HPV16 and BPV1 infection can be blocked by the dynamin inhibitor dynasore.

The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.

CuZn-SOD suppresses the bovine papillomavirus-induced proliferation of fibroblasts.

Eukaryotic cells continuously produce reactive oxygen species (ROS) and have mechanisms to control ROS levels. ROS have been shown to mediate cell proliferation and transformation. We studied the effect of CuZn-superoxide dismutase (CuZnSOD) on the focus-forming ability of bovine papillomavirus (BPV-1) wtDNA and hypertransforming mutant of its major oncoprotein E5, E5-17S. We found that CuZnSOD suppresses the focus-forming ability of BPV-1 wtDNA and E5 oncoprotein. Significantly fewer foci were detected in pCGCuZnSOD- and BPV-1 DNA-cotransfected cell culture compare to BPV-1 DNA-transfected cell culture (p<0.001). CuZnSOD decreases the rate of cell proliferation in both non-transformed C127 and BPV-1- and E5-transformed cell lines. CuZnSOD decelerates cell entry into the S phase of the cell cycle and has a suppressing effect on the actively dividing cells. As the transformed cells proliferate faster than normal cells when confluent, CuZnSOD inhibits the growth of foci. These results indicate that superoxide radicals may be involved in signaling for cell proliferation and that SOD suppresses cell proliferation.

Sarcoids in captive zebras (Equus burchellii): association with bovine papillomavirus type 1 infection.

Sarcoids were diagnosed in two captive zebras from different facilities. Zebra 1 (Equus burchellii boehmi) was a 4.5-yr-old, captive-born male that presented with a 9- by 7-cm inguinal mass. Seven months after surgical excision of the inguinal mass, the zebra presented with a similar lesion in the right upper eyelid that has relapsed repeatedly and has not responded to treatment including local cisplatin injections and cryosurgery. Zebra 2 (of undetermined taxon) was housed at a private wild animal farm. The zebra presented with a single, raised, 2.5- by 2.0- by 2.0-cm, ulcerated mass on the nose, and surgical excision was curative. Histologically, the three masses consisted of a dermal, compact, nonencapsulated, poorly demarcated neoplasm composed of well-differentiated spindle cells arranged in streams and whorls and accompanied by moderate epidermal hyperplasia with long rete pegs. On the basis of the morphologic resemblance to the unique equine cutaneous neoplasm, "sarcoid" was diagnosed. This is the first description of sarcoids in captive zebras. Association with bovine papillomavirus (BPV) type 1, as it occurs in horses, was demonstrated by polymerase chain reaction, nucleic acid sequencing, and in situ hybridization (ISH) on paraffin-embedded tissues from the inguinal mass of zebra 1. Sequencing revealed 98% identity of the 244-bp fragment with BPV type 1. The ISH for BPV type 1 DNA intensely stained the nuclei of neoplastic mesenchymal spindle cells. The sites and the clinical behavior of the sarcoids in these zebras are similar to those described in horses.

Two separate replication modes of the bovine papillomavirus BPV1 origin of replication that have different sensitivity to p53.

We have shown previously that transient amplificational replication of reporter plasmids that carry the papillomavirus origin of replication is efficiently blocked by p53 protein in several cell lines. We demonstrate now that the replication of stably maintained episomal bovine papillomavirus BPV1 URR (upstream regulatory region) reporter plasmid is not sensitive to p53. In addition, these two replication modes--initial transient amplificational replication and stable maintenance replication of essentially the same BPV1 URR reporter plasmid--can take place in the same cells, where amplificational replication does not interfere with the stable maintenance replication. These data suggest that BPV1 replicons could follow two clearly separable replication mechanisms during initial amplification and during stable extrachromosomal maintenance.

The bovine papillomavirus oncoprotein E5 retains MHC class I molecules in the Golgi apparatus and prevents their transport to the cell surface.

During papillomavirus infection, the E5 protein localizes in the cell Golgi apparatus and other endomembrane compartments. Cells transformed by E5 do not express major histocompatibility class I complex (MHC I) on the cell surface, while cells transformed by the other transforming proteins E6 and E7 do. In addition, the total amount of both MHC I protein and mRNA is reduced in E5-transformed cells. Here we show that expression of bovine papillomavirus E5 causes the retention of MHC I in the Golgi apparatus, thus preventing its transport to the cell surface. We ascribe this effect to a failure of acidification of the Golgi apparatus, as similar effects are observed in control cells treated with the ionophore monensin. Treatment of E5-transformed cells with either beta- or gamma-interferon increases the synthesis of MHC I, showing that inhibition of MHC I expression by E5 is not irreversible. However, even after interferon treatment, MHC I, although increased in quantity, is not transported to the cell surface. E5 therefore affects MHC I at several levels, but prevention of MHC I transport to the cell surface appears to be the dominant effect. Lack of surface MHC I would have profound consequences for presentation of viral peptides to the immune system.

