Koilocytosis
2008; Elsevier BV; Volume: 173; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.080280
ISSN1525-2191
AutoresEwa Krawczyk, Frank A. Suprynowicz, Xuefeng Liu, Yuhai Dai, Dan P. Hartmann, John A. Hanover, Richard Schlegel,
Tópico(s)Leprosy Research and Treatment
ResumoA long-recognized, pathognomonic feature of human papillomavirus (HPV) infection is the appearance of halo or koilocytotic cells in the differentiated layers of the squamous epithelium. These koilocytes are squamous epithelial cells that contain an acentric, hyperchromatic nucleus that is displaced by a large perinuclear vacuole. However, the genesis of the cytoplasmic vacuole has remained unclear, particularly because both HPV DNA replication and virion assembly occur exclusively in the nucleus. In clinical biopsies, koilocytosis is observed in both low- and high-risk HPV infections; therefore, in this study, we demonstrated that the E5 and E6 proteins from both low- and high-risk HPVs cooperate to induce koilocyte formation in human cervical cells in vitro, using both stable and transient assays. Both E5 and E6 also induce koilocytosis in human foreskin keratinocytes but not in primate COS cells. Deletion of the 20 C-terminal amino acids of E5 completely abrogates koilocytosis, whereas a 10-amino acid-deletion mutant retains ∼50% of its activity. Because the E6 protein from both the low- and high-risk HPVs is capable of potentiating koilocytosis with E5, it is apparent that the targeting of both p53 and PDZ proteins by E6 is not involved. Our data suggest new, cooperative functions for both the E5 and E6 proteins, hinting at additional targets and roles for these oncoproteins in the viral life cycle. A long-recognized, pathognomonic feature of human papillomavirus (HPV) infection is the appearance of halo or koilocytotic cells in the differentiated layers of the squamous epithelium. These koilocytes are squamous epithelial cells that contain an acentric, hyperchromatic nucleus that is displaced by a large perinuclear vacuole. However, the genesis of the cytoplasmic vacuole has remained unclear, particularly because both HPV DNA replication and virion assembly occur exclusively in the nucleus. In clinical biopsies, koilocytosis is observed in both low- and high-risk HPV infections; therefore, in this study, we demonstrated that the E5 and E6 proteins from both low- and high-risk HPVs cooperate to induce koilocyte formation in human cervical cells in vitro, using both stable and transient assays. Both E5 and E6 also induce koilocytosis in human foreskin keratinocytes but not in primate COS cells. Deletion of the 20 C-terminal amino acids of E5 completely abrogates koilocytosis, whereas a 10-amino acid-deletion mutant retains ∼50% of its activity. Because the E6 protein from both the low- and high-risk HPVs is capable of potentiating koilocytosis with E5, it is apparent that the targeting of both p53 and PDZ proteins by E6 is not involved. Our data suggest new, cooperative functions for both the E5 and E6 proteins, hinting at additional targets and roles for these oncoproteins in the viral life cycle. Epidemiological studies have shown that human papillomavirus (HPV) infection is the most common sexually transmitted disease, with nearly 80% of the female population being infected during their lifetime. More than 120 different HPV types have been isolated, of which more than 40 infect the genital mucosa.1Trottier H Franco EL The epidemiology of genital human papillomavirus infection.Vaccine. 2006; 24: 4-15Crossref Scopus (491) Google Scholar Some of these genital HPVs are low risk (eg, HPV-6 and -11) and are associated mainly with nonmalignant lesions. However, there are 12 to 15 high-risk HPVs that play a causative role in the development of cervical cancer and represent a major health threat worldwide.1Trottier H Franco EL The epidemiology of genital human papillomavirus infection.Vaccine. 2006; 24: 4-15Crossref Scopus (491) Google Scholar, 2Walboomers JM Jacobs MV Manos MM Bosch FX Kummer JA Shah KV Snijders PJF Peto J Meijer CJLM Munoz N Human papillomavirus is a necessary cause of invasive cervical cancer worldwide.J Pathol. 1999; 189: 12-19Crossref PubMed Scopus (6990) Google Scholar, 3zur Hausen H de Villiers E-M Human papillomaviruses.Annu Rev Microbiol. 1994; 48: 427-447Crossref PubMed Scopus (476) Google Scholar, 4zur Hausen H Papillomaviruses and cancer: from basic studies to clinical application.Nat Rev. 2002; 2: 342-350Crossref Scopus (3096) Google Scholar HPV-16 alone accounts for approximately half of all cervical cancers.3zur Hausen H de Villiers E-M Human papillomaviruses.Annu Rev Microbiol. 