The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification

Abstract

The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa "c" subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A 1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion.High-risk human papillomaviruses (HPVs) are the causative agent of cervical cancer (63) and HPV type 16 (HPV-16) is associated with a majority of cervical malignancies worldwide (13). HPV-16 encodes three oncoproteins: E5, E6, and E7. While the contributions of E6 and E7 to cellular immortalization and transformation have been characterized in detail (20), the role of HPV-16 E5 (16E5) is poorly understood (53). Nevertheless, a number of studies suggest that 16E5 does contribute to the development of cervical cancer. Most high-risk HPV types encode an E5 protein (48), and targeted expression of the three HPV-16 oncogenes in basal epithelial cells of transgenic mice (4) leads to a higher incidence of cervical cancer than does the expression of E6 and E7 alone (44). In addition, targeted epithelial expression of 16E5 (without E6 and E7) in transgenic mice induces skin tumors (21). It may be noteworthy that unlike high-risk HPV-18, which integrates into the host DNA and potentially disrupts E5 gene expression (20, 64), the HPV-16 genome often persists in episomal form in malignant lesions (12,16,24,36,42).Biological activities of 16E5 that may facilitate carcinogenesis include evading host immune detection by interfering with the transport of antigen-pre...

HPV-18 E6 mutants reveal p53 modulation of viral DNA amplification in organotypic cultures

“…For instance, compared with the E1^E4 message ( Table 1, species a), the E5 transcript (Table 1, species b) is relatively minor. This finding is in accordance with our understanding that E5 is a signal transduction regulatory protein (41), whereas E1E4 is an abundant cytoplasmic protein that interacts with cytokeratins (42). Interestingly, the E6 F4L mutant and the WT virus were virtually indistinguishable in abundance of each of the RNA species, whereas the other mutants had significantly reduced RNA levels (Fig.…”

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic

“…The oncogenic role of HPV16 E5 in epithelial transformation and malignant progression has been widely attributed to the ability of this viral protein to interfere with the endocytic pathway of signaling receptors, mainly of EGFR, either indirectly through inhibition of the endosomal acidification (Straight et al, 1995) and endosomal vesicle fusion (Suprynowicz et al, 2010), or directly through protein interactions with the receptor tyrosine kinases (Hwang et al, 1995;Zhang et al, 2005). As mentioned above, results obtained in previous studies from our group (Belleudi et al, 2006(Belleudi et al, , 2007 demonstrate that KGFR endocytosis on binding of the two alternative ligands, KGF or FGF10, is clathrindependent; however, once internalized the receptors follow two different intracellular endocytic pathways: while KGF targets the receptor to the lysosomal pathway, FGF10 induces KGFR recycling to the plasma membrane.…”

“…Alternatively, it may block the sorting from early to late endosomes perturbing the actin cytoskeleton (Thomsen et al, 2000). More recently, however, it has been proposed that 16E5 affects the EGF endocytic trafficking altering the endosome fusion through a pH-independent and cytoskeletonindependent mechanism (Suprynowicz et al, 2010). In addition, it has been also demonstrated that E5 protein is able to form a complex with the EGFR and to enhance its ligand-dependent signaling by disrupting EGFR/c-Cbl interaction and consequently decreasing the ubiquitin-mediated receptor degradation (Zhang et al, 2005).…”

Viroporins: structure, function and potential as antiviral targets

“…E5 induces anchorage-independent growth in culture (Leechanachai et al, 1992) and tumour formation in transgenic mouse models (Maufort et al, 2007), with expression detectable in human malignancies (Cavuslu et al, 1996;Hsieh et al, 2000;Sahab et al, 2012). E5 impairs endosomal maturation, thereby stabilizing epidermal growth factor receptor signalling, and leading to increased extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase activity Genther Williams et al, 2005;Leechanachai et al, 1992;Pedroza-Saavedra et al, 2010;Pim et al, 1992;Rodríguez et al, 2000;Straight et al, 1993;Suprynowicz et al, 2010;Tomakidi et al, 2000). However, understanding of the precise mechanism by which E5 mediates this is incomplete.…”