The identification of a functional
The identification of a functional leucine-rich NES mediating 8E7 nuclear export led us to analyze the interaction between 8E7 and CRM1 nuclear export receptor. HeLa cell lysates containing EGFP-8E7, EGFP–8cE7, EGFP-16E7–NES (as a positive control), or EGFP (as a negative control) were incubated with GST-CRM1 or GST immobilized on glutathione-Sepharose beads. Cell lysates and the eluted bound proteins were analyzed via immunoblotting with an anti-GFP antibody. Both EGFP-8E7 and EGFP–8cE7 bound to GST-CRM1 and not to GST itself (Fig. 4A, lanes 1, 2, 5, 6, 9 and 10), as well as the positive control EGFP-16E7–NES (Fig. 4A, lanes 3, 7 and 11). EGFP did not interact with either GST-CRM1 or GST (Fig. 4A, lanes 4, 8 and 12). These data indicate that HPV8 E7 oncoprotein interacts with CRM1 nuclear export receptor via its zinc-binding domain.
To investigate the role of the leucine-rich NES in the interaction between 8cE7 and CRM1 we performed isolation assays with all three NES mutants in comparison with EGFP–8cE7 and EGFP–8cE7 C91A. We found that both EGFP–8cE7 and EGFP–8cE7 C91A bound to GST-CRM1 but not to GST (Fig. 4B, lanes 1, 2, 7, 8, 13 and 14). Interestingly, all three NES mutants, EGFP–8cE7 C91A/I76A, EGFP–8cE7 C91A/F79A and EGFP–8cE7 C91A/L82A did not interact with GST-CRM1 (Fig. 4B, lanes 3, 4, 5, 9, 10 and 11). As expected, EGFP did not interact with either GST-CRM1 or GST (Fig. 4B, lanes 6, 12 and 18). These results indicate that these NES mutations inhibit the interaction of HPV8 E7 with CRM1 supporting the critical role of the order info amino acids, I76, F79 and L82 for the nuclear export function.
Overall, our results indicate that the cutaneous HPV8 E7 oncoprotein has a leucine rich NES (76IRTFQELLF84) within the zinc-binding domain that interacts with CRM1 export receptor and mediates its nuclear export. We have previously shown that the mucosal low risk HPV11 E7 has a leucine-rich NES (76IRQLQDLLL84) within the zinc-binding domain that interacts with CRM1 (McKee et al., 2013). We also found that the zinc-binding domain of HPV16 E7 containing the leucine-rich NES interacts with CRM1 (data not shown). Another cutaneous beta genus HPV5 E7 protein has also a potential leucine-rich NES (IRAFQQLL) within its zinc-binding domain that interacts with CRM1 export receptor (data not shown). This suggests that both the mucosal and cutaneous HPV E7 oncoproteins have evolved leucine-rich NESs that would mediate their nuclear export via a CRM1 pathway.
Overall, this work and a previous study (Onder and Moroianu, 2014) show that cutaneous HPV8 E7 shuttles between the nucleus and the cytoplasm and localizes predominantly in the nucleus with low levels in the cytoplasm, reflecting the contribution to the HPV8 E7 localization of the NLS and NES, respectively. The mucosal HPV16 E7 and HPV11 E7 oncoproteins also shuttle between the nucleus and the cytoplasm and this is in agreement with the fact that these HPV E7 oncoproteins have cellular targets in both cellular compartments.
Materials and methods
Acknowledgments We thank Dr. Jorgen Kjems for the generous gift of GST-CRM1 plasmid. We thank Paul Boucher for help with purification of GST-fusion proteins. This work was supported by a grant from the National Institutes of Health (R01 CA94898) to Junona Moroianu.
Introduction Ovarian cancer (OC) is one of the leading causes of death from gynecological malignancies , and its incidence has recently increased in China. Over 80% of ovarian cancers are of epithelial origin (EOC), are highly invasive, respond poorly to therapies, and are usually detected at advanced stages, resulting in poor prognoses . Early EOC usually has no obvious symptoms. In general, EOC is diagnosed when it is already in the late or metastatic stages. At present, no sufficiently accurate screening test has been proven effective in the early detection of EOC. An understanding of the biological mechanisms that regulate the progression of EOC is therefore critical for devising new treatment options and improving patient survival.