Grading immunohistochemical markers p16INK4a and HPV E4 identifies productive and transforming lesions caused by low- and high-risk HPV within high-grade anal squamous intraepithelial lesions.

OBJECTIVES: Because current guidelines recognise high-grade anal squamous intraepithelial lesions (HSILs) and low-grade SILs (LSILs), and recommend treatment of all HSILs although not all progress to cancer, this study aims to distinguish transforming and productive HSILs by grading immunohistochemical (IHC) biomarkers p16INK 4a (p16) and E4 in low-risk human papillomavirus (lrHPV) and high-risk (hr)HPV-associated SILs as a potential basis for more selective treatment. METHODS: Immunostaining for p16 and HPV E4 was performed and graded in 183 biopsies from 108 HIV-positive men who have sex with men. The causative HPV genotype of the worst lesion was identified using the HPV SPF10-PCR-DEIA-LiPA25 version 1 system, with laser capture microdissection for multiple infections. The worst lesions were scored for p16 (0-4) to identify activity of the hrHPV E7 gene, and panHPV E4 (0-2) to mark HPV production and life cycle completion. RESULTS: There were 37 normal biopsies, 60 LSILs and 86 HSILs, with 85% of LSILs caused by lrHPV and 93% of HSILs by hrHPV. No normal biopsy showed E4, but 43% of LSILs and 37% of HSILs were E4 positive. No differences in E4 positivity rates were found between lrHPV and hrHPV lesions. Most of the lesions caused by lrHPV (90%) showed very extensive patchy p16 staining; p16 grade in HSILs was variable, with frequency of productive HPV infection dropping with increasing p16 grade. CONCLUSIONS: Combined p16/E4 IHC identifies productive and nonproductive HSILs associated with hrHPV within the group of HSILs defined by the Lower Anogenital Squamous Terminology recommendations. This opens the possibility of investigating selective treatment of advanced transforming HSILs caused by hrHPV, and a 'wait and see' policy for productive HSILs. What's already known about this topic? For preventing anal cancer in high-risk populations, all patients with high-grade squamous intraepithelial lesions (HSILs) are treated, even though this group of lesions is heterogeneous, the histology is variable and regression is frequent. What does this study add? By adding human papillomavirus (HPV) E4 immunohistochemistry to p16 INK4a (p16), and grading expression of both markers, different biomarker expression patterns that reflect the heterogeneity of HSILs can be identified. Moreover, p16/E4 staining can separate high-risk HPV-associated HSILs into productive and more advanced transforming lesions, providing a potential basis for selective treatment.

Characterization of skin lesions induced by skin-tropic α- and ß-papillomaviruses in a patient with epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is a rare, lifelong, autosomal recessive skin disease associated with an unusual susceptibility to infections with ubiquitous ß-human papillomaviruses (ß-HPVs), and in some cases also skin-tropic α genotypes. In this case report, HPV infection patterns were correlated with pathology and clinical manifestations of skin lesions from a patient with EV, without loss-of-function mutations in the EVER genes. HPV infection was investigated by both polymerase chain reaction (PCR) and laser capture microdissection (LCM) PCR, alongside immunofluorescence for the viral proteins E4 and L1. Analysis of eyebrow hair bulbs revealed multiple ß-genus HPV infections, including HPV20 and HPV24, which were consistently found in all 11 skin lesions on the patient. Six lesions were also positive for the skin tropic α-genotype, HPV27. Clear-cut differences between two wart-like lesions, one caused by a skin-tropic α-genotype and the other by ß-genotypes (as detected by LCM PCR) are shown, including the high cellular proliferation rate in ß-HPV-induced lesions. Widespread expression of the early protein E4 was also evident in skin lesions positive for HPV20 by LCM PCR in both tumours and nearby intraepidermal proliferative areas. L1 expression was restricted to areas of intraepidermal proliferation showing productive infection. The patient's inability to control HPV infections is conclusive to the uncontrolled replication of few genotypes from both α and ß genera, which cause proliferative lesions with clear-cut clinical and histological features.

Immunosuppression facilitates the reactivation of latent papillomavirus infections.