1994; 48: 427-447Crossref PubMed Scopus (476) Google Scholar, 5Bosch FX de Sanjosé S Chapter 1: human papillomavirus and cervical cancer—burden and assessment of causality.J Natl Cancer Inst Monogr. 2003; 31: 3-13Crossref PubMed Scopus (729) Google Scholar, 6Baseman JG Koutsky LA The epidemiology of human papillomavirus infections.J Clin Virol. 2005; 32: 16-24Abstract Full Text Full Text PDF Scopus (697) Google Scholar Three HPV-16 genes possess transforming activity: E6, E7, and E5. The HPV-16 E6 gene encodes a 151-amino acid protein (16E6) that binds to the tumor suppressor protein, p53, and facilitates its degradation via ubiquitin-dependent pathways. This, together with inhibition of the pro-apoptotic protein, BAK, mediates resistance to apoptosis and chromosomal instability. 16E6 also transactivates the hTERT promoter, thereby increasing cellular telomerase activity and, in cooperation with the HPV-16 E7 protein (16E7), induces cellular immortalization.4zur Hausen H Papillomaviruses and cancer: from basic studies to clinical application.Nat Rev. 2002; 2: 342-350Crossref Scopus (3096) Google Scholar, 7Fehrmann F Laimins LA Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation.Oncogene. 2003; 22: 5201-5207Crossref PubMed Scopus (207) Google Scholar, 8Longworth MS Laimins LA Pathogenesis of human papillomaviruses in differentiating epithelia.Microbiol Mol Biol Rev. 2004; 68: 362-372Crossref PubMed Scopus (486) Google Scholar One of the primary functions of 16E7 is to inactivate the retinoblastoma tumor suppressor protein.7Fehrmann F Laimins LA Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation.Oncogene. 2003; 22: 5201-5207Crossref PubMed Scopus (207) Google Scholar, 8Longworth MS Laimins LA Pathogenesis of human papillomaviruses in differentiating epithelia.Microbiol Mol Biol Rev. 2004; 68: 362-372Crossref PubMed Scopus (486) Google Scholar In contrast to 16E6 and 16E7, biological functions of the HPV-16 E5 protein (16E5) are rather poorly understood.7Fehrmann F Laimins LA Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation.Oncogene. 2003; 22: 5201-5207Crossref PubMed Scopus (207) Google Scholar 16E5 is a small (83 amino acids), hydrophobic protein that localizes to membranes of the endoplasmic reticulum.9Conrad M Bubb VJ Schlegel R The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.J Virol. 1993; 67: 6170-6178Crossref PubMed Google Scholar, 10Disbrow GL Sunitha I Baker CC Hanover J Schlegel R Codon optimization of the HPV-16 E5 gene enhances protein expression.Virology. 2003; 311: 105-114Crossref PubMed Scopus (71) Google Scholar It exhibits weak transforming activity in vitro,7Fehrmann F Laimins LA Human papillomaviruses: targeting differentiating epithelial cells for malignant transformation.Oncogene. 2003; 22: 5201-5207Crossref PubMed Scopus (207) Google Scholar, 11Suprynowicz FA Campo MS Schlegel R Biological Activities of Papillomavirus E5 Protein.in: Campo MS Norfolk, Caister Academic Press, 2006: 97-113Google Scholar but induces epithelial tumors in transgenic mice.12Genther Williams SM Disbrow GL Schlegel R Lee D Threadgill DW Lambert PF Requirement of epidermal growth factor receptor for hyperplasia induced by E5, a high-risk human papillomavirus oncogene.Cancer Res. 2005; 65: 6534-6542Crossref PubMed Scopus (113) Google Scholar Documented intracellular binding targets for 16E5 include the 16-kDa subunit of the vacuolar H+-ATPase,9Conrad M Bubb VJ Schlegel R The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.J Virol. 1993; 67: 6170-6178Crossref PubMed Google Scholar, 13Rodríguez MI Finbow ME Alonso A Binding of human papillomavirus 16 E5 to the 16kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation.Oncogene. 2000; 19: 3727-3732Crossref PubMed Scopus (58) Google Scholar the heavy chain of HLA type I,14Ashrafi GH Haghshenas M Marchetti B Campo MS E5 protein of human papillomavirus 16 downregulates HLA class I and interacts with the heavy chain via its first hydrophobic domain.Int J Cancer. 2006; 119: 2105-2112Crossref PubMed Scopus (94) Google Scholar and ErbB4, a member of the epidermal growth factor receptor family.15Chen SL Lin ST Tsai TC Hsiao WC Tsao YP ErbB4 (JM-b/CYT-1)-induced expression and phosphorylation of c-Jun is abrogated by human papillomavirus type 16 E5 protein.Oncogene. 