The possible role of Stomoxys calcitrans in equine sarcoid transmission.

The association between bovine papillomavirus (BPV) and equine sarcoids is well established, but it is unclear how the virus spreads. Although evidence in support of viral spread through direct animal contact exists, this does not explain sarcoid development in isolated equids. BPV DNA has been detected in flies, which could indicate that these insects serve as a vector. This study aimed to investigate whether BPV-negative stable flies (Stomoxys calcitrans) become positive for BPV DNA after exposure to equine sarcoid or bovine papilloma tissue under experimental conditions and, if so, for how long. A total of 420 stable flies were caught alive and exposed to BPV positive equine sarcoid or bovine papilloma tissue. During the following week, dead flies were collected daily and BPV loads were determined by quantitative PCR. There was a significant rise in BPV load after tissue exposure both in sarcoid and papilloma exposed flies, but the viral load was higher and remained high for a longer time after exposure to papilloma tissue compared to sarcoid tissue. Within days, viral loads decreased again and became indifferent from loads before exposure. The results of these experiments indicate that BPV transmission by S. calcitrans seems possible and is more likely to occur after contact with bovine papillomas than with equine sarcoids. Transmission seems only possible shortly after tissue exposure. Further research could include experimental induction of sarcoids with BPV positive stable flies, or a repeat of the experiment with micro-dissection prior to PCR.

Phosphorylation of the Bovine Papillomavirus E2 Protein on Tyrosine Regulates Its Transcription and Replication Functions.

Papillomaviruses are small, double-stranded DNA viruses that encode the E2 protein, which controls transcription, replication, and genome maintenance in infected cells. Posttranslational modifications (PTMs) affecting E2 function and stability have been demonstrated for multiple types of papillomaviruses. Here we describe the first phosphorylation event involving a conserved tyrosine (Y) in the bovine papillomavirus 1 (BPV-1) E2 protein at amino acid 102. While its phosphodeficient phenylalanine (F) mutant activated both transcription and replication in luciferase reporter assays, a mutant that may act as a phosphomimetic, with a Y102-to-glutamate (E) mutation, lost both activities. The E2 Y102F protein interacted with cellular E2-binding factors and the viral helicase E1; however, in contrast, the Y102E mutant associated with only a subset and was unable to bind to E1. While the Y102F mutant fully supported transient viral DNA replication, BPV genomes encoding this mutation as well as Y102E were not maintained as stable episomes in murine C127 cells. These data imply that phosphorylation at Y102 disrupts the helical fold of the N-terminal region of E2 and its interaction with key cellular and viral proteins. We hypothesize that the resulting inhibition of viral transcription and replication in basal epithelial cells prevents the development of a lytic infection. IMPORTANCE: Papillomaviruses (PVs) are small, double-stranded DNA viruses that are responsible for cervical, oropharyngeal, and various genitourinary cancers. Although vaccines against the major oncogenic human PVs are available, there is no effective treatment for existing infections. One approach to better understand the viral replicative cycle, and potential therapies to target it, is to examine the posttranslational modification of viral proteins and its effect on function. Here we have discovered that the bovine papillomavirus 1 (BPV-1) transcription and replication regulator E2 is phosphorylated at residue Y102. While a phosphodeficient mutant at this site was fully functional, a phosphomimetic mutant displayed impaired transcription and replication activity as well as a lack of an association with certain E2-binding proteins. This study highlights the influence of posttranslational modifications on viral protein function and provides additional insight into the complex interplay between papillomaviruses and their hosts.

Quantitative analysis of infiltrating immune cells and bovine papillomavirus type 1 E2-positive cells in equine sarcoids.

