Genomic diversity and evolution of papillomaviruses in rhesus monkeys.

We are studying the diversity of and relationships among papillomaviruses (PVs) to understand the modes and timescales of PV evolution and in the hope of finding animal PVs that may serve as model systems for disease caused by human PVs (HPVs). Toward this goal, we have examined 326 genital samples from rhesus monkeys and long-tailed macaques with a PCR protocol optimized for detecting genital HPV types. In 28 of the rhesus monkey samples, we found amplicons derived from 12 different and novel PV genomes, RhPV-a to RhPV-m, with the likely taxonomic status of "type." The frequency with which novel RhPVs were detected suggests that rhesus monkeys may play host to PVs with a diversity similar to that of humans. In phylogenetic trees, all 12 of the different RhPVs and the previously described type RhPV-1 were members of the genital HPV supergroup and formed three minor branches distinct from the 11 branches formed by genital HPVs. We also identified a novel PV amplicon, MfPV-a, from a long-tailed macaque, a species belonging to the same genus as rhesus monkeys. MfPV-a turned out to be a close relative of five RhPVs. It appears that the evolution of primate lineages leading to the genus Macaca and to humans created transmission barriers for PVs, resulting in viral evolution closely linked to the host. Additional support for the linked-evolution hypothesis comes from considering the phylogenetic association of two other ape and monkey PVs with the genital HPVs, the supergroup formed by at least seven ungulate PVs, and the isolated phylogenetic position of the only known bird PV.

Genital papillomaviruses (PVs) and epidermodysplasia verruciformis PVs occur in the same monkey species: implications for PV evolution.

Portions of the genome from two different papillomaviruses (PVs) of the Abyssinian Colobus monkey were sequenced and phylogenetically analyzed. This revealed that the major evolutionary separation between genital PVs and epidermodysplasia verruciformis-associated PVs (EV-PVs) hitherto found only in human papillomaviruses (HPVs) also exists in animal PVs. The sequence of the long control region (LCR) of Colobus monkey PV type 2 (CgPV-2) reveals a small size and an arrangement of potential cis-responsive elements typical of the EV-HPVs; namely four binding sites for the viral E2 protein, with one of them being located within the L1 gene, a cluster of nuclear factor I (NFI)- and AP-1-binding sites and a 50-bp sequence upstream of the E6 gene consisting only of the nucleotides A and T. This level of conservation of functional elements within the highly variable LCR suggests that CgPV-2 could be adopted as a model for studying human skin cancer associated with EV-HPVs. Although isolated from the same monkey species, the other Colobus monkey PV, CgPV-1, is a typical genital PV as shown by E1 and L1 sequence comparisons. The presence of these two major phylogenetic divisions of PVs in both human and monkey hosts strongly suggests that this diversification predated the evolutionary split between monkeys and apes. In other words, at least two different groups of PVs have been evolving separately in their respective primate hosts for more than 22 million years with only moderate sequence changes since their genesis.

The E5 gene product of rhesus papillomavirus is an activator of endogenous Ras and phosphatidylinositol-3'-kinase in NIH 3T3 cells.

We examined the effect of two rhesus papillomavirus 1 (RhPV) oncogenes on cytokine-induced signal transduction pathways leading to the possible activation of Ras protein (p21ras) and phosphatidylinositol kinase. p21ras in both the activated (GTP-bound) and inactivated (GDP-bound) states were quantitated. NIH 3T3 cell lines expressing the RhPV 1 E5 gene or epidermal growth factor receptor cDNA had about a sixfold higher ratio of p21ras-bound GTP to p21ras-bound GDP as compared with parental NIH 3T3 cells or a cell line expressing the RhPV 1 E7 gene under normal culture conditions, yet expressed similar levels of p21ras. Quiescent cells had dramatically reduced levels of activated p21ras, except those containing RhPV 1 E7. Levels were restored by stimulation with epidermal growth factor or platelet-derived growth factor. Both epidermal growth factor and platelet-derived growth factor receptor of RhPV 1 E5- and E7-containing cells responded to cytokine stimulation. Endogenous phosphatidylinositol-3'-kinase was up-regulated in NIH 3T3 cells transformed with the E5 genes of RhPV 1 and bovine papillomavirus 1. These results suggest that E5 genes of papillomaviruses play a major role in the regulation of transduction pathways.

