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Papillomavirus L1 and L2, the major and minor capsid proteins, play significant roles in viral assembly, entry, and propagation. In the current study, we investigate the impact of L1 and L2 on viral life cycle and tumor growth with a newly established mouse papillomavirus (MmuPV1) infection model. MmuPV1 L1 knockout, L2 knockout, and L1 plus L2 knockout mutant genomes (designated as L1ATGko-4m, L2ATGko, and L1-L2ATGko respectively) were generated. The mutants were examined for their ability to generate lesions in athymic nude mice. Viral activities were examined by qPCR, immunohistochemistry (IHC), in situ hybridization (ISH), and transmission electron microscopy (TEM) analyses. We demonstrated that viral DNA replication and tumor growth occurred at both cutaneous and mucosal sites infected with each of the mutants. Infections involving L1ATGko-4m, L2ATGko, and L1-L2ATGko mutant genomes generally resulted in smaller tumor sizes compared to infection with the wild type. The L1 protein was absent in L1ATGko-4m and L1-L2ATGko mutant-treated tissues, even though viral transcripts and E4 protein expression were robust. Therefore, L1 is not essential for MmuPV1-induced tumor growth, and this finding parallels our previous observations in the rabbit papillomavirus model. Very few viral particles were detected in L2ATGko mutant-infected tissues. Interestingly, the localization of L1 in lesions induced by L2ATGko was primarily cytoplasmic rather than nuclear. The findings support the hypothesis that the L2 gene influences the expression, location, transport, and assembly of the L1 protein in vivo.

This is a photograph of Peyton Rous (Francis Peyton Rous, 1879–1970), who in 1909–11 made 2 seminal discoveries that are now the foundation blocks of modern virology and oncology. First, he discovered that a malignant tumor (a sarcoma in chickens) was transmissible; this was the first transmissible solid tumor discovered. Second, he found that the tumor-inducing factor could be passed through a Berkefeld ultrafilter known to retain bacteria. In the context of the times, this finding proved that the agent was a virus, the first of its kind. The virus he discovered is now known as Rous sarcoma virus and has since been studied in many laboratories around the world. Rous was born in 1879 in Baltimore, Maryland, USA, and raised by his widowed mother, who persevered in supporting his education. He received bachelor of arts and doctoral degrees from The Johns Hopkins University in Baltimore in 1900 and 1905, respectively. After teaching pathology at the University of Michigan, Ann Arbor, and studying morbid anatomy at Friedrichstadt Municipal Hospital in Dresden, Germany, in 1909, he took a position at the Rockefeller Institute for Medical Research in New York, New York, where he spent the rest of his life. Rockefeller was the place in the United States where virology first emerged as a distinct medical science; from the beginning of his career, Rous was surrounded by great scientists. Rous’ entry into tumor virology was fortuitous; the founding director of the Rockefeller Institute, Simon Flexner, had been interested in oncology but wanted to redirect his own work toward polio, which was becoming a major problem. Rous was hired to continue Flexner’s research into oncology, a subject about which Rous at first knew nothing. The beginning of the story behind Rous’ claim to fame is remarkable: a woman came to the Rockefeller Institute with a barred Plymouth Rock hen that had a large tumor on its breast. Rous later wrote, “In this paper is reported the first avian tumor that has proved transplantable to other individuals. It is a spindle-celled sarcoma of a hen, which has thus far been propagated to the fourth generation….” In his research, he found that only closely related chickens were susceptible, but in these chickens, continuous passage of cell-free material led to tumors that grew quickly, were more malignant than usual, and produced widespread metastases. Rous continued to study the phenomenon he had begun to unravel for many years, but understanding the mechanistic bases for the complex natural history of Rous sarcoma virus and related viruses had to wait until modern molecular and cellular biologic technologies became available in the 1960s. In 1934, Rous’ Rockefeller Institute colleague, Richard E. Shope, asked him to examine warts on jackrabbits that had definitively been shown to be caused by an ultrafilterable virus. This virus was Shope papillomavirus (rabbit papillomavirus). When Rous confirmed that the warts were benign tumors, he was reinvigorated in his intent to unravel the mysteries of viral oncology. Over the next 30 years, Rous and his colleagues showed that the benign tumors could progress to malignant carcinomas and that chemical carcinogens could interact with the virus—further discoveries that formed building blocks for modern virology. Today, we recognize that ≈20% of human cancers worldwide have infectious etiologies, for which preventive measures such as vaccines have great promise. Rous’ colleagues, including the scientists Rene Dubos and Charles B. Huggins, lauded Rous’ personal and professional qualities, writing that he was gifted with supreme intellectual powers, unfailing integrity and honesty, a remarkably intuitive sense for the science itself, great perseverance and work ethic, and an enormous zest for life. One can imagine with wonder Rous in his laboratory and in the legendary lunchroom of the Rockefeller Institute. Rous was duly honored for his masterful work, winning the National Medal of Science and membership in the National Academy of Sciences, the American Philosophical Society, the Royal Society, and other prestigious organization. In 1966, when he was 87 years old, Rous was awarded the Nobel Prize in Medicine, an honor he shared with Charles Huggins. After 55 years, the longest “incubation period” in the history of the Nobel Prizes, Rous’ discovery had finally been recognized with this honor. In the end, proof that Rous was just ahead of his time might be found in the several additional Nobel Prizes awarded since 1966 to virologists who further unraveled viral oncology. Figure Francis Peyton Rous (1879–1970), pictured in 1923, at age 44, in his laboratory at the Rockefeller Institute for Medical Research, New York, NY, USA. Source: National Library of Medicine.