Sarcoids are the most frequently observed skin tumours in equids and consist of cutaneous accumulations of transformed fibroblasts. Their aetiopathogenesis is closely linked to a presumably abortive infection by bovine papillomavirus (BPV) types 1 and 2. In cattle, dermal fibropapillomas induced by BPV1/2 usually regress spontaneously due to a local, cell-mediated, immune response; however, equids appear to lack an effective immune response to BPV1/2 and mechanisms of immune evasion have been postulated. As a consequence, equine sarcoids tend to persist and are prone to recur. In this study, cryosections were analysed by immunofluorescent staining and a high content analysis system to determine the presence and distribution of CD4(+), CD8(+), FoxP3(+), RORγt(-), CD206(+) and CD14(+) cells, along with expression of the BPV1 early regulatory protein E2. A higher density of cells was positive for BPV1 E2(+) within the transformed tissue than in perilesional tissue or normal skin of horses with sarcoids and control horses. The proportion of CD8(+) cytotoxic T cells was significantly increased in perilesional and lesional tissues, whereas CD4(+) T helper cells were present in higher density only in lesional tissue compared to normal skin from horses with and without sarcoids. The proportion of pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells was decreased in sarcoid tissue compared to perilesional, distant and control tissue. There were no significant differences in densities of CD4(+)FoxP3(+) RORγt(-) regulatory T cells between sarcoids and control tissues. Equine sarcoids are characterised by infiltrations of CD8(+) and CD4(+) T cells, with decreased representation by pro-inflammatory CD4(+)FoxP3(+)RORγt(+) regulatory T cells.

The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2.

The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins.

ERas protein is overexpressed and binds to the activated platelet-derived growth factor ß receptor in bovine urothelial tumour cells associated with papillomavirus infection.

Embryonic stem cell-expressed Ras (ERas) encodes a constitutively active form of guanosine triphosphatase (GTPase) that binds to and activates phosphatidylinositol 3 kinase (PI3K), which in turn phosphorylates and activates downstream targets such as Akt. The current study evaluated ERas regulation and expression in papillomavirus-associated urothelial tumours in cattle grazing on lands rich in bracken fern. ERas was found upregulated and overexpressed by PCR, real time PCR and Western blot. Furthermore, protein overexpression was also confirmed by immunohistochemistry. ERas was found to interact physically and colocalise with the activated platelet derived growth factor ß receptor (PDGFßR) by coimmunoprecipitation and laser scanning confocal investigations. Phosphorylation of Akt, a downstream effector both of ERas and PDGFßR, appeared to be increased in urothelial tumour cells. Altogether, these data indicate that ERas/PDGFßR complex could play a role in the pathogenesis of bovine papillomavirus-associated bladder neoplasia.

A novel murine model for evaluating bovine papillomavirus prophylactics/therapeutics for equine sarcoid-like tumours.

Equine sarcoids are highly recurrent bovine papillomavirus (BPV)-induced fibroblastic neoplasms that are the most common skin tumours in horses. In order to facilitate the study of potential equine sarcoid prophylactics or therapeutics, which can be a slow and costly process in equines, a murine model for BPV-1 protein-expressing equine sarcoid-like tumours was developed in mice through stable transfection of BPV-1 E5 and E6 in a murine fibroblast tumour cell line (K-BALB). Like equine sarcoids, these murine tumour cells (BPV-KB) were of fibroblast origin, were tumorigenic and expressed BPV-1 proteins. As an initial investigation of the preclinical potential of this tumour model for equine sarcoids prophylactics, mice were immunized with BPV-1 E5E6 Venezuelan equine encephalitis virus replicon particles, prior to BPV-KB challenge, which resulted in an increased tumour-free period compared with controls, indicating that the BPV-KB murine model may be a valuable preclinical alternative to equine clinical trials.

Increased FOXP3 expression in tumour-associated tissues of horses affected with equine sarcoid disease.

