. In contrast, residues R199H200are dispensable, which argues against the involvement of these two basic residues in nuclear localization of Bell. In other words, the NLS of Bell is not bipartite NLS. However, M199(R199H200→S199D200) mutation abrogates the ability of Bell to bind to LTR or IP promoter.To explore the specific sequences of PFV Bell NLS, we continue to conduct in-depth research. We inserted the peptide fragments of Bell into the EGFP-GST fusion protein and investigated their subcellular localization by performing fluorescence microscopy assay. For the first time, we accurately determine the Bell NLS sequences215PRQKRPR221, which accords with the consensus sequence K(K/R)X(K/R) of monopartite NLS. We further find that residues K218, R219, and R221are essential for nuclear localization of Bell by site mutation.After that, we go on to explore the nuclear import mechanism of Bell. The results of in vivo GST-Pulldown show that Bell truncated mutant215-223could interact with KPNA1, KPNA6, and KPNA7, which suggests the key role of KPNA1, KPNA6, and KPNA7in nucleocytoplasmic shuttling pathways of Bell. Finally, we determine that KPNA1, KPNA6, and KPNA7are the adaptor of Bell nuclear import using in vitro nuclear import assay, RNAi, et al.Collectively, in this study, we accurately define the Bell NLS sequences215PRQKRPR221, which is monopartite NLS, for the first time. At the same time, we have determined KPNA1, KPNA6, and KPNA7as the adaptor for Bell nuclear import. Moreover, we have found the key role of R199H200residues for Bell transactivity, that is R199h200residues involve in Bell directly binding to the promoter DNA.