2006; 26: 42-53Crossref PubMed Scopus (30) Google Scholar A common feature of both high- and low-risk papillomavirus infection is the appearance of koilocytes in the differentiated layers of squamous epithelium. Koilocytes are epithelial cells that contain an acentric, hyperchromatic, moderately enlarged nucleus that is displaced by a large perinuclear vacuole.16Hajdu SI A note from history: the link between koilocytes and human papillomaviruses.Ann Clin Lab Sci. 2006; 36: 485-487PubMed Google Scholar Although molecular methods are available for detecting papillomavirus infections of the female genital tract, the presence of koilocytes in Pap smears and cervical biopsies remains fundamental to pathological diagnosis.17Yamamoto LS Alves VA Maeda MY Longatto-Filho A Utagawa ML Eluf Neto J A morphological protocol and guide-list on uterine cervix cytology associated to papillomavirus infection.Rev Inst Med Trop Sao Paulo. 2004; 46: 189-193Crossref PubMed Google Scholar The viral genesis of koilocytes is somewhat enigmatic because most HPV proteins are localized in the nucleus where viral replication and assembly occur. In this article, we demonstrate that E5 from high-risk and low-risk HPV types cooperates with high- and low-risk E6 to induce koilocytosis in vitro. Retroviruses encoding 16E6 and 16E7 in the vector pBabePuro,18Morgenstern JP Land H Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1895) Google Scholar or encoding codon-optimized 16E5 or a C-terminal deletion mutant of codon-optimized 16E5 lacking the last 10 amino acids [16E5 (−10)],19Disbrow GL Hanover JA Schlegel R Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH.J Virol. 2005; 79: 5839-5846Crossref PubMed Scopus (68) Google Scholar 16E6, HPV-6b E5 (6bE5), and HPV-6b E6 (6bE6) in the vector pLXSN, were generated using the Phoenix cell system.20Pear WS Nolan GP Scott ML Baltimore D Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci USA. 1993; 90: 8392-8396Crossref PubMed Scopus (2291) Google Scholar Both 16E5 and 6bE5 proteins were N-terminally tagged with the AU1 epitope, DTYRYI.21Lim PS Jenson AB Cowsert L Nakai Y Lim LY Jin XW Sundberg JP Distribution and specific identification of papillomavirus major capsid protein epitopes by immunocytochemistry and epitope scanning of synthetic peptides.J Infect Dis. 1990; 162: 1263-1269Crossref PubMed Scopus (72) Google Scholar AU1 epitope-tagged or untagged wild-type E6 constructs have been described before.22Liu X Yuan H Fu B Disbrow GL Apolinario T Tomaic V Kelley ML Baker CC Huibregtse J Schlegel R The E6AP ubiquitin ligase is required for transactivation of the hTERT promoter by the human papillomavirus E6 oncoprotein.J Biol Chem. 2005; 280: 10807-10816Crossref PubMed Scopus (97) Google Scholar To construct E6 mutants, overlapping polymerase chain reaction was used to replace cysteine 136 of both 6bE6 and 16E6 with glycine; primers containing the mutant codon are as follows: 6bE6C136G mutant, 5′-TGTACGTGGAAGGGTCGCGGCCTACACTGCTGGACAAC-3′ and 5′-GTTGTCCAGCAGTGTAGGCCGCGACCCTTCCACGTACA-3′; 16E6C136G mutant, 5′-GGTCGGTGGACCGGTCGAGGTATGTCTTGTTGCAGATC-3′ and 5′-GATCTGCAACAAGACATACCTCGACCGGTCCACCGACC-3′. All constructs were confirmed by sequencing. Primary human ectocervical cells (HECs) were derived from cervical tissue after hysterectomy for benign uterine disease as described,23Baege AC Berger A Schlegel R Veldman T Schlegel R Cervical epithelial cells transduced with the papillomavirus E6/E7 oncogenes maintain stable levels of oncoprotein expression but exhibit progressive, major increases in hTERT gene expression and telomerase activity.Am J Pathol. 2002; 160: 1251-1257Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar and were immortalized by infection with a 16E6/16E7-encoding retrovirus and selection in the presence of puromycin (0.5 μg/ml). Stable 16E5-expressing cell lines were generated from immortalized HECs by infection with retroviruses encoding 16E5, 16E5 (−10) or the empty pLXSN expression vector, and selection in the presence of geneticin G418 (100 μg/ml). Nonimmortalized HECs expressing 16E6 or 16E7 alone were generated by infection with E6- or E7-encoding retroviruses and selection. HEC lines and HECs were grown at 37°C and 5% CO2 in keratinocyte growth medium (Invitrogen, Carlsbad, CA), supplemented with gentamicin sulfate (10 μg/ml). To maintain constant levels of 16E5 and 16E5 (−10) expression, G418 was administered to cell cultures at alternating passages. Human foreskin keratinocytes (HFKs) were isolated from neonatal foreskins as described previously and cultured in keratinocyte growth medium.24Schlegel R Phelps WC Zhang YL Barbosa M Quantitative keratinocyte assay detects two biological activities of human papillomavirus DNA and