Recent studies suggest that regulatory T cells (Tregs) are associated with disease severity and progression in papilloma virus induced neoplasia. Bovine papilloma virus (BPV) is recognised as the most important aetiological factor in equine sarcoid (ES) disease. The aim of this study was to compare expression levels of Treg markers and associated cytokines in tissue samples of ES-affected equids with skin samples of healthy control horses. Eleven ES-affected, and 12 healthy horses were included in the study. Expression levels of forkhead box protein 3 (FOXP3), interleukin 10 (IL10), interleukin 4 (IL4) and interferon gamma (IFNG) mRNA in lesional and tumour-distant samples from ES-affected horses, as well as in dermal samples of healthy control horses were measured using quantitative reverse transcription polymerase chain reaction (PCR). Expression levels were compared between lesional and tumour-distant as well as between tumour-distant and control samples. Furthermore, BPV-1 E5 DNA in samples of ES-affected horses was quantified using quantitative PCR, and possible associations of viral load, disease severity and gene expression levels were evaluated. Expression levels of FOXP3, IL10 and IFNG mRNA and BPV-1 E5 copy numbers were significantly increased in lesional compared to tumour-distant samples. There was no difference in FOXP3 and cytokine expression in tumour-distant samples from ES- compared with control horses. In tumour-distant samples viral load was positively correlated with IL10 expression and severity score. The increased expression of Treg markers in tumour-associated tissues of ES-affected equids indicates a local, Treg-induced immune suppression.

Interactions between E6, FAK, and GIT1 at paxillin LD4 are necessary for transformation by bovine papillomavirus 1 E6.

UNLABELLED: Bovine papillomavirus 1 E6 interacts with two similar proteins that regulate cell attachment and cell migration called paxillin (PXN) and HIC-5 (also known as HIC5, ARA55, HIC-5, TSC-5, and TGFB1I1). Despite the similarity between HIC-5 and paxillin, paxillin is required for E6 to transform mouse embryo fibroblasts while HIC-5 is not. Using mutants of paxillin, we found that dynamic competitive interactions between E6, focal adhesion kinase, and the GIT1 ARF-GAP protein for binding to paxillin are required but not sufficient for transformation by E6. Using mutants of paxillin and chimeric proteins between HIC-5 and paxillin, we demonstrate that a critical difference between HIC-5 and paxillin is within the LIM domains of paxillin that do not directly interact with E6. Mutational analysis indicates that at least six distinct domains of paxillin are required for E6 transformation. IMPORTANCE: Papillomaviruses cause epitheliomas in vertebrates through the actions of virus-encoded oncoproteins. Despite the immense diversity of papillomavirus types, our understanding of the mechanisms by which the virus-encoded E6 oncoproteins contribute to cell transformation is restricted to human papillomavirus types that are associated with cancer. Bovine papillomavirus 1 (BPV-1) E6 has served as a model system for studies of E6 structure and function. This study examines the mechanisms by which BPV-1 E6 association with the cellular focal adhesion adapter protein paxillin contributes to cell transformation and extends our knowledge of the diverse mechanisms by which papillomaviruses transform host cells.

Development of a DNA-based vaccine strategy against bovine papillomavirus infection, involving the E5 or L2 gene.

Papillomaviruses are known to cause tumor lesions, generally benign, in epithelial tissues of diverse organisms; these lesions may progress to cancer under suitable conditions. Bovine papillomavirus (BPV) can cause urinary bladder cancer and cancer of the upper gastrointestinal tract. Furthermore, BPV1 and BPV2 are implicated in the development of tumors in equids. Many studies with animal models clearly demonstrate that DNA vaccines are very effective tools in controlling viral infections, providing strong humoral and cellular immune responses. In this study, we have described the development of two vaccine constructs for the control of diseases caused by BPV. The 1st strategy is prophylactic and is based on the L2 gene; the 2nd is therapeutic and is based on the E5 gene. Vaccine constructs were obtained and evaluated in vitro in mammalian cells. The results show the occurrence of E5 and L2 transcription and viral protein production. These results confirm the functionality of the vaccine constructs in mammalian cells. This is the 1st step in the development of a DNA-based vaccine strategy for the control and/or treatment of diseases caused by BPV.

Bovine papillomavirus type 2 infection and a series of mesenchymal tumors of the urinary bladder in cattle.