At mucosal sites, papillomavirus genomes can persist in the epithelial basal layer following immune-mediated regression. Subsequent T-cell depletion stimulates a 3- to 5-log increase in the viral copy number, to levels associated with productive infection. Reappearance of microlesions was rare within the short time frame of our experiments but was observed in one instance. Our studies provide direct evidence that immunosuppression can trigger the reactivation of latent papillomavirus genomes, as previously proposed in humans.

Deregulation of E-cadherin by human papillomavirus is not confined to high-risk, cancer-causing types.

BACKGROUND: E-cadherin is a tumour suppressor protein, which is normally expressed on keratinocytes and antigen-presenting Langerhans cells (LCs) in the epidermis. We have previously shown that E-cadherin is lost from tissues infected with the high-risk cancer-causing human papillomavirus (HPV) type 16. OBJECTIVES: To test if E-cadherin dysregulation is associated with the cancer risk of the infecting HPV and to establish if it is conserved among HPVs in the α, ß, γ and µ genera. METHODS: Forty-seven lesions infected with low- or high-risk HPV types spanning four HPV genera were stained for E-cadherin, P-cadherin and CD1a to detect LCs. RESULTS: Surface E-cadherin was reduced in tissues infected with members of the α4, α7 and α9 species and the γ and µ genera but was equivalent to normal epidermis in the ß only-infected lesions tested and patchy in α10-infected tissues. There was a direct relationship between atypical E-cadherin expression and a significant reduction in LCs. Expression of P-cadherin, a protein that is increased in the E-cadherin constitutive knockout mouse, was increased in lesions with reduced E-cadherin. CONCLUSIONS: These data show that E-cadherin dysregulation by HPV is widely conserved across the majority of HPV genera. E-cadherin expression was reduced or lost in epidermis irrespective of the cancer risk of the infecting HPV type or the ability of the virus to degrade retinoblastoma protein or p53. A correlation between dysregulated E-cadherin and reduced numbers of LCs supports viral regulation of surface E-cadherin contributing to viral evasion of the host immune system.

Inhibition of HPV-16 E7 oncogenic activity by HPV-16 E2.

Human papillomavirus (HPV) E7 is essential in inducing S-phase progression in differentiating epithelial cells. We have previously shown that HPV-16 E7 activity can be controlled by a direct interaction with the viral transcriptional activator E2, thereby inhibiting transforming potential of E7. We have extended these analyses to show that E2 induces a generalized re-localization of E7 within the cell nucleus, one potential consequence of which is the inhibition of E7-induced degradation of pRb. Most importantly, we show that E2 can also inhibit the ability of E7 to induce centrosome abnormalities, thus preventing aberrant mitoses. Taken together, these studies highlight the central importance of E2 in controlling the functions of E7, independently of the ability of E2 to regulate transcription.

The human papillomavirus type 11 E1/\E4 protein is not essential for viral genome amplification.

An abundant human papillomavirus (HPV) protein E1/\E4 is expressed late in the virus life cycle in the terminally differentiated layers of epithelia. The expression of E1/\E4 usually coincides with the onset of viral DNA amplification. However, the function of E1/\E4 in viral life cycle is not completely understood. To examine the role of E1/\E4 in the virus life cycle, we introduced a single nucleotide change in the HPV-11 genome to result in a truncation of E1/\E4 protein without affecting the E2 amino acid sequence. This mutated HPV-11 genome was introduced into a human foreskin keratinocyte cell line immortalized by the catalytic subunit of human telomerase, deficient in p16(INK4a) expression, and previously shown to support the HPV-11 life cycle when grown in organotypic raft culture. We have demonstrated that E1/\E4 is dispensable for HPV-11 viral DNA amplification in the late stages of the viral life cycle.

Detection of viral DNA and E4 protein in basal keratinocytes of experimental canine oral papillomavirus lesions.