Chimeric human papillomavirus virus-like particles (HPV cVLP) are currently being explored as a therapeutic vaccination strategy against cervical cancer. HPV cVLP are being explored as a result of their interaction with and activation of dendritic cells, a potent antigen-presenting cell. However Langerhans cells, another type of antigen-presenting cell, can interact with HPV cVLP especially during mucosal routes of vaccine administration. Langerhans cells are not activated by HPV cVLP, utilize a different endocytosis mechanism than DC for HPV cVLP uptake, do not initiate an immune response toward HPV cVLP derived antigens, and are potentially immunosuppressive after interaction with HPV cVLP. Taken together, these findings indicate that the overall effectiveness of HPV cVLP as a therapeutic vaccine may be reduced. Bovine papillomavirus (BPV) VLP, cotton-tail rabbit papillomavirus (CRPV) VLP, and HPV VLP immune complexes (IC), which are taken up via similar endocytosis mechanisms in DC and LC, activate both cell types. DC and LC incubated with these VLP upregulate surface activation markers and increase secretion of IL-12 p70. The activated cells are then able to initiate an immune response against chimeric VLP-derived antigens. These data indicate that other therapeutic vaccination strategies based on using chimeric BPV VLP, chimeric CRPV VLP, or chimeric HPV VLP immune complexes may be more effective in generating an immune response against HPV-induced diseases such as cervical cancer.

A DNA vaccine encoding the major capsid protein L1 of cottontail rabbit papillomavirus (CRPV) was constructed and administered intracutaneously (i.c.) to rabbits as supercoiled plasmids bound to gold beads using a specialized delivery device (“gene gun”). L1 DNA-vaccinated rabbits developed cellular proliferative responses to CRPV virus-like particles and developed high titered antibodies with neutralizing activity to CRPV. Following experimental challenge with CRPV, all of the L1 DNA-vaccinated rabbits, vs none of the controls, were protected from papilloma formation. These results demonstrate that i.c. vaccination of rabbits with the L1 papillomavirus capsid gene can induce antibodies that protect against subsequent papillomavirus infection.

Leukocyte proliferation responses to cotton-tail rabbit papillomavirus (CRPV) were measured in vitro with fresh whole blood as well as with ammonium chloride lysis-separated leukocytes. The antigens used were (1) CRPV particles produced in the athymic( (nu/nu) mouse xenograft system and (2) purified bacterial fusion proteins of the CRPV major and minor capsid proteins L1 and L2. CRPV-infected domestic rabbits with persistent papillomas or after papilloma regression, as well as uninfected controls were studied. There was a clearcut difference between infected and uninfected animals. We demonstrated antigen-specific leukocyte proliferation to at least one CRPV antigen in 12 of 21 infected rabbits but there was no positivity in 9 control animals (P = 0.004). There was whole-blood reactivity preferentially to intact CRPV particles in regressors. Specific but weak leukocyte proliferation against CRPV particles was detected in 6 of 9 regressor rabbits (66%) but only in 1 of 12 progressors (8%; P = 0.0158). This trend of greater reactivity to intact CRPV particles in regressors as compared with progressors was not seen with peripheral blood leukocytes isolated by ammonium chloride lysis. We conclude that specific leukoproliferative responses against capsid CRPV proteins exist in rabbits experimentally infected with CRPV.