This report describes the histopathology of two hundred and fifty-three mesenchymal tumors of the urinary bladder in cattle grazing on lands rich in bracken fern. Approximately 80% were hemangiomas and angiosarcomas. Hemangioma (capillary, cavernous, and large vessels) was the most frequent mesenchymal tumor and was more common than angiosarcoma. Although the appearance of endothelial cells can vary remarkably, epithelioid angiosarcomas, often containing multinucleated cells, were the most frequent malignant vascular tumors. Hemangiopericytoma and tumors of muscle and soft connective tissue origin, alone and/or in association with tumor-like lesions, were less frequently seen. Furthermore, forty-five cases of intravascular papillary endothelial hyperplasia (IPEH), a lesion not previously reported in the urinary bladder of cattle, were also described. Bovine papillomavirus type-2 DNA was amplified in tumor samples. Forty vascular tumors were investigated by dual-labeling immunofluorescence, and, for the first time, a coexpression of E5 and platelet-derived growth factor ß receptor (PDGF ß R) was shown to occur. The results show that the BPV-2 E5 oncoprotein binds to the activated form of the PDGF ß receptor thus playing an important role in mesenchymal as well as epithelial carcinogenesis of the urinary bladder. Furthermore, these findings demonstrate that BPV-2 infects both epithelial and mesenchymal cells.

BAG3 protects bovine papillomavirus type 1-transformed equine fibroblasts against pro-death signals.

In human cancer cells, BAG3 protein is known to sustain cell survival. Here, for the first time, we demonstrate the expression of BAG3 protein both in equine sarcoids in vivo and in EqS04b cells, a sarcoid-derived fully transformed cell line harbouring bovine papilloma virus (BPV)-1 genome. Evidence of a possible involvement of BAG3 in equine sarcoid carcinogenesis was obtained by immunohistochemistry analysis of tumour samples. We found that most tumour samples stained positive for BAG3, even though to a different grade, while normal dermal fibroblasts from healthy horses displayed very weak staining pattern for BAG3 expression. By siRNA technology, we demonstrate in EqS04b the role of BAG3 in counteracting basal as well as chemical-triggered pro-death signals. BAG3 down-modulation was indeed shown to promote cell death and cell cycle arrest in G0/G1. In addition, we found that BAG3 silencing sensitized EqS04b cells to phenethylisothiocyanate (PEITC), a promising cancer chemopreventive/chemotherapeutic agent present in edible cruciferous vegetables. Notably, such a pro-survival role of BAG3 was less marked in E. Derm cells, an equine BPV-negative fibroblast cell line taken as a normal counterpart. Altogether our findings might suggest a mutual cooperation between BAG3 and viral oncoproteins to sustain cell survival.

Bovine papillomavirus type 2 infection and microscopic patterns of urothelial tumors of the urinary bladder in water buffaloes.

Microscopic patterns of thirty-four urothelial tumors of the urinary bladder of water buffaloes from the Marmara and Black Sea Regions of Turkey are here described. All the animals grazed on lands rich in bracken fern. Histological diagnosis was assessed using morphological parameters recently suggested for the urinary bladder tumors of cattle. Papillary carcinoma was the most common neoplastic lesion (22/34) observed in this study, and low-grade carcinoma was more common (seventeen cases) than high-grade carcinoma (five cases). Papilloma, papillary urothelial neoplasm of low malignant potential (PUNLMP), and invasive carcinomas were less frequently seen. Carcinoma in situ (CIS) was often detected associated with some papillary and invasive carcinomas. De novo (primary) CIS was rare representing 3% of tumors of this series. A peculiar feature of the most urothelial tumors was the presence in the tumor stroma of immune cells anatomically organized in tertiary lymphoid organs (TLOs). Bovine papillomavirus type-2 (PV-2) E5 oncoprotein was detected by molecular and immunohistochemistry procedures. Early protein, E2, and late protein, L1, were also detected by immunohistochemical studies. Morphological and molecular findings show that BPV-2 infection contributes to the development of urothelial bladder carcinogenesis also in water buffaloes.

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.

Genetic analysis of the E2 transactivation domain dimerization interface from bovine papillomavirus type 1.