We studied experimental canine oral papillomavirus (COPV) infection by in situ hybridization and immunohistochemistry of weekly biopsies. After 4 weeks, viral DNA in rete ridges suggested a keratinocyte stem cell target. Abundant viral DNA was seen in E4-positive cells only. E4 was predominantly cytoplasmic but also nuclear, being concentrated in the nucleoli during wart formation. Infected cells spread laterally along the basal layer and into the parabasal layers, accompanied by E7 transcription and increased mitoses. Most of the lower epithelium was positive for viral DNA, but, in mature warts, higher levels of E4 expression and genome amplification occurred in only sporadic superficial cells. L1 expression was late and in only a subset of E4-positive cells. During regression, viral DNA was less abundant in deep epithelial layers, suggesting downregulation of replication prior to replacement of infected cells from beneath. Detection of viral DNA in post-regression tissue indicated latent infection.

The E1E4 protein of human papillomavirus type 16 associates with a putative RNA helicase through sequences in its C terminus.

Human papillomavirus type 16 (HPV16) infects cervical epithelium and is associated with the majority of cervical cancers. The E1E4 protein of HPV16 but not those of HPV1 or HPV6 was found to associate with a novel member of the DEAD box protein family of RNA helicases through sequences in its C terminus. This protein, termed E4-DBP (E4-DEAD box protein), has a molecular weight of 66,000 (66K) and can shuttle between the nucleus and the cytoplasm. It binds to RNA in vitro, including the major HPV16 late transcript (E1E4. L1), and has an RNA-independent ATPase activity which can be partially inhibited by E1E4. E4-DBP was detectable in the cytoplasm of cells expressing HPV16 E1E4 (in vivo and in vitro) and could be immunoprecipitated as an E1E4 complex from cervical epithelial cell lines. In cell lines lacking cytoplasmic intermediate filaments, loss of the leucine cluster-cytoplasmic anchor region of HPV16 E1wedgeE4 resulted in both proteins colocalizing exclusively to the nucleoli. Two additional HPV16 E1E4-binding proteins, of 80K and 50K, were identified in pull-down experiments but were not recognized by antibodies to E4-DBP or the conserved DEAD box motif. Sequence analysis of E4-DBP revealed homology in its E4-binding region with three Escherichia coli DEAD box proteins involved in the regulation of mRNA stability and degradation (RhlB, SrmB, and DeaD) and with the Rrp3 protein of Saccharomyces cerevisiae, which is involved in ribosome biogenesis. The synthesis of HPV16 coat proteins occurs after E1E4 expression and genome amplification and is regulated at the level of mRNA stability and translation. Identification of E4-DBP as an HPV16 E1E4-associated protein indicates a possible role for E1E4 in virus synthesis.

Synthesis of viral DNA and late capsid protein L1 in parabasal spinous cell layers of naturally occurring benign warts infected with human papillomavirus type 1.

We investigated human papillomavirus type 1 (HPV1)-specific transcription, viral DNA replication, and viral protein expression in naturally occurring benign tumors by in situ hybridization, 5-bromodeoxyuridine (BrdU) incorporation, and immunohistochemistry and obtained results different from other HPV-infected benign tumors characterized so far. Moderate amounts of transcripts with a putative coding potential for E6/E7, E1, and E2 were demonstrated from the first subrabasal cell layer throughout the stratum spinosum and granulosum. In addition very large amounts of E4 and L1 transcripts were present in the same epithelial layers. This finding was substantiated by the demonstration of L1 and E4 protein already in the bottom-most spinous cell layer. Furthermore massive amplification of the viral DNA as measured by BrdU incorporation and different methods of in situ hybridization took place in the lowest 5 to 10 suprabasal cell layers. These findings are in contrast to the assumption that late gene expression and viral DNA synthesis are restricted to the more differentiated cell layers of the epithelium and point to differences in the regulation of the vegetative life cycle between different papillomavirus types.

The identification of a conserved binding motif within human papillomavirus type 16 E6 binding peptides, E6AP and E6BP.