C an A V irus C ause C ancer ? A L ook into the H istory and S ignificance of O ncoviruses Niema Rwazavian T he I mportance of O ncoviruses Cancer, a disease caused by unregulated cell growth, is often attributed to chemical carcinogens (e.g. tobacco), hormonal imbalances (e.g. high levels of estrogen), or genetics (e.g. breast cancer susceptibility gene 1). While cancer can originate from any number of sources, many people fail to recognize another important etiology: oncoviruses, or cancer-causing (van Epps 2005). Although many in the scientific community were unconvinced of the role of viruses in cancer, research on the subject nevertheless continued. In 1933, Richard Shope discovered the first mammalian oncovirus, cottontail rabbit papillomavirus (CRPV), which could infect cottontail rabbits, and in 1936, John Bittner discovered the mouse mammary tumor virus (MMTV), which could be transmitted from mothers to pups via breast milk (Javier and Butle 2008). By the 1960s, with the additional “…despite limited awareness, oncoviruses are discovery of the murine leukemia (MLV) in mice and the SV40 nevertheless important because they represent virus in rhesus monkeys, researchers virus over 17% of the global cancer burden.” began to acknowledge the possibility that viruses could be linked to human cancers as well. The growing concern viruses. Oncoviruses are often overlooked because over the possibility of human cancer viruses lead to current preventative measures against cancer focus the creation of the U.S. Special Virus Cancer Program on modifiable lifestyle risk factors, such as diet and in 1964, which was devoted to the search for human exercise. Despite limited awareness, oncoviruses oncoviruses. are nevertheless important because they represent over 17% of the global cancer burden (Parkin 2006). discovered the first mammalian oncovirus, cottontail Studies have demonstrated that if these pathogens rabbit papillomavirus (CRPV), which could infect were eliminated, there would be 23.6% fewer cases of cancer in developing countries and 7.7% in developed countries, leading to a Preduction of 1.5 million and 390,000 cases of cancer per year, respectively (Parkin 2006). Given their significant yet surprising role in human cancer, oncoviruses are considered to be the second-most important risk factor after tobacco consumption for cancer development in humans (zur Hausen 1991). Currently, six viruses have been proven to cause over ten types of cancer in humans (see Table T he H istory of O ncovirus R esearch The discovery of oncoviruses dates back to the early 20th century, when in 1908 Oluf Bang and Vilhelm Ellerman, researchers at the University of Copenhagen, demonstrated that leukemia could be induced in healthy chickens by treating them with a filterable extract containing the avian leukemia virus (Javier and Butle 2008). Despite their results, the discovery of the first known tumor virus was credited to Payton Rous, who proved that the Rous sarcoma virus caused cancer in Plymouth Rock chickens B S J Figure 1. An electron micrograph of two Epstein Barr Virus virons. The dark sphere in the middle contains the virus’ genome. cottontail rabbits, and in 1936, John Bittner discovered the mouse mammary tumor virus (MMTV), which could be transmitted from mothers to pups via breast B erkeley S cientific J ournal • I nfectious D isease • F all 2010 • V olume 14 • I ssue 1 • 19