Papillomavirus microbicidal activities of high-molecular-weight cellulose sulfate, dextran sulfate, and polystyrene sulfonate.

The high-molecular-weight sulfated or sulfonated polysaccharides or polymers cellulose sulfate, dextran sulfate, and polystyrene sulfonate were tested for microbicidal activity against bovine papillomavirus type 1 (BPV-1) and human papillomavirus type 11 (HPV-11) and type 40 (HPV-40). In vitro assays included the BPV-1-induced focus-forming assay and transient infection of human A431 cells with HPVs. The compounds were tested for microbicidal activity directly by preincubation with virus prior to addition to cell cultures and indirectly by addition of virus to compound-treated cells and to virus-coated cells to test inactivation of the virus after virus-cell binding. The data indicated that all three compounds showed direct microbicidal activity with 50% effective concentrations between 10 to 100 microg/ml. These concentrations were nontoxic to cell cultures for both assays. When a clone of C127 cells was tested for microbicidal activity, approximately 10-fold-less compound was required to achieve a 50% reduction in BPV-1-induced foci than for the uncloned parental C127 cells. Pretreatment of cells with compound prior to addition of virus also demonstrated strong microbicidal activity with dextran sulfate and polystyrene sulfonate, but cellulose sulfate required several orders of magnitude more compound for virus inactivation. Polystyrene sulfonate prevented subsequent infection of HPV-11 after virus-cell binding, and this inactivation was observed up to 4 h after addition of virus. These data indicate that the polysulfated and polysulfonated compounds may be useful nontoxic microbicidal compounds that are active against a variety of sexually transmitted disease agents including papillomaviruses.

Association of bovine papillomavirus type 1 with microtubules.

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane.

Xenograft model for identifying chemotherapeutic agents against papillomaviruses.

The report describes the establishment and characterization of a mouse xenograft transplantation model for the study of papillomavirus infection of bovine skin. Calf scrotal skin was inoculated with bovine papillomavirus type 2 before grafting it to the dorsum of severe combined immunodeficient mice. The grafted skin contained epidermis, dermis, and a thin layer of fat. After 5 months the induced warts not only showed histological features of papillomavirus infections but also tested positive for viral DNA and papillomavirus capsid antigen. The formation of infectious virions was demonstrated by inoculation of new transplants with crude extract from the induced warts as well as in a cell culture focus assay. Topical application of bromovinyl-2'-deoxyuridine led to a reduction in viral DNA content in the developing wart. This small-animal xenograft model should be useful for characterizing antiviral compounds and providing an understanding of the regulation of papillomavirus infections.

n-Butyrate mediated inhibition of papovavirus DNA replication in vivo and in cell culture: a mechanistic approach.

n-Butyrate, an inhibitor of G1-to-S transition inhibits papovavirus DNA replication in cell culture. To explore the efficacy of n-butyrate in vivo and to better understand its mechanism, we studied the effect of n-butyrate on viral DNA replication in mice acutely infected with polyomavirus and in the papovavirus-infected cells in culture. Newborn mice treated with n-butyrate stop growing and become runted. When infected with polyomavirus, these mice show a strong overall inhibition of viral DNA. However, a notable exception to this was the continued viral DNA replication in the differentiated mouse keratinocytes and renal epithelial cells as determined by in situ hybridization. n-Butyrate significantly inhibited viral DNA replication in the cultured IDL cells, and in polyomavirus-infected C2C12 myoblasts based on Southern blot analysis and in situ hybridization. DNA polymerase alpha (but not DNA polymerase beta) and the characteristic nuclear expression of PCNA were both inhibited in the n-butyrate treated IDL and C2C12 cells. n-Butyrate, therefore, inhibited host and viral DNA synthesis in the undifferentiated cells.

A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses.

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.

Calcium is required in reassembly of bovine papillomavirus in vitro.

