The E5 protein of the human papillomavirus type 16 modulates composition and dynamics of membrane lipids in keratinocytes.

The E5 protein of the human papillomavirus type 16 is a small protein found associated to membranes, mainly in the Golgi apparatus, and expressed in the early stages of viral infection. Its expression modifies the cell response towards growth factors and stress exposures, and also blocks the surface expression of MHC molecules. A global explanation for these multiple effects is hitherto not available. Here we present data showing that the expression of HPV16-E5 increases the amount of free cholesterol readily extractable from the plasma membrane, without altering the total cholesterol content. In addition, HPV16-E5 modifies the composition of the cell membranes, increasing the synthesis rate of phosphatidylcholine and phosphatidylserine, while diminishing that of phosphatidylglycerol. We propose that these changes in the lipid composition of the membrane are the central effect of HPV16-E5 on the cell. The multiple and apparently disconnected effects of HPV16-E5 on tyrosine-kinase receptors, induction of the apoptosis and impairment of MHC trafficking could follow the initial alteration on the membrane composition.

The human papillomavirus type 16 E5 protein modulates ERK1/2 and p38 MAP kinase activation by an EGFR-independent process in stressed human keratinocytes.

The human papillomavirus type 16 E5 gene product has been shown to upregulate the activation of MAP kinases ERK1/2 and cellular proliferation promoted by EGF in a ligand-dependent process. We now report the growth factor-independent modulation of MAP kinases by HPV 16 E5 in human keratinocytes. After treatment with 600 mM sorbitol or low concentrations of anisomycin, E5-expressing cells upregulate the activation of ERK1/2. In addition, E5 enhances p38 activation after anisomycin but not after sorbitol treatment, but it has no effect on MAP kinases activation after shocking the cells with 300 mM sodium chloride. The E5-mediated, sorbitol-dependent increase in ERK1/2 activation is EGF-independent and is only partially inhibited by tyrphostin AG1478, which is known to inhibit specifically EGF receptor activation.

The human papillomavirus type 16 E5 protein modulates phospholipase C-gamma-1 activity and phosphatidyl inositol turnover in mouse fibroblasts.

The human papillomavirus type 16 E5 (HPV16-E5) protein is a membrane protein that has been associated with malignant growth. The protein affects growth factor-mediated signal transduction in a ligand-dependent manner. We show now that E5 expression in A31 fibroblasts results in an increased level of diacylglycerol (DAG) and inositol phosphates. Immunoprecipitation of phospholipase C-gamma-1 (PLC-gamma-1) with specific antibodies and immunoblotting with anti-phosphotyrosine antibodies reveal a large increase in tyrosine phosphorylation of the enzyme in E5-expressing cells compared to control vector-transfected cells. This activation of tyrosine phosphorylation is growth factor independent. In addition, an enhanced formation of phosphatidic acid (PA) was observed in E5 cells. This increase did not result from activation of phospholipase D (PLD), although the enzyme was activatable by treatment with phorbol ester Thus, a phosphohydrolase-mediated DAG synthesis from PLD-produced PA can be excluded. The observed effects were not further enhanced by EGF showing that the presence of the growth factor is not necessary for maintaining permanent activation of PLC-gamma-1 in E5-expressing cells. The DAG- and inositol phosphate-mediated signal cascade within the cells is thus effectively uncoupled from external control via EGF and its receptor in the presence of E5 protein.

The human papillomavirus type 16 E5-protein modulates ligand-dependent activation of the EGF receptor family in the human epithelial cell line HaCaT.

The E5 open reading frame of the human papillomavirus type 16 encodes a transmembrane protein associated with the Golgi, ER, and plasma membranes. We have analyzed the effect of E5 expression on the activation of the EGF receptor family. We find that expression of the E5-protein strongly enhances EGFR activation in a ligand-dependent manner. This activation takes place immediately after addition of ligand, demonstrating that increased tyrosine phosphorylation cannot solely be due to an impaired downregulation of the receptors. Furthermore, this activation is not a result of impaired activity of EGFR-specific phosphatase through the E5-protein, as demonstrated by using inhibitors specifically blocking EGFR activation. In addition, treatment with EGF results in an enhanced activation of the ErbB2 receptor in E5-expressing cells. This superactivation must be a result of heterodimer formation between EGFR and ErbB2, since EGF is not a ligand for ErbB2. Finally, treatment of E5-expressing cells with HB-EGF shows no increased phosphorylation of the ErbB4 receptor, suggesting a specific effect of E5 on the activation of the different members of the EGFR family.