Publisher Summary This chapter focuses on the naturally occurring viral diseases of rabbits. Poxviruses cause several important diseases in domestic and wild mammals and birds. Infection with poxviruses usually results in relatively mild disease involving the skin of the infected animals, however, generalized and often fatal disease may also occur, for example, in myxomatosis in rabbits. Close antigenic relationships exist among many poxviruses derived from different animal species. In spite of close antigenic relationships, the poxviruses of rabbits that produce distinct disease syndromes are discussed as separate entities. Virus of the genus Papillomavirus cause papillomas in various animal species and includes two rabbit viruses, the cottontail rabbit papillomavirus and the rabbit oral papillomavirus. The genus Polyomavirus contains one virus of rabbits, the rabbit kidney vacuolating virus. Papillomatosis of wild cottontail rabbits is characterized by the presence of horny warts, usually on the neck, shoulders, or abdomen. The warts begin as red raised areas at the site of infection, grow to become typical papillomas with rough rounded surfaces, and may later develop into large, keratinized horny growths. The rabbit kidney vacuolating virus was isolated in primary rabbit kidney cell cultures from papillomas of cottontail rabbits collected in Kansas. This virus causes vacuolar cytopathic effects in cell cultures. The virus resembles the cottontail rabbit (Shope) papil lomavirus but is a distinct virus. It does not produce papillomas when inoculated into rabbits and does not immunize rabbits against rabbit papillomavirus.

In contrast to antivirion antisera raised against (Shope) rabbit papillomavirus (RPV) and human plantar wart papillomavirus (HPV-1), sera of rabbits bearing Vx7 carcinoma, a transplantable tumor deriving from a RPV-induced papilloma, permit the detection of an antigenic relationship between these viruses. Sera containing anti-RPV antibodies were collected between the 116th and 165th transplant from 10 rabbits selected for their large intramuscular tumors of long duration or for their purposely grafted tumors (both intramuscularly and intraperitoneally). When tested by immunofluorescence using plantar wart sections, seven of these sera detected an antigen whose location corresponds to that of HPV-1 capsid antigens. The reactivity of fluorescein-conjugated IgG, obtained from the most cross-reactive serum, was abolished by incubation with alkali-disrupted HPV-1 virions but not with intact HPV-1 particles. In contrast to an anti-HPV-1 virion antiserum, this Vx7 serum reacted with the main HPV-1 structural polypeptide (MW, 54,000) after SDS disruption and iodination of HPV-1 particles, as shown by radioimmunoprecipitation. Furthermore, one of the two anti-disrupted HPV-1 virion antisera and the two-anti-HPV-1 polypeptide antisera studied reacted with RPV antigens, as shown by immunofluorescence using cottontail rabbit wart sections. Moreover, one of the cross-reactive Vx7 sera gave a precipitin line continuous to that given by an anti-HPV-1 virion antiserum when tested by immunodiffusion using HPV-1 virions as antigens. This was further observed for four of the 110 additional Vx7 sera tested. The specificity of this reaction was confirmed by immune electron microscopy experiments. Similarly, an anti-HPV-1 polypeptide antiserum reacted with RPV virions. The results indicate the existence of two kinds of antigenic determinants shared by RPV and HPV-1 structural polypeptides; some are masked in intact particles and others are located on the virion surface but in a form usually unable to elicit the formation of antibodies.

A restriction endonuclease map was constructed for rabbit papillomavirus DNA. Analysis of the viral genome by cleavage with specific endonucleases, when compared with bovine papillomavirus types 1 and 2 genome maps, revealed similarities among the three genomes. A number of cleavage sites and their relative order on each genome were found to be conserved.

Human papillomavirus (HPV) 8 induces skin tumors which are at high risk for malignant conversion. The nucleotide sequence of HPV8 has been determined and compared to sequences of papillomaviruses with different oncogenic potential. The general organization of the HPV8 genome is similar to that of other types. Highly conserved, genus-specific sequences were found in open reading frames (ORFs) E1, E2, and L1. In ORFs E6, E7, and L2, HPV8 is more distantly related, but it was possible to differentiate subgenera in which HPV8 belonged to the HPV1-cottontail rabbit papillomavirus group. Sequences within ORF E4 and part of ORF L2 are rather type specific. HPV8 stands out by several unique features: the considerably reduced size of the noncoding region (397 base pairs), with a seemingly low potential for forming complex secondary structures; a cluster of putative promoter elements in the 3' half of ORF E1; an RNA polymerase III promoter-like sequence close to the C terminus of ORF E2; and of particular interest, the homology between the putative protein encoded by ORF E4 and the Epstein-Barr virus nuclear antigen 2 protein, which may reflect similar mechanisms in virus-mediated transformation.