The bovine papillomavirus type 1 (BPV1) E2 protein binds as a dimer to the viral genome to promote its transcription, replication and maintenance in keratinocytes. Although BPV1 E2 dimerizes primarily through its DNA-binding domain, it was shown previously that its transactivation domain (TAD) can also dimerize in vitro through formation of a disulfide bond between cysteine 57 (C57) of adjacent monomers and of an ion pair between arginine 172 (R172) and aspartic acid 175 (D175). The function of this TAD dimerization interface in vivo remains unknown. Here, we report the effects of substituting C57, R172 and D175 by alanine on the transactivation activity of BPV E2 as well as on its ability to support viral DNA replication using a novel luciferase-based assay. Results for this mutational analysis suggest that the TAD dimerization interface is not essential for either process but may contribute to the DNA replication activity of BPV1 E2.

Equine sarcoids: Bovine Papillomavirus type 1 transformed fibroblasts are sensitive to cisplatin and UVB induced apoptosis and show aberrant expression of p53.

Bovine papillomavirus type 1 infects not only cattle but also equids and is a causative factor in the pathogenesis of commonly occurring equine sarcoid tumours. Whilst treatment of sarcoids is notoriously difficult, cisplatin has been shown to be one of the most effective treatment strategies for sarcoids. In this study we show that in equine fibroblasts, BPV-1 sensitises cells to cisplatin-induced and UVB-induced apoptosis, a known cofactor for papillomavirus associated disease, however BPV-1 transformed fibroblasts show increased clonogenic survival, which may potentially limit the therapeutic effects of repeated cisplatin treatment. Furthermore we show that BPV-1 increases p53 expression in sarcoid cell lines and p53 expression can be either nuclear or cytoplasmic. The mechanism and clinical significance of increase/abnormal p53 expression remains to be established.

DNA hypomethylation and oxidative stress-mediated increase in genomic instability in equine sarcoid-derived fibroblasts.

It is widely accepted that equine sarcoid disease, the most common skin associated neoplasm in equids, is induced by bovine papillomavirus (BPV-1). Although BPV-1 DNA has been found in almost all examined sarcoids so far, its detailed impact on the horse's host cell metabolism is largely unknown. We used equine fibroblast cell lines originating from sarcoid biopsies to study BPV-1-associated changes on DNA methylation status and oxidative stress parameters. Sarcoid-derived fibroblasts manifested increased proliferation in vitro, transcriptional rDNA activity (NORs expression) and DNA hypomethylation compared to control cells. Cells isolated from equine sarcoids suffered from oxidative stress: the expression of antioxidant enzymes was decreased and the superoxide production was increased. Moreover, increased ploidy, oxidative DNA damage and micronuclei formation was monitored in sarcoid cells. We postulate that both altered DNA methylation status and redox milieu may affect genomic stability in BPV-1-infected cells and in turn contribute to sarcoid pathology.

Pathological study of non-neoplastic urinary bladder lesions in cattle and buffaloes: a preliminary report.

A total of 236 urinary bladders (94 cattle and 142 buffaloes) collected from Bareilly, Uttar Pradesh, India, were studied for spontaneous lesions. These adult animals belonged to Institute's organized dairy farm and rural areas in the Rohilkhand region of Uttar Pradesh. Grossly, congestion, hemorrhages, and cystoliths in urinary bladders were diagnosed. Histopathologically, the major conditions diagnosed were acute cystitis, 44 (18.64%), including, congestion, hemorrhages, sub-acute cystitis; chronic cystitis, 74 (31.35%), including chronic cystitis un-complicated type, lymphocytic cystitis, plasmolymphocytic cystitis, follicular cystitis, hyperplasia, nodular/acinar hyperplasia, and cystolithiasis; and nothing unusual diagnosed, 118 (50.00%). Similar types of pathological conditions were diagnosed in both species of animals with exception of follicular cystitis and nodular/acinar hyperplasia which was diagnosed respectively only in buffaloes and cystoliths in cows. In addition, a good number of 17/25 (68%) urinary bladder samples tested were found positive for presence of bovine papillomavirus type-2 (BPV-2) by polymerase chain reaction. These included eight cases of acute cystitis, an equal number of cases of chronic cystitis, and one normal bladder. BPV-2 is known as potential source of enzootic bovine hematuria along with other co-factors in enzootic areas. Lesions of zoonotic significance, like tuberculosis, etc., were not diagnosed. None of the observed lesions represented conditions, which, by themselves, would warrant carcass condemnation in buffaloes.

p38 mitogen-activated protein kinase is crucial for bovine papillomavirus type-1 transformation of equine fibroblasts.