identifies viral types associated with cervical carcinoma.EMBO J. 1988; 7: 3181-3187Crossref PubMed Scopus (193) Google Scholar HFK cell strains were generated by infection with retroviruses encoding 16E6 and/or 16E7, or the empty pLXSN expression vector. COS cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 U/ml penicillin G, and 100 μg/ml streptomycin sulfate (Invitrogen). HECs and HEC lines, HFKs, and COS cells were grown as described in 25-cm2 or 75-cm2 tissue culture flasks. Before infection, the growth medium was aspirated and retroviruses mixed with polybrene (1 μl/ml) were added to the cells for 1.5 hours at 37°C while shaking. After infection, this mixture was removed and growth medium added to the cells. AU1 epitope-tagged E5 proteins were detected in RIPA lysates by immunoprecipitation and Western blotting,25Suprynowicz FA Disbrow GL Simic V Schlegel R Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?.Virology. 2005; 332: 102-113Crossref PubMed Scopus (27) Google Scholar using 4 μg of rabbit anti-AU1 polyclonal antibody for immunoprecipitation, and 1:5000-diluted rabbit anti-AU1 for immunoblotting (Covance, Princeton, NJ). Additional Western blots were labeled with 1:10,000-diluted anti-β-actin mouse monoclonal antibody (Sigma, St. Louis, MO), anti-p53 (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-pRb (1:1000 dilution; Cell Signaling, Beverly, MA), as described.26Suprynowicz FA Sparkowski J Baege A Schlegel R E5 oncoprotein mutants activate phosphoinositide 3-kinase independently of platelet-derived growth factor receptor activation.J Biol Chem. 2000; 275: 5111-5119Crossref PubMed Scopus (28) Google Scholar, 27Suprynowicz FA Baege A Sunitha I Schlegel R c-Src activation by the E5 oncoprotein enables transformation independently of PDGF receptor activation.Oncogene. 2002; 21: 1695-1706Crossref PubMed Scopus (21) Google Scholar HECs and HFKs were grown on four-chamber microscope slides in keratinocyte growth medium, fixed in 4% (w/v) paraformaldehyde, and stained with hematoxylin and eosin (H&E) according to standard histological procedures. Alternatively, HECs were fixed, permeabilized in 0.1% (w/v) saponin, and blocked with 10% normal goat serum before staining with hematoxylin and labeling with Covance anti-AU1 mouse monoclonal antibody (1:1500 dilution of ascites) and goat anti-mouse IgG (peroxidase conjugate; DakoCytomation, Glostrup, Denmark). A BH-2 microscope (Olympus, Center Valley, PA) was used for visualization and microphotography, and an Axioskop microscope (Zeiss, Thornwood, NY) was used for counting cells. While investigating the effect of 16E5 on plasma membrane ganglioside content,28Suprynowicz FA Disbrow GL Krawczyk E Simic V Lantzky K Schlegel R HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells.Oncogene. 2008; 27: 1071-1078Crossref PubMed Scopus (68) Google Scholar we observed that a subpopulation of E5-expressing, immortalized HECs displayed the unique features of koilocytosis. As mentioned previously, koilocytosis is a diagnostic marker for papillomavirus infection and can be observed morphologically in both histological sections of HPV-infected cervix and in Pap smears. For example, in cervical biopsies, koilocytes can be detected in the differentiated layers of HPV-infected squamous epithelium (Figure 1A, left). In Pap smears, cytoplasmic vacuolization (Figure 1A, middle) is even more prominently displayed. We found morphologically similar, perinuclear vacuolization in cultured monolayers of 16E6/16E7-immortalized HECs expressing 16E5 (Figure 1A, right). This suggested that the phenomenon of koilocytosis might be associated with the expression of a specific subset of HPV-16 oncoproteins. We therefore fixed and stained 16E6/16E7-immortalized HEC lines (with and without the 16E5 gene) with H&E. Koilocytes were readily detected in the 16E5-expressing HEC lines compared to cells harboring the empty pLXSN expression vector (Figure 1B). Importantly, immunohistochemistry of these HEC lines (using an anti-AU1 antibody to detect the epitope-tagged E5 protein) revealed that 16E5 was present in the koilocytotic vacuolar membranes (Figure 2, bottom row). 16E5 was also noted in the cytoplasm, which we believe reflects its endoplasmic reticulum localization that is visualized more precisely by immunofluorescence microscopy.28Suprynowicz FA Disbrow GL Krawczyk E Simic V Lantzky K Schlegel R HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells.Oncogene. 