The expression levels of the human papillomavirus type 16 E7 correlate with its transforming potential.

The transforming potential of the human papillomavirus (HPV) type 16 has been defined largely in the E7, E6, and E5 oncoproteins, with the major transforming capability residing in the E7 gene. In this paper, we found that in cooperation with the activated ras, the HPV16 E7 gene when expressed in a retroviral vector could fully transform baby rat kidney (BRK) cells in transfections, whereas the same construct could only immortalize the BRK cells following retroviral infection. This inability to transform correlated with the low levels of E7 gene RNA expression in the viral infected cells, which harbor a lower number of copies of the E7 gene constructs. Cotransfection of the expression vector FV2E7, which gives high levels of E7 gene expression, and activated ras lead to rapid and efficient morphological transformation of BRK cells which grew easily in soft agar and induced large tumors in athymic nude mice. In contrast, cotransfections of the expression vector FV1E7, which gives lower levels of E7 gene expression, produced much lower numbers of transformed colonies which took longer to form, showed a retarded growth on soft agar, and induced smaller tumors in nude mice. Under these conditions, colonies of immortalized, but morphologically untransformed cells formed in large numbers. These results indicate that the transforming potential is directly correlated to the expression levels of the oncoprotein and that a threshold level of the E7 oncoprotein may be required before the cells can be fully transformed. This supports the hypothesis that the transformation processes include at least two separate and continuous steps which first lead to immortalization and then to metastasis, in agreement with the clinical progression of genital tumors from benign to malignancy. Such a progression may involve enhanced expression of the oncoproteins.

Serological and molecular evidence of rhesus papillomavirus type 1 infections in tissues from geographically distinct institutions.

We have previously demonstrated the presence of rhesus monkey papillomavirus type 1 (RhPV-1), from molecular and pathological evidence, in a mating group within a single institution. We have now also obtained a number of fresh or archival tissues of rhesus monkeys from other geographically distinct institutions. Using PCR amplification, we observed two animals from one of these institutions and five animals from another which demonstrated RhPV-1 DNA sequences. In addition we molecularly cloned the E7, E2, E4, L2 and L1 genes of RhPV-1 into bacterial expression vectors. The fusion gene products were used to test for serological response to RhPV-1 antigens by Western blot analysis. Responses were observed in up to 52% of the animals tested. While some serologically positive animals were also RhPV-1 DNA-positive, most were not.

The E6 gene of human papillomavirus type 16 is sufficient for transformation of baby rat kidney cells in cotransfection with activated Ha-ras.

The transforming potential of the E6 open reading frame (ORF) of the human papillomavirus type 16 was investigated with transformation assays in cotransfections with an activated ras gene. The E6 ORF driven by the heterologous CMV promoter could fully transform baby rat kidney cells (BRK) in cooperation with ras. The transformed cells grew in soft agar and induced tumors in athymic nude mice. The E6 ORF with mutations at the splicing donor site, which only encodes the full length E6 but not E6*s, could also fully transform the BRK cells at a similar efficiency as the wild-type E6 ORF, indicating that the full-length E6 was sufficient for the transformed phenotype.

Topical CTC-96 accelerates wart growth in rabbits infected with cottontail rabbit papillomavirus.

CTC-96, a cobalt containing complex, was tested as a putative topical therapeutic agent for the treatment of papillomavirus-induced tumors in our cottontail rabbit papillomavirus (CRPV)-rabbit model system. Following experimental infection of domestic rabbits with CRPV, CTC-96 was applied to infection sites twice daily, 5 days a week for a total of 8 weeks. Two levels of concentrations of aqueous CTC-96 were compared to placebo control-treated animals. With increasing dose of CTC-96 we observed tumors earlier, larger, and more often across eight infected sites on each animal.

Pilot trial of ribavirin for the treatment of laryngeal papillomatosis.