Papillomaviruses are small DNA viruses which infect and induce benign warts and sometimes malignant tumours in the epithelium of the skin or mucosa. The viruses do not replicate in conventional tissue culture systems and little is known about the requirements for virus assembly. We investigated the effect of ethylene glycol-bis(aminoethyl ether)-tetraacetic acid (EGTA) and dithiothreitol (DTT) treatment on the stability of bovine papillomavirus type 1 (BPV-1) particles in vitro. Removal of calcium ions by 11 mM EGTA at pH 8.0 together with reduction of disulfide bonds by 15 mM DTT destabilized BPV particles. Electron microscopy examination of treated particles showed that the BPV particles had been disrupted to capsomeres. Addition of exogenous calcium ions to the disruption buffer prevented virus destabilization. Adding calcium to the disrupted BPV particles resulted in the reassembly of disrupted particles. The reassembled particles were morphologically similar to intact BPV virions. We further quantified the efficiency of reassembly by focus formation assay. We recorded 500-fold less infectivity for reassembled BPV and 4-fold less haemagglutination activity compared to untreated BPV, pointing towards a decrease in the amount of reassembled particles recovered.

Intercapsomeric disulfide bonds in papillomavirus assembly and disassembly.

In order to analyze bonding contacts that stabilize the virion or promote capsid assembly, bovine papillomavirus (BPV) virions were subjected to buffer conditions known to disrupt polyomavirus virions. At physiologic ionic strength, incubation with dithiothreitol (DTT), EGTA, or DTT plus EGTA did not disrupt BPV virions as determined by electron microscopy. However, incubation of virions with DTT rendered the BPV L1 protein susceptible to trypsin cleavage at its carboxy terminus and rendered the genome susceptible to digestion with DNase I. When DTT-treated BPV virions were analyzed by analytical ultracentrifugation, they sedimented at 230S compared with 273S for untreated virions, suggesting a capsid shell expansion. Incubation with EGTA had no effect on trypsin or DNase I sensitivity and only a small effect upon the virion S value. A single cysteine residue conserved among BPV and human papillomavirus (HPV) L1 proteins resides within the trypsin-sensitive carboxy terminus of L1, which is required for capsid assembly. A recombinant HPV type 11 L1 protein, which was purified after expression in Escherichia coli and which has a Cys-to-Gly change at this position (Cys424), formed pentamers; however, unlike the wild-type protein, these mutant pentamers could no longer assemble in vitro into capsid-like structures. These results indicate an important role for interpentamer disulfide bonds in papillomavirus capsid assembly and disassembly and suggest a mechanism of virus uncoating in the reducing environment of the cytoplasm.

Early phase in the infection of cultured cells with papillomavirus virions.

The fate of full bovine papillomavirus (BPV) virions and virus-like particles after binding to C127 or CV-1 cells was studied by electron microscopy and indirect immunofluorescence. After incubation at 4 degrees for 1 hr, BPV virions were found to be bound to the plasma membrane, and most viruses were absorbed by the cells after 30 min incubation at 37 degrees. Ninety minutes after the virions had been bound to the plasma membrane, the uptake of the virions was completed and most of the antigen was found to be localized in the nucleus. The viruses were transported in phagosomes and the uptake and transportation could be inhibited by cytochalasin B and taxol, suggesting the possible involvement of microfilaments and microtubules in the virus particle uptake and transportation. The capsid proteins could be detected for about 14 hr, until degradation and deposit of the viral antigen in the Golgi complexes. Although binding to the plasma membrane and uptake of virions into large cytoplasmic vesicles could be monitored by electron microscopy, no complete virions were observed in the nucleus of infected cells despite a very strong nuclear fluorescent staining for both L1 and L2 proteins. This may indicate that disintegration of the virions occurs in the cytoplasm and the L1/L2 proteins migrate to the nucleus via their nuclear localization signals.

Inactivation of papillomavirus by low concentrations of povidone-iodine.