2008; 27: 1071-1078Crossref PubMed Scopus (68) Google ScholarFigure 216E5 localizes to perinuclear vesicles in koilocytotic cells. HECs were fixed, stained with hematoxylin, and reacted with peroxidase-conjugated anti-AU1 antibody (brown). Scale bar = 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above studies demonstrated that the stable expression of 16E5 induces koilocytosis in HECs immortalized with 16E6 and 16E7. Although E5 clearly is essential for this phenomenon, it was unclear whether 16E5 alone is sufficient to induce koilocytosis or whether there might be collaborative interactions with 16E6 and 16E7. To test this possibility, we developed an acute assay for koilocytosis in which various cervical cells are infected with retroviruses that encode wild-type (wt) or mutant 16E5 oncoproteins. As shown in Figure 3A, expression of HPV-16 E6, E7, or E5 alone was insufficient to generate koilocytes in HECs. In addition, the combined expression of 16E6 and 16E7 was ineffective; similar to the results obtained with immortalized pLXSN-HECs. However, co-expression of 16E5 and 16E6 resulted in levels of koilocytes that were nearly 10-fold higher than the 16E5/E6/E7 HEC line. It is also important to note that the AU1 epitope tag on 16E5 had no effect on its frequency of koilocyte formation. 16E5 and 16E5 (no AU1) induced very similar levels of koilocytosis (Figure 3A). This cooperative effect was not observed with 16E5 and 16E7. In fact, 16E7 dramatically suppressed koilocytosis when co-expressed with 16E5 and 16E6 (Figure 3A). Koilocytes were reduced by ∼50% in 16E6-HECs expressing a mutant 16E5 lacking the C-terminal 10 amino acids, 16E5 (−10). This mutant is also defective for endosome alkalinization19Disbrow GL Hanover JA Schlegel R Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH.J Virol. 2005; 79: 5839-5846Crossref PubMed Scopus (68) Google Scholar and epidermal growth factor receptor activation.13Rodríguez MI Finbow ME Alonso A Binding of human papillomavirus 16 E5 to the 16kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation.Oncogene. 2000; 19: 3727-3732Crossref PubMed Scopus (58) Google Scholar Importantly, deletion of the 20 C-terminal amino acids produced mutant 16E5 (−20) that was completely defective for koilocyte formation (Figure 3A). This suggests the existence of additional E5 targets that are separate from those regulating V-ATPase function and epidermal growth factor receptor signaling. Because low-risk HPVs also induce koilocytosis in vivo,29Sjö NC Heegaard S Prause JU von Buchwald C Lindeberg H Human papillomavirus in conjunctival papilloma.Br J Ophthalmol. 2001; 85: 785-787Crossref PubMed Scopus (84) Google Scholar we used the acute transduction assay (with various HECs) to screen the biological activity of E5 from low-risk HPV-6b (6bE5). As with high-risk 16E5, we observed koilocytes when 6bE5 was co-expressed with 16E6 (Figure 3B). It should be noted that wild-type and mutant E5 proteins were expressed at similar levels in 16E6-HECs after acute retrovirus infection (Figure 3C). Thus, the decreased koilocyte-inducing activity of 16E5 (−10) and the complete defectiveness of 16E5 (−20) are not because of lower expression of these proteins but rather to altered activity. In addition, 16E6 and 16E7 are functional in these cells, because 16E6 degrades p53 and 16E7 triggers the loss of pRb (Figure 3D). To evaluate the ability of various combinations of low- and high-risk oncoproteins to induce koilocytosis, we used a modified acute assay. Rather than infecting 16E6-HECs with different E5 proteins, we co-infected primary HECs with two different retroviruses (encoding either E5 or E6) and screened for koilocytes (Figure 4A). This modified assay generated koilocytosis with approximately half the efficiency of the previous assay but now permitted us to assay both E6 as well as E5 mutant proteins. Interestingly, all combinations of low- and high-risk E5 and E6 proteins collaborated to induce koilocytes, suggesting the retention of conserved functions in these variant and biologically distinct proteins. In addition, we showed that addition of the AU1 epitope to the HPV-16 E6 protein did not alter its ability to induce koilocytes (Figure 4A, compare lanes 1 and 2). Addition of the AU1 epitope to the HPV-6b E6 protein induced a slight (50%) decrease in its activity. We also demonstrated the requirement for E6 in this assay by measuring the activity of a mutant in which a conserved C-terminal cysteine residue (amino acid 136) was changed to glycine. Mutations at this site have been shown to abrogate many E6 activities, presumably because of gross alterations in protein conformation.30Kanda T Watanabe S Zanma S Sato H Furuno A Yoshiike K Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif.Virology. 