A 16-mer peptide library was screened using the yeast two-hybrid system to identify peptides which specifically interact with the human papillomavirus type 16 (HPV-16) E6 protein. Four different peptides were identified, three of which contained an E-L-L/V-G motif. A fifth E6 binding peptide, derived from the putative tumour suppressor protein tuberin, was identified during a two-hybrid screen of a HeLa cDNA expression library. This peptide contained a D-I-L-G motif. Homology to the peptides was found within the E6 binding proteins E6-AP and E6-BP. A synthetic peptide containing the ELLG motif blocked the interaction of E6 with both E6-AP and E6-BP. The data suggest that E6 interacts through a structurally similar binding domain present within a number of cellular proteins.

Characterization of events during the late stages of HPV16 infection in vivo using high-affinity synthetic Fabs to E4.

HPV late gene expression is initiated as an infected basal cell migrates through the differentiating layers of the epidermis, resulting in the onset of vegetative viral DNA replication and the expression of viral late proteins. We have used a large synthetic immunoglobulin library displayed on phage (diversity 6.5 x 10(10) phage) to isolate three Fabs (TVG405, 406, and 407) which recognize distinct epitopes on the E4 late protein of HPV16. A C-terminal monoclonal (TVG404) was generated by hybridoma technology, and N-terminal polyclonal antiserum was prepared by peptide immunization (alpha N-term). The most potent antibody (TVG405) had an affinity for E4 of approximately 1.0 nM. All antibodies recognized the protein in paraffin-embedded archival material, allowing us to map events in the late stages of virus infection. Expression of E4 in vivo does not coincide with synthesis of the major virus coat protein L1, but precedes it by 1 or 2 cell layers in premalignant lesions caused by HPV16 and by up to 20 cell layers in HPV63-induced warts. In higher grade lesions associated with HPV16, E4 is produced in the absence of L1. By contrast, vegetative viral DNA replication and E4 expression correlate exactly and in some lesions begin as the infected epithelial cell leaves the basal layer. Differentiation markers such as filaggrin, loricrin, and certain keratins are not detectable in E4-positive cells, and nuclear degeneration is delayed. HPV16 E4 has a filamentous distribution in the lower epithelial layers, but associates with solitary perinuclear structures in more differentiated cells. Antibodies to the N-terminus of the protein stained these structures poorly. Our findings are compatible with a role for the HPV16 E4 protein in vegetative DNA replication or in modifying the phenotype of the infected cell to favor virus synthesis or virus release. The Fabs will be of value in the evaluation of model systems for mimicking HPV infection in vitro.

Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates.

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.

Sequence close to the N-terminus of L2 protein is displayed on the surface of bovine papillomavirus type 1 virions.

The bovine papillomavirus type 1 (BPV1) L2 protein purified from Escherichia coli was used as an antigen to produce monoclonal antibodies (MAbs). A total of 26 individual clones which recognized the BPV1 L2 protein were obtained. Using infectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles. Binding of the MAbs to BPV1 was confirmed by immunoelectron microscopy. A set of 92 13-mer peptides overlapping by 8 amino acids spanning the entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies. Seventeen linear epitopes were identified. Our results revealed that a sequence toward the N-terminus of the L2 protein (aa 61-123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid. Although the polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity for the 3 L2-specific monoclonal antibodies which bound to the BPV1 particles. This suggests that extra binding sites may be needed for neutralization. This study prompted us to propose a model about how L1 and L2 proteins may interact during infectious papillomavirus assembly.

Analysis of HPV1 E4 complexes and their association with keratins in vivo.

The HPV1 E4 gene encodes a family of abundant nonstructural late proteins whose role in the virus life cycle is unknown. Their localization to keratin filaments when expressed in cultured epithelial cells has suggested a possible involvement in virus release by disturbing the integrity of the infected cell. Here we show that in naturally occurring HPV1-induced tumors, the majority of the E4 protein (>95%) exists as complexes which do not contain keratins. The identification of discrete species of 34K, 58K, 70K, 88K, and 105K suggests that these are simple multimers of the 17K monomer, with very little of the soluble E4 being present in complexes larger than 140K. The truncated 10/11K E4 species, which comprise the C-terminal domain of E4, exist as trimers and dimers in vivo. Less than 5% of the E4 was present as complexes greater than 140K, and these were found to be insoluble. The 34K (dimer) and 58K (putative trimer) E4 complexes were components of these larger structures. Neither E4 monomers nor E4 complexes could be shown to interact directly with keratins or with keratin filaments although formation of the 105K E4 complex was abolished (and formation of the 58K species enhanced) when keratins were present during E4 synthesis in vitro. We conclude that while E1-E4 may transiently associate with keratins during synthesis, the two proteins do not stably associate via a direct interaction. The majority of the HPV1 E4 protein in established tumors in vivo is neither filament associated nor contained in inclusion granules, but exists predominantly as soluble cytoplasmic multimers.