BACKGROUND AND OBJECTIVES: A recent report by Hermonat et al showed that nonoxynol 9 is completely inactive against bovine papillomavirus, which is very closely related to human papillomavirus. Finding a vaginal microbicide active against human papillomavirus to reduce the risk of sexual transmission of human papillomavirus would be desirable. GOAL OF THIS STUDY: To determine whether povidone-iodine is active in vitro against bovine papillomavirus. METHODS: A bovine papillomavirus-1 stock prepared by extraction of a fibropapilloma was treated with various concentrations of povidone-iodine. The virus/povidone-iodine samples were incubated at 37 degrees C for 15 minutes and then placed on contact-inhibited cells of mouse fibroblast line C127 in 10-cm tissue culture dishes for the transformation assay. At 2 weeks post-infection, oncogenic foci induced by bovine papillomavirus appeared and were counted after the cells were fixed with 4% formaldehyde and stained with methylene blue. RESULTS: Approximately 90% inactivation of papilloma virus was demonstrated with exposure to 0.1% povidone-iodine, and 99.9% inactivation was seen at 0.3%. CONCLUSIONS: The concentrations of povidone-iodine that were effective in this study are lower than concentrations in available over-the-counter preparations of povidone-iodine. Additional research is needed to verify whether papillomavirus is susceptible to other, more acceptable agents. Clinical trials may be warranted to determine whether povidone-iodine or other agents would reduce the rate of sexual transmission of the human papillomavirus strains associated with cervical cancer.

In vitro evaluation of phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential treatment for genital warts.

Papillomaviruses induce benign proliferative lesions, such as genital warts, in humans. The E2 gene product is thought to play a major role in the regulation of viral transcription and DNA replication and may represent a rational target for an antisense oligonucleotide drug action. Phosphorothioate oligonucleotides complementary to E2 mRNAs were synthesized and tested in a series of in vitro bovine papillomavirus (BPV) and human papillomavirus (HPV) models for the ability to inhibit E2 transactivation and virus-induced focus formation. The most active BPV-specific compounds were complementary to the mRNA cap region (ISIS 1751), the translation initiation region for the full-length E2 transactivator (ISIS 1753), and the translation initiation region for the E2 transrepressor mRNA (ISIS 1755). ISIS 1751 and ISIS 1753 were found to reduce E2-dependent transactivation and viral focus formation in a sequence-specific and concentration-dependent manner. ISIS 1755 increased E2 transactivation in a dose-dependent manner but had no effect on focus formation. Oligonucleotides with a chain length of 20 residues had optimal activity in the E2 transactivation assay. On the basis of the above observations, ISIS 2105, a 20-residue phosphorothioate oligonucleotide targeted to the translation initiation of both HPV type 6 (HPV-6) and HPV-11 E2 mRNA, was designed and shown to inhibit E2-dependent transactivation by HPV-11 E2 expressed from a surrogate promoter. These observations support the rationale of E2 as a target for antiviral therapy against papillomavirus infections and specifically identify ISIS 2105 as a candidate antisense oligonucleotide for the treatment of genital warts induced by HPV-6 and HPV-11.

High rotational mobility of DNA in animal cells and its modulation by histone acetylation.

DNA rotational mobility in a bovine papilloma virus (BPV)-based minichromosome, autonomously replicating in mouse cells, was studied using topoisomer analysis in temperature shift experiments. It was found that in live cells the average number of topological turns increased by six in the course of temperature shift through a range of 37 degrees C. This comprised approximately 85% of the total potential mobility of naked plasmid DNA. DNA rotation in isolated nuclei was found to be 3.5-4.0 turns per 37 degrees C in 100 mM NaCl - much higher than in all experiments with animal cells reported thus far. In low salt mobility was considerably lowered. Attempts to extract minichromosomes from nuclei allowed isolation of no more than 10% of minichromosomal DNA, with could indicate a very high proportion of transcriptionally active minichromosomes in the intracellular population. Growing cells in the presence of sodium butyrate resulted not only in an increase in the level of plasmid superhelicity and a decrease of its transcription (as we report in the accompanying publication) but also reduced rotational mobility of plasmid DNA threefold (from 6 to 2 turns per 37 degrees C). The decrease in DNA rotational mobility after butyrate treatment was also partially manifested in isolated nuclei (especially at lower ionic strength). To check whether histone acetylation is directly responsible for DNA immobilization, we performed in vitro acetylation of histones using acetyl adenylate. This resulted in severe DNA immobilization in experiments using both up and down temperature shifts.

Transforming growth factor-beta expression in fibropapillomas induced by bovine papillomavirus type 1, in normal bovine skin, and in BPV-1-transformed cells.

