Annunziata Corteggio

Papillomavirus E5: the smallest oncoprotein with many functions

Papillomaviruses (PVs) are established agents of human and animal cancers. They infect cutaneous and mucous epithelia. High Risk (HR) Human PVs (HPVs) are consistently associated with cancer of the uterine cervix, but are also involved in the etiopathogenesis of other cancer types. The early oncoproteins of PVs: E5, E6 and E7 are known to contribute to tumour progression. While the oncogenic activities of E6 and E7 are well characterised, the role of E5 is still rather nebulous. The widespread causal association of PVs with cancer makes their study worthwhile not only in humans but also in animal model systems. The Bovine PV (BPV) system has been the most useful animal model in understanding the oncogenic potential of PVs due to the pivotal role of its E5 oncoprotein in cell transformation. This review will highlight the differences between HPV-16 E5 (16E5) and E5 from other PVs, primarily from BPV. It will discuss the targeting of E5 as a possible therapeutic agent.

Productive Infection of Bovine Papillomavirus Type 2 in the Placenta of Pregnant Cows Affected with Urinary Bladder Tumors

Papillomaviruses (PVs) are believed to be highly epitheliotropic as they usually establish productive infections within stratified epithelia. In vitro, various PVs appear to complete their entire life-cycle in different trophoblastic cell lines. In this study, infection by and protein expression of bovine papillomavirus type 2 (BPV-2) in the uterine and chorionic epithelium of the placenta has been described in four cows suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. E5 oncoprotein was detected both by Western blot analysis and immunohistochemically. It appears to be complexed and perfectly co-localized with the activated platelet-derived growth factor ß receptor (PDGFßR) by laser scanning confocal microscopy. The activated PDGFßR might be involved in organogenesis and neo-angiogenesis rather than in cell transformation during pregnancy. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection has been detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the uterine and chorionic epithelium. Trophoblastic cells appear to be the major target for L1 protein expression. Finally, the early protein E2, required for viral DNA replication and known to be expressed during a productive infection, has been detected by Western blot and immunohistochemically. Electron microscopic investigations detected viral particles in nuclei of uterine and chorionic epithelium. This study shows that both active and productive infections by BPV-2 in the placenta of pregnant cows can occur in vivo.

Inoculation of young horses with bovine papillomavirus type 1 virions leads to early infection of PBMCs prior to pseudo-sarcoid formation

Bovine papillomavirus types 1 and 2 (BPV-1 and BPV-2) are known to induce common equine skin tumours, termed sarcoids. Recently, it was demonstrated that vaccination with BPV-1 virus-like particles (VLPs) is safe and highly immunogenic in horses. To establish a BPV-1 challenge model for evaluation of the protective potential of BPV-1 VLPs, four foals were injected intradermally with infectious BPV-1 virions and with viral genome-based and control inocula, and monitored daily for tumour development. Blood was taken before inoculation and at weekly intervals. BPV-1-specific serum antibodies were detected by a pseudo-virion neutralization assay. Total nucleic acids extracted from tumours, intact skin and PBMCs were tested for the presence of BPV-1 DNA and mRNA using PCR and RT-PCR, respectively. Intralesional E5 oncoprotein expression was determined by immunofluorescence. Pseudo-sarcoids developed exclusively at sites inoculated with virions. Tumours became palpable 11-32 days after virion challenge, reached a size of ≤20 mm in diameter and then resolved in ≤6 months. No neutralizing anti-BPV-1 serum antibodies were detectable pre- or post-challenge. BPV-1 DNA was present in lesions but not in intact skin. In PBMCs, viral DNA was already detectable before lesions were first palpable, in concentrations correlating directly with tumour growth kinetics. PBMCs from two of two foals also harboured E5 mRNA. Immunofluorescence revealed the presence of the E5 protein in tumour fibroblasts, but not in the apparently normal epidermis overlying the lesions. Together with previous findings obtained in horses and cows, these data suggest that papillomavirus infection may include a viraemic phase.