Enhancement of EGF- and PMA-mediated MAP kinase activation in cells expressing the human papillomavirus type 16 E5 protein.

In this report we demonstrate that cells expressing the human papillomavirus type 16 E5 open reading frame (HPV16-E5) show a greatly enhanced transcription of the immediate early genes after EGF or PMA treatment compared to control cells. This enhancement is due to amplification of the signal transduction pathways in response to growth factors or phorbol esters. Upon short-time EGF treatment of the E5-expressing cells we observed an increase in the activation of EGF receptors, resulting in a stronger activation of MAP kinases ERK1/2 compared to control-transfected cells. We also observed that in E5-expressing cells, treatment with PMA results in an increase in membrane-associated PKC activity, and a superactivation of the ERK1/2 MAP kinases. This superactivation is PKC-dependent, since pretreatment of the cells with the PKC inhibitor Ro 31-8220 inhibits MAP kinase activation and early gene transcription almost completely. Furthermore, treatment with genistein strongly reduces the PMA-mediated superactivation of ERK1/2 kinases, demonstrating a PKC-mediated, tyrosine kinase-dependent pathway in the superinduction of MAP kinase activation. Thus, HPV16-E5 effects superactivation of MAP kinases over at least two different pathways, a PKC-mediated, and another, receptor tyrosine-kinase mediated, PKC-independent one.

Human papillomavirus type 16 E5 protein (review).

The association of certain human papillomavirus (HPV) types with malignancies of the anogenital tract is well established. The virus type most frequently associated with cellular transformation is HPV 16, as has been shown in epidemiological studies. Its transforming capacity has also been demonstrated in many in vitro cell transformation experiments. The most potent oncogenes of HPV 16 are the E6 and E7 proteins, but the E5 protein, whose homologue is the main oncogene of bovine papillomavirus, has recently been identified as an oncogene also for HPV. On the basis of epidemiological and clinical data from tumor material as well as from in vitro data it has been suggested, that the HPV 16 E5 protein would have a function at the early stages of cervical carcinogenesis. The E5 protein enhances growth factor-mediated signal transduction to the nucleus and consequently augments cellular proliferation. Expression of the E5 protein enables the infected cell to escape growth control provided by surrounding cells by inhibiting gap junctional intercellular communication in epithelial cells. This viral oncogene seems to interfere with the control mechanisms of cellular growth and proliferation and thus facilitate the function of the E6 and E7 proteins and further steps towards epithelial cell transformation.

Human papillomavirus type 16 E5 protein affects cell-cell communication in an epithelial cell line.

The human papillomavirus type 16 (HPV16) E5 protein is considered to have weak oncogenic properties, and its function in infected human keratinocytes is unknown. HPV16 E5 protein has been found to localize to the Golgi apparatus and the plasma membrane. To analyze the effect of E5 on plasma membrane properties, cells from the human keratinocyte cell line HaCaT were transfected with the HPV16 E5 open reading frame under the control of an inducible promoter. The gap junction-mediated cell-cell communication of E5- and vector-transfected cells was analyzed by microinjection of Lucifer yellow to measure dye coupling of the cells. A strong impairment of dye transfer in E5-transfected cells but not in vector-transfected cells was observed, with more than 80% dye transfer inhibition 40 min after injection. This impairment correlated with dephosphorylation of connexin 43, the major gap junctional protein in HaCaT cells. Furthermore, the dye coupling inhibition was not the result of differentiation of the E5-expressing cells, since no overexpression of cytokeratin 1 or filaggrin, markers of HaCaT cell differentiation, could be observed. These results therefore strongly suggest a correlation between expression of the HPV16 E5 open reading frame, impairment of gap junction-mediated dye coupling, and dephosphorylation of connexin 43.