The antiviral drug ribavirin was used as an adjunct to laser surgery for the treatment of patients with laryngeal papillomatosis (LP). An uncontrolled clinical trial for four patients with ribavirin treatment at a daily dose of 23 mg/kg was performed. Three adults received drug prior to laser surgery and continuing orally for 6 months. One infant was treated for 3 months. Two adults achieved complete remissions for at least 2 consecutive months, and both patients developed only minimal recurrent disease in 4 months of follow-up. The other adult and the child sustained a partial response and an increased interval between the required surgeries. Ribavirin caused only a mild, reversible reduction in hemoglobin and reticulocytosis. This preliminary trial shows that ribavirin may be an effective therapy in combination with surgery for LP in a larger controlled clinical trial.

The products of the E5, E6, or E7 open reading frames of RhPV 1 can individually transform NIH 3T3 cells or in cotransfections with activated ras can transform primary rodent epithelial cells.

Rhesus papillomavirus (RhPV) type 1 was recently shown to cooperate with the activated ras oncogene to transform primary rodent epithelial cells at a level comparable to HPV 16. In similar cotransfection studies, subgenomic portions of RhPV 1 driven by either their natural or a strong heterologous promoter were used in primary baby rat kidney cells to demonstrate that transforming properties of RhPV 1 could be localized individually to the E5, E6, and E7 open reading frames. Fully transformed cells were observed when either E5 or E7 were downstream of a strong heterologous promoter. Similarly, either E6 or E6 and E7 downstream of the native promoter fully transformed these cells as determined by immortalization, anchorage independent growth and tumorigenicity studies.

Cellular transformation by a unique isolate of human papillomavirus type 11.

Infection with human papillomavirus type 11 (HPV 11) is associated with benign epithelial proliferations and rarely with malignant and metastasizing tumors. Because of the biological diversity displayed in tissues infected with HPV 11, we have examined the capacity of various isolates of HPV 11 to transform cultured cells and compared their molecular differences by DNA sequence analysis. Five isolates of HPV 11 were examined for their ability to transform primary neonatal rat kidney epithelial cells and NIH 3T3 mouse fibroblasts in DNA transfection experiments using calcium phosphate precipitation. Included in these studies are the prototype isolate from a laryngeal papilloma (HPV 11P); HPV 11VC from a verrucous carcinoma of the penis; HPV 11Epi from the viral episomes of a primary squamous cell carcinoma; and two integrated genomes (HPV 11Int 1 and HPV 11Int 2) of the metastases. Only HPV 11VC cotransfected with the oncogene Ha-ras transformed neonatal rat kidney epithelial cells with an efficiency comparable to that of HPV 16 DNA. HPV 11VC DNA alone transformed NIH 3T3 cells. Analysis of the DNA sequence of HPV 11P and 11VC revealed 16 single nucleotide changes in the upstream regulatory region and open reading frames E1, E2, E4, and E5, five resulting in amino acid substitutions. This is the first demonstration of cellular transformation by a natural isolate HPV 11 DNA in vitro and illustrates that minimal changes in the DNA sequence of certain viruses confer oncogenicity to what are normally nontransforming viruses.

Ribavirin mitigates wart growth in rabbits at early stages of infection with cottontail rabbit papillomavirus.

The challenge to develop antiviral agents effective against DNA viruses such as human papillomavirus (HPV) has been dependent on finding an animal model which mimics the human forms of the disease. We have used an existing model system for the purpose of measuring the effect of antiviral drugs on the inhibition of growth of these lesions. This was based upon domestic rabbits which efficiently grow cutaneous papillomas (warts) when infected with cottontail rabbit papillomavirus (CRPV). One agent which had shown significant success in achieving these goals was ribavirin. Ribavirin was administered intradermally shortly prior to infection at multiple sites with CRPV. Following daily injections of this drug for eight weeks, we have shown a dose-dependent response which had markedly reduced the number of warts, the time of first appearance of warts and reduced the tumor mass as compared to placebo-treated control animals. At the highest dose of ribavirin tested, 30 mg/kg/day, compared to controls, the average reduction in the number of warts was 52%, the average time of first appearance of warts was 49% longer, and the average mass of the warts was reduced by 98%. No detectable antibodies to CRPV were observed in any of the animals. The only side effects which were observed was focal alopecia, and a decrease in body growth upon prolonged treatment, both of which were completely reversible. Pharmacokinetic studies established the metabolism of ribavirin over a 24-h period of time. Ribavirin administered beginning 12 or 30 days post-infection, while not reducing the number of warts, slightly retarded the growth of warts as determined by date of first appearance of warts and mass of warts.