BPV-1 infection is not confined to the dermis but also involves the epidermis of equine sarcoids.

In equids, bovine papillomaviruses of type 1 (BPV-1) and less frequently type 2 induce common, locally aggressive skin tumours termed sarcoids. Whereas BPV infection in cattle usually involves the epidermis and is productive in this skin layer, infection in equids is currently thought to be abortive, with virus solely residing as multiple episomes in dermal fibroblasts. Based on recent observations that do not agree with this assumption, we hypothesised that BPV also infects equid epidermis and is active in this skin layer. To test this hypothesis, we conducted a proof-of-principle study on eight distinct sarcoids. Presence of viral DNA was addressed by qualitative and quantitative BPV-1 PCR from microdissected sarcoid epidermis, and by subsequent amplicon sequencing. Viral activity was assessed by screening sarcoid epidermis for BPV-1 protein expression using immunohistochemistry (IHC) or immunofluorescence (IF). Virus-free equine skin served as negative control throughout the assays. BPV-1 DNA was demonstrated in all sarcoid epidermis samples, with viral DNA loads ranging between 2 and 195 copies/cell. Identical BPV-1 E5 genes were identified in epidermis and dermis of each of two sarcoids, yet different E5 variants were found in individual lesions. IHC/IF revealed the presence of E5 and E7 protein in sarcoid epidermis, and L1 capsomers in the squamous layer of one lesion. These findings indicate that BPV infection also involves the epidermis, where it may occasionally be productive.

Bovine papillomavirus E5 and E7 oncoproteins in naturally occurring tumors: are two better than one?

Bovine papillomaviruses (BPVs) are oncogenic DNA viruses, which mainly induce benign lesions of cutaneous and/or mucosal epithelia in cattle. Thirteen (BPV 1–13) different viral genotypes have been characterized so far. BPVs are usually species-specific but BPV 1/2 may also infect equids as well as buffaloes and bison and cause tumors in these species. BPV-induced benign lesions usually regress, however occasionally they develop into cancer particularly in the presence of environmental carcinogenic co-factors. The major transforming protein of BPV is E5, a very short hydrophobic, transmembrane protein with many oncogenic activities. E5 contributes to cell transformation through the activation of the cellular β receptor for the platelet-derived growth factor (PDGFβ-r), it also decreases cell surface expression of major histocompatibility complex class I (MHCI) causing viral escape from immunosurveillance, and plays a role in the inhibition of the intracellular communication by means of aberrant connexin expression. E7 is considered as a weak transforming gene, it synergies with E5 in cell transformation during cancer development. E7 expression correlates in vivo with the over-expression of β1-integrin, which plays a role in the regulation of keratinocytes proliferation and differentiation. Additionally, E7 is involved in cell-mediated immune responses leading to tumour rejection, in anoikis process by direct binding to p600, and in invasion process by upregulation of Matrix metalloproteinase1 (MMP-1) expression. Studies on the role of BPV E5 and E7 oncoproteins in naturally occurring tumours are of scientific value, as they may shed new light on the biological role of these two oncogenes in cell transformation.

Bovine papillomavirus: new insights into an old disease.

Bovine papillomaviruses (BPVs) are small DNA tumoral viruses able to induce benign cutaneous and/or mucosal epithelial lesions. Generally, the benign tumours affecting the skin or mucosa spontaneously regress, but under special circumstances, the defence system may be overwhelmed, thus leading to cancer, especially in the presence of immunosuppressant and mutagen agents from bracken fern. To date, thirteen different BPV genotypes have been associated with skin and mucosal tumours in cattle, and out of these, only four types (BPV-1, -2, -5 and -13) cross-infect other species. Recent investigations in vivo have revealed new insights into the epidemiology and pathogenesis of this viral infection. This review briefly discusses viral epidemiology, will give data on BPV genome structure and viral genes and will describe the cellular events and new aspects of both cutaneous and mucosal tumours in large ruminants. Finally, some aspects of active immunization will be described.