1991; 185: 536-543Crossref PubMed Scopus (52) Google Scholar In both the 6b and 16 E6 proteins, mutation of the terminal cysteine residue abrogated the induction of koilocytosis. To verify that the defective nature of the E6 cysteine mutants was not because of decreased protein expression, we examined the levels wild-type and mutant E6 proteins by Western blotting (Figure 4B). The cysteine mutants of 16E6 and 6bE6 were present at levels similar to the respective wild-type proteins (Figure 4B, compare lanes 2 with 3 and lanes 4 with 5). Although koilocytes are present in cervical HPV infections, they also are observed at other anatomical sites in different epithelial cell types. To investigate whether koilocytosis can be induced in another HPV target cell, the HFK, we transduced these cells with combinations of 16E6, 16E7 and empty pLXSN expression vector followed by 16E5 (Figure 5A). Koilocytosis was induced in the HFKs, but only when both 16E5 and 16E6 were present (Figure 5, A and B). As in HECs, 16E7 greatly suppressed the frequency of koilocytosis induced by E5 and E6. In contrast to HECs and HFKs, koilocytes did not form in COS cells (a primate kidney cell line) when 16E5 and 16E6 were co-expressed, indicating that not all cell lines provide suitable environments for koilocytosis. In contrast to the well-characterized E6 and E7 oncoproteins of HPV-16, biological activities of the 16E5 oncoprotein and its role in the HPV replication cycle are not well understood. The biological phenotypes of E5 are myriad and vary with the cell types used for assays. Briefly, E5 has been shown to alkalinize endosome pH,19Disbrow GL Hanover JA Schlegel R Endoplasmic reticulum-localized human papillomavirus type 16 E5 protein alters endosomal pH but not trans-Golgi pH.J Virol. 2005; 79: 5839-5846Crossref PubMed Scopus (68) Google Scholar augment epidermal growth factor signaling pathways,31Crusius K Auvinen E Steuer B Gaissert H Alonso A The human papillomavirus type 16 E5-protein modulates ligand-dependent activation of the EGF receptor family in the human epithelial cell line HaCaT.Exp Cell Res. 1998; 241: 76-84Crossref PubMed Scopus (119) Google Scholar alter membrane lipid composition,28Suprynowicz FA Disbrow GL Krawczyk E Simic V Lantzky K Schlegel R HPV-16 E5 oncoprotein upregulates lipid raft components caveolin-1 and ganglioside GM1 at the plasma membrane of cervical cells.Oncogene. 2008; 27: 1071-1078Crossref PubMed Scopus (68) Google Scholar stimulate anchorage-independent growth,25Suprynowicz FA Disbrow GL Simic V Schlegel R Are transforming properties of the bovine papillomavirus E5 protein shared by E5 from high-risk human papillomavirus type 16?.Virology. 2005; 332: 102-113Crossref PubMed Scopus (27) Google Scholar and interfere with gap junction communication.32Tomakidi P Cheng H Kohl A Komposch G Alonso A Connexin 43 expression is downregulated in raft cultures of human keratinocytes expressing the human papillomavirus type 16 E5 protein.Cell Tissue Res. 2000; 301: 323-327Crossref PubMed Scopus (25) Google Scholar Similarly, there have been several cell proteins identified as targets for 16E5, including the V-ATPase,9Conrad M Bubb VJ Schlegel R The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.J Virol. 1993; 67: 6170-6178Crossref PubMed Google Scholar, 13Rodríguez MI Finbow ME Alonso A Binding of human papillomavirus 16 E5 to the 16kDa subunit c (proteolipid) of the vacuolar H+-ATPase can be dissociated from the E5-mediated epidermal growth factor receptor overactivation.Oncogene. 2000; 19: 3727-3732Crossref PubMed Scopus (58) Google Scholar MHC complexes,14Ashrafi GH Haghshenas M Marchetti B Campo MS E5 protein of human papillomavirus 16 downregulates HLA class I and interacts with the heavy chain via its first hydrophobic domain.Int J Cancer. 2006; 119: 2105-2112Crossref PubMed Scopus (94) Google Scholar and ErbB4.15Chen SL Lin ST Tsai TC Hsiao WC Tsao YP ErbB4 (JM-b/CYT-1)-induced expression and phosphorylation of c-Jun is abrogated by human papillomavirus type 16 E5 protein.Oncogene. 