Isolation of a peptide antagonist to the thrombin receptor using phage display.

The thrombin receptor on platelets is an integral membrane protein and is cleaved by thrombin to expose a "tethered ligand" that binds to and triggers the receptor. Here we have explored the power of phage selection technology to make a peptide antagonist of this receptor using platelets directly for the selection. To focus the selection to the thrombin receptor, we eluted the phage with a peptide agonist of the thrombin receptor. A repertoire (1 x 10(7) phage clones) displaying peptide sequences based on the sequence of the tethered ligand, was constructed and selected by binding to the platelets. After several rounds of selection, we identified phage clones that were able to immunoprecipitate the thrombin receptor from platelets and the encoded peptides were sequenced. This revealed some features in common with the tethered ligand, in particular an arginine residue followed by a proline. Several of the peptides were synthesized chemically and one of the peptides was shown to antagonise platelet aggregation triggered by the agonist peptide, and to inhibit serotonin release and tyrosine phosphorylation triggered by either thrombin or the agonist peptide. Anti-aggregatory activity was about ten-fold higher than that of previously reported peptide antagonists of the thrombin receptor.

Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the E1--E4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPV1, HPV2, HPV4, or HPV6. The E1--E4 protein migrates as a 10K species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPV1 in benign warts. In naturally occurring HPV16-induced tumors the E1--E4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPV16 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPV16 E1--E4 and should complement those already available to E7 and L1 for the screening of frozen sections of clinical biopsies and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer.

Identification of the nuclear localization signal of human papillomavirus type 16 L1 protein.

Human papillomavirus type 16(HPV16) L1 and L2 capsid proteins can be detected only in the nucleus of infected cells. For other nuclear proteins, specific sequences of basic amino acids(aa) termed nuclear localization signals (NLS) direct the protein from the cytoplasm to the nucleus. We used a series of deletion and substitution mutations of the HPV16 L1 protein, produced by recombinant vaccinia virus (rVV), to identify NLS within HPV16 L1 and showed that HPV16 L1 contains two NLS sequences, each containing basic aa clusters. One NLS consisted of 6 basic amino acids (KRKKRK from aa 525 to 530) at the carboxy terminal end of L1. The other NLS contained 2 basic aa clusters(KRK from aa 510 to 512 and KR at aa 525, 526) separated by 12 amino acids. Mutations in either NLS did not alter nuclear localization of L1 when the other remained intact, but mutations to both prevented nuclear localization of L1. The L1 NLS could be overridden by introduction of a membrane binding sequence at the amino terminal end of the protein. A databases search showed that all sequenced papillomaviruses are predicted to have L1 and L2 capsid proteins with sequences of basic amino acids homologous with one or both NLS of HPV16 L1.

Specific interaction between HPV-16 E1-E4 and cytokeratins results in collapse of the epithelial cell intermediate filament network.

The human papillomaviruses (HPV) are associated specifically with epithelial lesions, ranging from benign warts to invasive carcinoma. The virus encodes three late proteins, which are produced only in terminally differentiating keratinocytes, two of which are structural components of the virion. The third, E1-E4, is derived primarily from the E4 open reading frame, which represents a region of maximal divergence between different HPV types. E1-E4 does not seem to be a component of the virus particle or to be needed for transformation in vitro, but accumulates in the cytoplasm, where in certain benign lesions it can comprise 20-30% of total cell protein. We show here that expression of the HPV-16 E1-E4 protein in human keratinocytes (the natural host cell for HPV infection) results in the total collapse of the cytokeratin matrix. Tubulin and actin networks are unaffected by E1-E4, as are the nuclear lamins.