Two conserved amino acids differentiate the biology of high-risk and low-risk HPV E5 proteins.

The high-risk alpha human papillomaviruses (HPVs) are responsible for 99% of cervical cancers. While the biological functions of the HPV E6 and E7 oncoproteins are well-characterized, the function of E5 has remained elusive. Here, we examined gene expression changes induced by E5 proteins from high-risk HPV-16 and low-risk HPV-6b in multiple pools of primary human keratinocytes. Surprisingly, microarray analysis revealed that over 700 genes were significantly regulated by HPV-6b E5, while only 25 genes were consistently and significantly regulated by HPV-16 E5 in three biological replicates. However, we observed that more than thousand genes were altered in individual sample compared with vector. The gene expression profile induced by 16E5 in primary genital keratinocytes was very different from what has been previously published using immortalized HaCaT cells. Genes altered by HPV-16 E5 were unaffected by HPV-6b E5. Our data demonstrate that E5 proteins from the high- and low-risk HPVs have different functions in the HPV-host cell. Interestingly, conversion of two amino acids in HPV-16 E5 to the low-risk HPV-6b sequence eliminated the induction of high-risk related cellular genes.

Quantitative measurement of human papillomavirus type 16 e5 oncoprotein levels in epithelial cell lines by mass spectrometry.

The high-risk human papillomavirus type 16 (HPV-16) E5 protein (16E5) induces tumors in a transgenic mouse model and may contribute to early stages of cervical carcinogenesis. Although high-risk E5 expression is generally thought to be lost during the progression to cervical carcinoma following integration of HPV DNA into the host genome, episomal viral DNA has been documented in a subset of HPV-16-positive malignant lesions. Numerous studies have shown that transcripts that could potentially encode 16E5 are present in cervical biopsy specimens and cervical cancer cell lines, but the presence of E5 protein has been demonstrated in only two reports that have not been corroborated. In the present study, we show that trypsin cleavage of 16E5 generates a unique four-amino-acid C-terminal peptide (FLIT) that serves as a marker for E5 expression in transfected cells and epithelial cell lines containing integrated and episomal HPV-16 DNA. Following trypsin cleavage, reversed-phase chromatography and mass spectrometry (MS) were used to detect FLIT. Immunoprecipitation assays using a newly generated anti-16E5 antibody confirmed that 16E5 was solely responsible for the FLIT signal, and deuterated FLIT peptide provided an internal standard that enabled us to quantify the number of 16E5 molecules per cell. We show that 16E5 is expressed in the Caski but not in the SiHa cervical cancer cell line, suggesting that 16E5 may contribute to the malignant phenotype of some cervical cancers, even in cells exclusively containing an integrated HPV genome.

The HPV-16 E5 protein represses expression of stress pathway genes XBP-1 and COX-2 in genital keratinocytes.

The HPV-16 E5 protein resides in membranes of the endoplasmic reticulum (ER) and modulates cell growth and viral replication. In order to help define its biological activities, we analyzed E5-induced changes in human keratinocyte gene expression. Our studies identified the downregulation of spliced XBP-1 transcripts, a key player in the ER stress response, as a biochemical marker of E5 expression. IRE1alpha, the endoribonuclease responsible for XBP-1 RNA splicing, was also downregulated. Furthermore, cDNA microarray analysis revealed the repression of COX-2, another member of the ER stress pathway. In contrast, these genes were not altered either by the low-risk HPV-6b E5, or a C-terminal HPV-16 E5 mutant, in which the histidine and alanine residues (conserved in high-risk HPVs) were replaced with tyrosine and isoleucine (conserved in low-risk HPVs). HPV-16 E5 was also able to lower COX-2 mRNA levels in cells co-expressing E6/E7, suggesting that it might exert similar activity during viral replication. Interestingly, the E6/E7 genes were independently able to lower COX-2 transcripts compared to vector cells, indicating that multiple pathways of COX-2 repression exist. COX-2 downregulation by E5 could be overcome by thapsigargin or tunicamycin treatments, which initiate ER stress via calcium fluxes and abnormal protein glycosylation respectively, making it unlikely that E5 specifically tempers these pathways. Overall, our data indicate that E5 represses the cellular ER stress response and suggest a potential role for E5 during productive HPV infection.

The human papillomavirus type 16 E5 oncoprotein inhibits epidermal growth factor trafficking independently of endosome acidification.

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.

Membrane orientation of the human papillomavirus type 16 E5 oncoprotein.

The E5 protein of human papillomavirus type 16 is a small, hydrophobic protein that localizes predominantly to membranes of the endoplasmic reticulum (ER). To define the orientation of E5 in these membranes, we employed a differential, detergent permeabilization technique that makes use of the ability of low concentrations of digitonin to selectively permeabilize the plasma membrane and saponin to permeabilize all cellular membranes. We then generated a biologically active E5 protein that was epitope tagged at both its N and C termini and determined the accessibility of these termini to antibodies in the presence and absence of detergents. In both COS cells and human ectocervical cells, the C terminus of E5 was exposed to the cytoplasm, whereas the N terminus was restricted to the lumen of the ER. Finally, the deletion of the E5 third transmembrane domain (and terminal hydrophilic amino acids) resulted in a protein with its C terminus in the ER lumen. Taken together, these topology findings are compatible with a model of E5 being a 3-pass transmembrane protein and with studies demonstrating its C terminus interacting with cytoplasmic proteins.