2006; 26: 42-53Crossref PubMed Scopus (30) Google Scholar However, there is no single target that seems to explain the many properties ascribed to 16E5 and it is possible that E5 may have several cell targets for inducing its effects. In the present study, we demonstrate that HPV E5 and E6 proteins cooperate to induce koilocytosis. Koilocytes are epithelial cells containing a hyperchromatic nucleus that is acentrically displaced by perinuclear vacuole(s),16Hajdu SI A note from history: the link between koilocytes and human papillomaviruses.Ann Clin Lab Sci. 2006; 36: 485-487PubMed Google Scholar and these morphological alterations are used by pathologists to help identify HPV-infected epithelial cells in Pap smears. In clinical biopsies, koilocytosis is observed in both low- and high-risk HPV infections. Correspondingly, we found that the E5 and E6 proteins from low- and high-risk HPVs induce koilocytosis in vitro, using both transient and stable expression conditions. The koilocytes observed in our experiments are very similar to those observed in biopsies and Pap smears, although they exhibit some qualitative differences (Figure 1A). For example, although koilocytes consisting of a single vacuole surrounding the nucleus can be observed, there is a greater proportion of koilocytes with multivesiculation in vitro. This is most evident with the 16 E5 (−10) mutant that is partially defective for koilocytosis. We presume that this might reflect incomplete vesicle fusion, but we have not yet performed a thorough investigation of the time course of vesicle formation/fusion with the in vitro system. The difference between in vivo and in vitro koilocytosis might have several origins: i) the expression of E5 in our cells may not be as high as during a productive infection; ii) the cells that we study are not differentiated; and iii) our experiments use only two viral oncogenes rather than the complete viral genome and might not be expected to mimic precisely the morphological changes occurring during productive viral infection. In natural infections, koilocytosis is only observed in the uppermost, well-differentiated layers of stratified squamous epithelium and it is in these layers (and specifically in koilocytes) that the viral capsid proteins are expressed and assembled into infectious virions. In contrast, we have been studying koilocytosis under conditions that do not favor terminal differentiation (ie, low calcium medium) and this might explain the low level (<3%) of koilocyte formation. However, even when the complete HPV genome is expressed in organotypic (raft) culture systems that favor keratinocyte differentiation, koilocytosis is rarely observed.33Genther SM Sterling S Duensing S Munger K Sattler C Lambert PF Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle.J Virol. 2003; 77: 2832-2842Crossref PubMed Scopus (141) Google Scholar, 34Aaltonen LM Wahlström T Rihkanen H Vaheri A A novel method to culture laryngeal human papillomavirus-positive epithelial cells produces papilloma-type cytology on collagen rafts.Eur J Cancer. 1998; 34: 1111-1116Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Additionally, we found that the frequency of koilocytosis in cultured HEC lines does not increase under conditions that acutely induce differentiation (Ca2+ and 10% fetal bovine serum for 24, 48, or 72 hours) (data not shown). This might reflect the differentiation insensitivity of retroviral promoters controlling E5 and E6 expression or might also be the consequence of a need for more complete differentiation conditions. Regardless, the selective presence of koilocytes in the differentiated layers of the stratified squamous epithelium may reflect increased expression of E5 and/or E6 in these cells. Compatible with this suggestion is the hypothesis that HPVs use a leaky ribosome-scanning mechanism to translate proteins from polycistronic mRNAs in vivo and, as a consequence, the E5 protein is primarily synthesized in differentiating cells because of a shift of E5 from the fourth to second open reading frame of HPV transcripts.8Longworth MS Laimins LA Pathogenesis of human papillomaviruses in differentiating epithelia.Microbiol Mol Biol Rev. 2004; 68: 362-372Crossref PubMed Scopus (486) Google Scholar More directly, the BPV-1 E5 protein has been shown to be expressed highly in the uppermost layers of stratified squamous epithelium in papillomas productively replicating BPV-1.35Burnett S Jareborg N DiMaio D Localization of bovine papillomavirus type 1 E5 protein to transformed basal keratinocytes and permissive differentiated cells in fibropapilloma tissue.Proc Natl Acad Sci USA. 1992; 89: 5665-5669Crossref PubMed Scopus (59) Google Scholar It is unclear why the E5 and E6 proteins fail to induce koilocytes in COS cells, but there are obviously many possible reasons for this, including differences in the species of origin, cell type, and the presence of SV40-T antigen (which