Transforming properties of Felis catus papillomavirus type 2 E6 and E7 putative oncogenes in vitro and their transcriptional activity in feline squamous cell carcinoma in vivo

Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting in upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC.

Molecular epidemiology of bovine papillomatosis and the identification of a putative new virus type in Brazilian cattle

Bovine papillomaviruses (BPVs) are a diverse group of double-stranded DNA viruses, of which 12 viral types have been detected and characterized so far. However, there is still a limited understanding of the diversity of BPV. Several putative new BPVs have been detected and some of these have been recently characterized as new viral types. However, only a very limited amount of information is available on the pathology associated with these novel viral types yet this information could be of significant value in improving our understanding of the biology of BPV. The objective of this study was to examine some of the epidemiological features of cutaneous bovine papillomatosis in Brazilian cattle, in particular to establish the relationship between BPV types isolated from beef and dairy cattle herds and the lesions they cause. Seventy-two cutaneous lesions were collected from 60 animals. Histopathological, PCR and sequencing assays were conducted to characterize the lesions and detect the BPV types responsible. Phylogenetic analysis was carried out using the maximum likelihood method. BPV types 1-6 and 8-10 were found, as well as a putative new BPV type that belongs to the Deltapapillomavirus genus. The tumors were all classified as fibropapillomas. This is believed to be the first record of BPV types 3 and 10 associated with fibropapillomas. These results confirm that there is a wide range of BPV types that infect cattle, and that an understanding of this diversity is necessary for improved methods of therapeutic treatment.

Productive Infection of Bovine Papillomavirus Type 2 in the Urothelial Cells of Naturally Occurring Urinary Bladder Tumors in Cattle and Water Buffaloes

Background Papillomaviruses (PVs) are highly epitheliotropic as they usually establish productive infections within squamous epithelia of the skin, the anogenital tract and the oral cavity. In this study, early (E) and late (L) protein expression of bovine papillomavirus type 2 (BPV-2) in the urothelium of the urinary bladder is described in cows and water buffaloes suffering from naturally occurring papillomavirus-associated urothelial bladder tumors. Methods and Findings E5 protein, the major oncoprotein of the BPV-2, was detected in all tumors. L1 DNA was amplified by PCR, cloned and sequenced and confirmed to be L1 DNA. The major capsid protein, L1, believed to be only expressed in productive papillomavirus infection was detected by Western blot analysis. Immunohistochemical investigations confirmed the presence of L1 protein both in the cytoplasm and nuclei of cells of the neoplastic urothelium. Finally, the early protein E2, required for viral DNA replication and known to be a pivotal factor for both productive and persistent infection, was detected by Western blot and immunohistochemically. Electron microscopic investigations detected electron dense particles, the shape and size of which are consistent with submicroscopic features of viral particles, in nuclei of neoplastic urothelium. Conclusion This study shows that both active and productive infections by BPV-2 in the urothelium of the bovine and bubaline urinary bladder can occur in vivo.

Bovine papillomavirus E7 oncoprotein binds to p600 in naturally occurring equine sarcoids.

Studies regarding the functions of the bovine papillomavirus (BPV) E7 oncoprotein in vivo are lacking and no E7-mediated mechanism underlying mesenchymal carcinogenesis is known. Here, we show that the interaction between the 600 kDa retinoblastoma protein-associated factor (p600) and BPV E7, described in vitro in cultured cells, takes place in vivo in naturally occurring equine sarcoids. In these cancers we detect the expression of E7 and p600, and demonstrate that E7 and p600 co-localize and physically interact. Furthermore, intracellular signals involved in p600 functional activity are found not to be overexpressed, suggesting a different functional activity of p600 in naturally occurring carcinogenesis. Our results demonstrate, for the first time, that E7-p600 interaction occurs during the natural history of BPV-induced equine tumours, suggesting an important role for E7 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E7.