There is substantial evidence to suggest that transforming growth factor-beta (TGF-beta) plays an important role in wound healing and tissue repair as well as in carcinogenesis. It has also been observed that naturally occurring bovine papillomavirus type 1 (BPV-1)-induced bovine fibropapillomas occur predominantly at traumatized sites of the body, suggesting that humoral factors released in wounds might be important for papillomavirus infection. We have therefore investigated the possible role of TGF-beta 1 in BPV-1 infections. Two antipeptide antibodies which recognize different epitopes in the N-terminus of TGF-beta 1 were used to localize TGF-beta 1 in bovine fibropapillomas and normal bovine skin using immunohistochemical methods. Staining by anti-LC(1-30) is intracellular in suprabasal keratinocytes of the epidermis as well as the hair follicles and sebaceous glands and correlates with known sites of TGF-beta 1 mRNA synthesis. Anti-CC(1-30) staining is extracellular in the immediately underlying dermis. Neither the pattern nor intensity of TGF-beta 1 staining was affected by BPV-1 infection. C127 cells and BPV-1-transformed C127 cells were compared for TGF-beta 1 mRNA expression and secretion of TGF-beta 1 peptide. Although the levels of messenger RNA and secreted TGF-beta 1 peptide were similar in both cell types, five- to six-fold greater amounts of TGF-beta-like activity per cell was detected in media conditioned by the uninfected cells. TGF-beta 1 treatment had no effect on the growth rate of either cell type or on BPV-1 gene expression in the transformed cells.

Characterization of bovine papillomavirus type 1-transformed clones which show distinct transformed phenotypes.

Seventeen independent cell clones were isolated from C127 cells transformed by bovine papillomavirus type 1 (BPV-1). Transformants showed differing degrees of expression of the transformed phenotype as monitored by saturation density, doubling time, growth in medium with a low serum concentration and colony-forming efficiency in soft agar. The degree of expression of the transformed phenotype did not correlate with either the BPV-1 copy number or levels of BPV-1-specific RNA in the transformed cell clones. A characteristic transformed cell clone, T1c, showed the lowest degree of expression of the transformed phenotype but contained the highest copy number of BPV-1 DNA and the highest level of BPV-1-specific mRNA. When we analysed different transformants by two-dimensional gel electrophoresis, we found that a set of six proteins changed quantitatively. Changes in the expression of these proteins were most consistent in clones expressing the greatest number of parameters of transformation, e.g. clone T4a. These data indicate that changes in the expression of cellular genes may correlate with the degree of expression of the transformed phenotype.

Changes in DNA copy numbers of bovine papillomavirus type 1 after termination of retinoic acid treatment.

The number of bovine papillomavirus type 1 (BPV) DNA copies [plasmid pdBPV-1 (142-6)] was examined in transformed C127 cells of an RIII mouse during exposure to all-trans-retinoic acid (RA) and after its withdrawal. RA treatment of a transformed cell line reduced the number from approximately 60 copies to an average of less than one copy per cell within 5 weeks. The composition of the RA-treated cell population was heterogeneous with respect to BPV DNA copies: 89.7% of the cells had no detectable copies, 8.6% had one copy, 1.7% had fewer than five copies, and one in 13,000 cells carried more than 10 copies. The low number of BPV DNA copies in the RA-treated cell population did not increase when the cells were subcultured before reaching confluence. RA-treated cell populations that contained less than one BPV DNA copy lost the transformed phenotype. However, a small fraction of cells (1 in 13,000) with greater than or equal to 10 BPV DNA copies retained the capacity to develop into transformed colonies. The relevance of these results to the regression of papillomavirus, DNA-carrying human lesions after exposure to retinoids and the redevelopment of these lesions after withdrawal of retinoids is discussed.

Differential regulation of papilloma virus early gene expression in transformed fibroblasts and carcinoma cell lines.

Treatment of bovine papilloma virus (BPV) 1-transformed mouse fibroblasts with cycloheximide led to a 10-fold increase in the amount of viral transcripts, after as little as 1 h of protein synthesis inhibition. Northern blots revealed no qualitative changes in the RNA pattern. Nuclear run-on experiments showed about a 7-fold increase in specific transcriptional activity after cycloheximide treatment. The half-life of BPV1 mRNA was twice as long as in untreated controls. These results indicate that both RNA synthesis and degradation of viral RNA are controlled by labile proteins. Cycloheximide stimulation turned out to be independent of the BPV1 E2 gene activity which enhances viral transcription. Cycloheximide treatment had no effect on the amount of human papilloma virus (HPV) 18 transcripts in cervical carcinoma derived HeLa and C4-1 cells. Transcription of HPV16 in the cervical carcinoma line SiHa was likewise unaffected. The differential regulation of transcription in transformed fibroblasts and cancer-derived cells, and the significance for malignant conversion are discussed.