Implication of human papillomavirus in postirradiation dysplasia.

The presence of human papillomavirus (HPV) DNA and association of condylomata acuminata (CA) in the biopsy tissues of postirradiation dysplasia (PRD) of the cervix and/or vagina from 17 patients who previously had radiation therapy for malignancies of the uterine cervix, vagina, and endometrium were evaluated with DNA in situ hybridization. Eight of 17 patients (47.1%) had HPV DNA identified in the lesions of postirradiation dysplasia (PRD). Five of eight cases (62.5%) contained HPV DNA of more than one type. Type 16 HPV DNA (HPV-16) was the most frequently identified type. Several PRD lesions also contained HPV-6, HPV-18, HPV-31, and/or HPV-33 DNA. Eleven patients (64.7%) showed CA in the vicinity of PRD. In two cases, different types of HPV were found in the lesions of PRD and contiguous CA. The frequency of the cases containing HPV DNA, the types of HPV, and the distribution pattern of silver grains in the preparations of in situ hybridization over the nuclei of cells of PRD were very similar to those found in naturally occurring dysplasia. Based on these findings, persistent or repeat HPV infection was the most likely etiologic factor of PRD, which might be facilitated by immunosuppression due to pelvic irradiation.

Expression of cytokine receptors and markers of differentiation in human papillomavirus-infected cervical tissues.

Human papillomavirus infection of the uterine cervix is associated with a spectrum of benign, premalignant, and malignant epithelial lesions, a process that appears to require the coordinated effects of secondary cellular and environmental events. We have used flow cytometry and immunohistochemistry to examine the expression of the cellular markers for proliferation (interleukin-1, epidermal growth factor receptor, and transferrin receptor) and the markers of cellular differentiation (filaggrin and low-molecular-weight cytokeratin) in normal and human papillomavirus--infected human cervical tissues representing the natural range of human papillomavirus--induced disease. The results were correlated with the histologic grade of disease, human papillomavirus type, cellular deoxyribonucleic acid content, and cell cycle status. Interleukin-1 and transferrin receptor were slightly increased in high-grade dysplasias and in squamous cell carcinomas. Filaggrin expression was found to be inversely related and cytokeratin and epidermal growth factor receptor expression directly related to the degree of neoplasia. These findings indicate that cytokeratin and epidermal growth factor receptor are useful markers of cell proliferation in human papillomavirus--infected tissues and that their expression may directly increase as a result of infection.

Rhesus papillomavirus type 1 cooperates with activated ras in transforming primary epithelial rat cells independent of dexamethasone.

Rhesus Papillomavirus type 1 (RhPV-1) was recently cloned from a rhesus monkey lymph node metastasis of a penile squamous cell carcinoma. In this paper, we demonstrate that RhPV-1 cooperates with the activated ras oncogene to transform primary cells at a level comparable to human papillomavirus type 16. The viral DNAs were cloned such that their expression was under the control of their natural promoter elements. Unlike human papillomavirus type 16, RhPV-1 DNA cooperated with ras independently of the hormone dexamethasone. However, dexamethasone did have a positive influence on the ability of some RhPV-1 cotransformed cells to grow in soft-agar assays. The transformed cells are highly tumorigenic in vivo in nude mice.

Characterization of the complete RhPV 1 genomic sequence and an integration locus from a metastatic tumor.

