We have previously characterized the leucine rich NESs locat
We have previously characterized the leucine-rich NESs located within the zinc-binding domains of mucosal high risk HPV16 E7 (76IRTLEDLLM84) and low risk HPV11 E7 (76IRQLQDLLL84) mediating their nuclear export in a CRM1 dependent manner (Knapp et al., 2009, McKee et al., 2013). The zinc-binding domain of cutaneous HPV8 E7 oncoprotein also contains a leucine-rich sequence (76IRTFQELLF84) that has homology with these previously characterized NESs of mucosal HPV E7 proteins. To determine if HPV8 E7 oncoprotein can also undergo nuclear export we used transient transfections in HeLa N1-Methylpseudouridine synthesis with plasmids containing enhanced green fluorescent protein (EGFP) fused to 8cE743–103 and mutants, and Ratjadone A (RJA), a specific nuclear export inhibitor that blocks CRM1-mediated nuclear export of proteins containing leucine-rich NESs. RJA inhibition functions the same way as Leptomycin B (LMB): it covalently modifies a cysteine residue in the NES-binding pocket of CRM1, thereby inhibiting NES-binding (Hutten and Kehlenbach, 2007, Meissner et al., 2004). We analyzed the effect of RJA on the localization of two cysteine mutants, EGFP–8cE7 CC60AA and EGFP–8cE7 C91A, that we have previously shown to have a pancellular localization, in contrast with the nuclear localization of the wild type EGFP–8cE7 (Onder and Moroianu, 2014). As a positive control for RJA, we used EGFP-16E7–NES, which contains a fusion of 16E7 with the strong leucine-rich NES of HIV Rev at its C-terminus (Knapp et al., 2009). Wild type EGFP-16E7 localizes to the nucleus, and addition of the NES of Rev changes its localization to cytoplasmic (Knapp et al., 2009). HeLa cells were transfected with EGFP–8cE7, EGFP–8cE7 CC60AA, EGFP–8cE7 C91A or EGFP-16E7–NES and examined by confocal microscopy 24h post transfection. The RJA treatment was performed after 20h transfection for 4h. Quantitative analysis of five independent experiments showed a change from 89.9±2.62% pancellular localization for EGFP–8cE7 CC60AA mutant in the absence of RJA to 89.6±1.86% nuclear localization in the presence of RJA (Fig. 1A, panels E and G; Fig. 1B). Also, the pancellular localization for EGFP–8cE7 C91A mutant in 88.4±1.98% cells in the absence of RJA changed to 88.9±2.32% nuclear localization in the presence of RJA (Fig. 1A, panels I and K; Fig. 1B). In the presence of RJA inhibitor the nuclear export of CC60AA and C91A mutants is inhibited which allows the accumulation of these mutants (with weakened NLS) in the nucleus at the steady state analyzed in the transfection assays. The nuclear localization of EGFP–8cE7 wild type remained largely unchanged after RJA treatment, with only a small increase in the percentage of cells with nuclear localization (Fig. 1A, panels A and C; Fig. 1B). As expected, the cytoplasmic localization of the EGFP-16E7–NES control changed to nuclear in the majority of transfected cells after RJA treatment (Fig. 1B). These data suggest the presence of a functional NES that is masked by a stronger NLS in the C-terminal domain of cutaneous HPV8 E7 oncoprotein.Similar to HPV16 E7 and HPV11 E7, HPV8 E7 protein has a leucine-rich NES sequence, 76IRTFQELLF84, where the bold characters represent the hydrophobic amino acids that may be critical for the NES function. To characterize this NES sequence and the amino acids that are essential for its proper function, three NES mutants were generated in the context of EGFP–8cE7 C91A cysteine mutant: I76A, F79A and L82A. The C91A mutant was chosen for generating the three nuclear export mutants because it has only a minimal mutation that weakens the NLS of HPV8 cE7 and the RJA export inhibitor rescued its nuclear localization (Figs. 1A and 1B). HeLa cells were transfected with EGFP–8cE7, EGFP–8cE7 C91A, EGFP–8cE7 C91A/I76A, EGFP–8cE7 C91A/F79A, or EGFP–8cE7 C91A/L82A plasmids and the intracellular localizations of the expressed EGFP fusion proteins were analyzed 24h after transient transfections using confocal fluorescence microscopy (Fig. 2). As expected, the majority of cells expressing EGFP–8cE7 showed mostly nuclear localization (Fig. 2, panels A and C, and Fig. 3), whereas EGFP–8cE7 C91A mutant exhibited pancellular localization (Fig. 2, panels D and F, and Fig. 3). Interestingly, the localization of EGFP–8cE7 C91A/I76A, EGFP–8cE7 C91A/F79A, and EGFP–8cE7 C91A/L82A NES mutants was changed to mostly nuclear (Fig. 2, panels G and I; J and L; M and O). Quantitative analysis of four independent experiments showed that all these three NES mutants exhibited predominant nuclear localization in the majority of cells similar as the wild type EGFP–8cE7 expressing-cells (Fig. 3). These data suggest that cutaneous HPV8 E7 has a functional leucine-rich NES within its C-terminal domain and that the hydrophobic amino acid residues, I76, F79 and L82 are essential for its nuclear export function.