has functional similarities to the HPV E7 protein that inhibits koilocytosis).36Münger K Heselmeyer K Henson DE Gazdar A The Molecular Pathogenesis of Cervical Cancer: The Role of Human Papillomaviruses. IOS Press, Amsterdam1999: 97-111Google Scholar It is also unclear why the E7 oncoprotein decreases the incidence of koilocytes in our study. In an infected cell, HPV-16 E6 and E7 are produced from polycistronic mRNAs8Longworth MS Laimins LA Pathogenesis of human papillomaviruses in differentiating epithelia.Microbiol Mol Biol Rev. 2004; 68: 362-372Crossref PubMed Scopus (486) Google Scholar and hence both proteins should be expressed. However, changes in cell differentiation could certainly affect the efficiency of splicing the E6/E7 regions and therefore alter the ratio of E6 and E7. In addition, it is possible that the translation of E7 mRNA or stability of E7 protein might be altered as a consequence of keratinocyte differentiation. The koilocytes formed in our experiments do not appear to be apoptotic or senescent cells. The E5-expressing, immortalized HECs do not exhibit the pyknotic and shrunken nuclei usually observed in apoptotic cells.37Zamzami N Kroemer G Condensed matter in cell death.Nature. 1999; 401: 127-128Crossref PubMed Scopus (96) Google Scholar Additionally, we have found that koilocytosis is promoted by the E6 oncoprotein, which is known to inhibit apoptosis.38Finzer P Aguilar-Lemarroy A Rösl F The role of human papillomavirus oncoproteins E6 and E7 in apoptosis.Cancer Lett. 2002; 188: 15-24Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Chakrabarti O Krishna S Molecular interactions of “high risk” human papillomaviruses E6 and E7 oncoproteins: implications for tumour progression.J Biosci. 2003; 28: 337-348Crossref PubMed Scopus (32) Google Scholar, 40Li TT Zhao LN Liu ZG Han Y Fan DM Regulation of apoptosis by the papillomavirus E6 oncogene.World J Gastroenterol. 2005; 11: 931-937PubMed Google Scholar E7, in contrast, strongly inhibits koilocyte formation in our study but promotes apoptosis in keratinocytes.38Finzer P Aguilar-Lemarroy A Rösl F The role of human papillomavirus oncoproteins E6 and E7 in apoptosis.Cancer Lett. 2002; 188: 15-24Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 39Chakrabarti O Krishna S Molecular interactions of “high risk” human papillomaviruses E6 and E7 oncoproteins: implications for tumour progression.J Biosci. 2003; 28: 337-348Crossref PubMed Scopus (32) Google Scholar Senescence-associated β-galactosidase, a diagnostic marker for cellular senescence, was detectable in only a very small number (<0.02%) of nonimmortalized HECs, and we found that koilocytes were no more likely to be senescent than nonkoilocytotic cells (data not shown). The role of perinuclear vacuolization in viral replication is unclear. Cytoplasmic vacuolization could contribute to keratinocyte fragility and the release of viral-laden nuclei from HPV lesions. In fact, it has been suggested that papillomavirus egress in the upper epithelial layer is facilitated by disturbing keratin integrity and assembly of the cornified envelope.41Doorbar J The papillomavirus life cycle.J Clin Virol. 2005; 32S: 7-15Abstract Full Text Full Text PDF Scopus (573) Google Scholar However, although we believe that E5 might have a significant effect on the viral life cycle in vivo, it has already been shown to have a modest effect on viral replication in raft keratinocyte cultures.33Genther SM Sterling S Duensing S Munger K Sattler C Lambert PF Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle.J Virol. 2003; 77: 2832-2842Crossref PubMed Scopus (141) Google Scholar, 34Aaltonen LM Wahlström T Rihkanen H Vaheri A A novel method to culture laryngeal human papillomavirus-positive epithelial cells produces papilloma-type cytology on collagen rafts.Eur J Cancer. 1998; 34: 1111-1116Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Finally, we have also identified E5 and E6 mutants that are defective for inducing koilocytosis in vitro and anticipate that they may be useful in identifying specific pathways and cell targets for these viral oncoproteins. It will also be important to define the common properties of the low- and high-risk E6 proteins and how they contribute to the pathological changes in the cell cytoplasm. We thank Drs. Elke A. Jarboe and Christopher P. Crum, Brigham and Women's Hospital, Boston, MA, for the photo of koilocytosis in a Pap smear; and Tigest Seyoum for excellent technical assistance with histological staining.
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