Equine sarcoids represent the most common skin tumours in equids worldwide, characterized by extensive invasion and infiltration of lymphatics, rare regression and high recurrence after surgical intervention. Bovine papillomavirus type-1 (BPV-1) and less commonly BPV-2 are the causative agents of the diseases. It has been demonstrated that BPV-1 viral gene expression is necessary for maintaining the transformation phenotype. However, the underlying mechanism for BPV-1 transformation remains largely unknown, and the cellular factors involved in transformation are not fully understood. Previously mitogen-activated protein kinase (MAPK) signalling pathway has been shown to be important for cellular transformation. This study investigated the role of p38 MAPK (p38) in the transformation of equine fibroblasts by BPV-1. Elevated expression of phosphorylated p38 was observed in BPV-1 expressing fibroblasts due to the expression of BPV-1 E5 and E6. The phosphorylation of the MK2 kinase, a substrate of p38, was also enhanced. Inhibition of p38 activity by its selective inhibitor SB203580 changed cell morphology, reduced the proliferation of sarcoid fibroblasts and inhibited cellular invasiveness, indicating the indispensable role of p38 in BPV-1 transformation of equine fibroblasts. These findings provide new insights into the pathogenesis of equine sarcoids and suggest that p38 could be a potential target for equine sarcoid therapy.

In vivo analysis of the cell cycle dependent association of the bovine papillomavirus E2 protein and ChlR1.

It has been shown that the genomes of episomally maintained DNA viruses are tethered to host cell chromosomes during cell division, facilitating maintenance in dividing cells. The papillomavirus E2 protein serves this mechanism of viral genome persistence by simultaneously associating with chromatin and the viral genome during mitosis. Several host cell proteins are reported to be necessary for the association of E2 with chromatin including the cohesion establishment factor ChlR1. Here we use fluorescence resonance energy transfer (FRET) technology to confirm the interaction between BPV-1 E2 and ChlR1. Furthermore, we use synchronised live cells to study the temporal nature of this dynamic protein interaction and show that ChlR1 and E2 interact during specific phases of the cell cycle. These data provide evidence that the association of E2 with ChlR1 contributes to a loading mechanism during DNA replication rather than direct tethering during mitotic division.

PBMCs are additional sites of productive infection of bovine papillomavirus type 2.

Bovine papillomavirus type 2 (BPV-2) is an oncogenic virus infecting both epithelial and mesenchymal cells. Its life cycle, similar to other papillomaviruses (PVs), appears to be linked to epithelial differentiation. Human and bovine PVs have been known to reside in a latent, episomal form in PBMCs; therefore, it is believed that blood cells, like all mesenchymal cells, function as non-permissive carriers. Here, for the first time in veterinary and comparative medicine, the BPV-2 E5 oncoprotein and the major structural L1 capsid protein, known to be expressed only in productive infections, were shown to occur in defined subsets of PBMCs. E5 oncoprotein was detected in sorted T- and B-cells as well as in monocytes by flow cytometry and Western blot analysis. However, CD4(+) and CD8(+) lymphocytes appeared to be the main circulating targets of the virus, thus possibly representing the most important reservoir of active BPV-2 in blood. L1 protein was identified by flow cytometry in a population of blood cells recognized as lymphocytes by morphological scatter properties. Western blot analysis was performed on lysates obtained from the sorted subpopulations of PBMCs and detected L1 protein in CD4(+) and CD8(+) cells only. Thus, this study showed that CD4(+) and CD8(+) lymphocytes are permissive for BPV-2 and are new, hitherto unknown sites of productive PV infection. In light of these observations, the life cycle of PVs needs to be revisited to gain novel insights into the epidemiology of BPV infection and the pathogenesis of related diseases.