The complete nucleotide sequence of the rhesus papillomavirus type 1 (RhPV 1) genome was determined. The genome is 8026 nucleotides in length and has a genomic organization similar to that of other characterized papillomaviruses. Sequence comparison of RhPV 1 to other papillomaviruses found similarities closest to HPV 16, a sexually transmitted human virus with a high oncogenic potential. Slight differences in the glucocorticoid responsive elements may explain disparate reliance upon added dexamethasone for transformation in vitro of these two papillomaviruses. In addition, a previously described DNA clone consisting of contiguous RhPV 1 and cellular sequences was partially sequenced. The disruption of the RhPV 1 genome due to integration occurred within the L1 open reading frame of RhPV 1, and no significant similarities were observed between the adjacent cellular sequences and information in various data banks.

A rhesus monkey model for sexual transmission of a papillomavirus isolated from a squamous cell carcinoma.

Recently we molecularly cloned and characterized a papillomavirus from a lymph node metastasis of a primary penile carcinoma found in a rhesus monkey; this virus species, rhesus papillomavirus type 1 (RhPV-1), is similar to oncogenic human papillomaviruses (HPVs), such as HPV-16 or HPV-18, in that the RhPV-1 DNA was found to be integrated in the tumor cell DNA. To compare the sexual transmission and oncogenic nature of RhPV-1 with these HPVs, we undertook an extensive retrospective study of a group of rhesus monkeys whose sexual mating and offspring histories were known. These animals had mated directly with the index male mentioned above or were secondarily exposed to this virus through intermediate sexual partners. This study combines cytological, histopathological, and several complementary hybridization and DNA amplification techniques on multiple tissue samples to demonstrate the sexually transmitted nature of RhPV-1. The oncogenic potential of RhPV-1 is suggested in several of the infected animals by the presence of various degrees of neoplasia including squamous cell cancer of the cervix.

Human papillomavirus type 27: detection of a novel human papillomavirus in common warts of a renal transplant recipient.

The cloning and partial characterization of the genome of human papillomavirus type 27 (HPV-27) is described. Hybridization analyses reveal that this is a new HPV type, with the strongest homology to HPV-2.

Human papillomavirus DNA in glandular dysplasia and microglandular hyperplasia: presumed precursors of adenocarcinoma of the uterine cervix.

Presumed precursors of adenocarcinoma of the uterine cervix were investigated with specific techniques to identify human papillomavirus (HPV) DNA. The presence of HPV DNA in 36 lesions of glandular dysplasia and 16 lesions of microglandular hyperplasia of the uterine cervix was studied by in situ hybridization using 3H-labeled HPV 16 and HPV 18 DNA probes. Only two of 36 lesions (6%) of glandular dysplasia contained HPV 18 DNA, although 64% of coexisting adenocarcinoma in situ, microinvasive adenocarcinoma, and cervical squamous intraepithelial neoplasia III lesions contained HPV 18 and/or HPV 16 DNA. Two lesions of HPV 18 DNA-positive glandular dysplasia coexisted with adenocarcinoma in situ that contained the same type of HPV DNA. None of the microglandular hyperplasia lesions contained HPV 16 DNA or HPV 18 DNA. These results suggest that, if HPV infection is an initial step toward carcinogenesis, it is unlikely that glandular dysplasia and microglandular hyperplasia are precursor lesions of adenocarcinoma of the uterine cervix. A large proportion of glandular dysplasia may represent reactive lesions of endocervical columnar epithelium. Two lesions of HPV 18 DNA-positive glandular dysplasia may represent well-differentiated components of adenocarcinoma in situ of the uterine cervix.

Human papillomavirus-16 DNA in a cutaneous invasive cancer.

A middle-aged man with a 20-year history of a warty lesion on his finger was diagnosed histologically as having an invasive carcinoma at that location. Molecular analysis of the lesion showed that human papillomavirus (HPV) DNA was present in this tumor. Under stringent hybridization conditions, only HPV-16 DNA, which is commonly associated with genital neoplasia or cutaneous bowenoid lesions, was detected. Two-dimensional gel electrophoresis revealed that various sections of the tumor contained mostly episomal viral DNA; but in one case, low levels of integrated HPV-16 DNA were detected as well.