Harald Zur Hausen

Human Papillomaviruses and Their Possible Role in Squamous Cell Carcinomas

The view that common warts (verrucae vulgares), juvenile warts (verrucae planae), and genital warts (condylomata acuminata) are caused by the same virus and that differences in appearance are the result of variations in location and host reactivity has been shared by dermatologists for several decades (Frey, 1924; Waelsch and Habermann, 1924; Young, 1964; Rowson and Mahy, 1967; Nasemann, 1974; Lever and Schaumburg-Lever, 1975). This has probably reduced the interest in specific types of human papillomas.

Characterization of the DNA of a defective human parvovirus isolated from a genital site

An apparently helper-dependent parvovirus was isolated from a penile flat condylomatous lesion by inoculating the material into a culture of human fibroblasts, and by coinfecting these cells with adenovirus type 12. Upon addition of an adenovirus helper, the virus could be readily propagated in human KB cells. Analysis of its DNA revealed that it shares structural similarities with the DNA of the four defective parvoviruses (adeno-associated viruses, AAV) described so far, but that the cleavage pattern obtained after digesting the DNA with restriction enzymes is different from that of AAV 1-AAV 4. Nucleic acid hybridization data further support the assumption that the isolated defective parvovirus is not identical with the other four serotypes. When conditions of high stringency were used, only weak cross-reactivity was seen with the DNAs of AAV 1-AAV 4, whereas the latter showed strong cross-hybridization with each other. The organization of the DNA of the newly isolated parvovirus was investigated by mapping the cleavage sites of several restriction enzymes. It is proposed to designate the new isolate as AAV 5.

Papillomavirus infections and human genital cancer

Human papillomas are induced by at least eight distinct types of papillomaviruses. They are listed in Table 1. Condylomata acuminata, human genital warts, represent a group of fibropapillomatous benign tumors with exuberant exophytic growth which are induced by a papillomavirus (reviewed in [l]). They are mainly located at the foreskin of the penis, the glans, the vulva, the introitus vaginae, and intravaginally (see review [l]). They have also been reported on the cervix uteri, perianally, and even within the urethra [2-41. Epidemiological studies demonstrate a venereal mode of transmission [5]. They are prevalent in groups of high sexual promiscuity, such as homosexuals [3, 61 and prostitutes, and are by no means a rare disease. According to British statistics [7] they comprise 6.1% of all reported cases of venereally transmitted diseases (Table 2). Malignant conversion of condylomata acuminata has been reported repeatedly (reviewed in [l]). Anecdotal reports were published mainly from cases of long duration but exceptionally also from young individuals with a rather recent history of genital warts (reviewed in [l]). Malignant tumors developed within genital warts of the vulva, the penis, and also of the vagina. The role of the virus found electron microscopically in a few nuclei of the benign condylomata acuminata in the induction of such malignant tumors has not been established. Our own investigations on a possible role of human genital wart virus in genital cancer were initiated after a number of unsuccessful attempts to demonstrate herpes simplex type II DNA in biopsies of such tumors by nucleic acid hybridization [8]. Since epidemiological features of genital cancer point in a number of parameters (reviewed in [9]), such as dependence on the number of sexual partners, early onset of sexual relations, existence of marital clusters, and correlation in the incidence between cervical and penile cancer [9-151, to an infectious etiology, we started to analyze additional candidate viruses for their possible involvement in this disease. The presence of a virus belonging to a group of clearly oncogenic agents in genital warts and occasional reports on their malignant conversion stimulated the interest in these viruses. Initial experiments were designed to clarify the question of whether the papillomavirus found in genital warts is identical with papillomaviruses observed in other skin papillomas. This led to the identification of distinct types of papil-

Lymphotropic papovaviruses isolated from African green monkey and human cells

A lymphotropic papovavirus was isolated from a lymphoblastoid cell line of African green monkey (AGM) cells which also contained a herpesvirus and a paramyxovirus-like agent. The papovavirus was analyzed by restriction endonuclease cleavage; its biochemical and serological crossreactivity with SV40 and host range have been determined. Thus far, only B-lymphoblasts of primate and human origin have been found to be susceptible to infection. Although more than 50% of the tested monkey sera were reactive with antigens of this virus, all human sera tested failed to react. Cleavage patterns and hybridization studies with the viral DNA indicate that the virus represents a novel member of the papovavirus group that is characterized by its lymphotropic host range. Papovavirus particles were also demonstrated in a human lymphoblastoid cell line (CCRF-SB) originally derived from a leukemic child. These cells revealed nuclear fluorescence when tested with human sera, but failed to react with AGM sera. Although characterization of this agent has not yet been completed, available evidence suggests that it represents another lymphotropic papovavirus which seems to be spread within the human population.

Induction of mutations within the host cell genome by partially inactivated herpes simplex virus type 1.

Abstract Neutral red or ultraviolet inactivated herpes simplex virus type 1 proved to be mutagenic when infecting cells of the human rhabdomyosarcoma line RD-176. This was shown by the induction of resistance to 8-azaguanine of the infected cells indicating a mutation in the hypoxanthine-guanine-phosphoribosyltransferase gene. In some experiments the mutagenic effect was comparable to the mutation rate induced by the chemical carcinogen 4-nitroquinoline-l-oxid.

The Role of Viruses in Human Tumors

Publisher Summary This chapter discusses the role of viruses in the induction of certain human tumors. A number of viruses have been identified which induce tumors under experimental and under natural conditions. Various DNA- as well as RNA-containing agents proved to be oncogenic. The analysis of several representative tumor viruses revealed at least two consistent features in the mechanism of tumor induction by viruses. These viruses implant at least one functioning gene into their respective host cell that may become covalently linked to host cell DNA. Expression of this persisting gene is required in order to maintain the transformed state of this cell. Tumors develop as a consequence of failing host cell control of early functions (effector functions) of persisting oncogenic viruses. This may affect the expression less than the functional control of the effector gene product. The control of late functions remains either unaffected under these conditions, or the persisting genome is defective in late functions. Tumors may also arise in host nonadapted to the control of early functions, although they suppress late functions of the same genome. Tumor viruses of this group are defined as agents that adapted to induce host cell DNA synthesis during their evolution in order to promote their own replication.

Partial characterization of a new type of bovine papilloma viruses

Abstract Papilloma virus isolates from 13 individual bovine cutaneous warts were characterized by monospecific rabbit antisera and were shown to fall into two groups without detectable crossreactivity when tested by immune electron microscopy or complement fixation. cRNA transcribed from representatives of both groups did not hybridize with DNA from heterologous isolates. The two types of BPV also differed in the electrophoretic mobility of their proteins and in the molecular weight of their DNA (4.5 × 106 and 4.9 × 106, respectively). One isolate with DNA of 4.9 × 106 MW and one isolate with DNA of 4.5 × 106 MW were analyzed by cleavage of their DNA with the restriction endonucleases BamHI, EcoRI, HindII, HindlH, and HaeHI and physical maps were established. The two genomes differed completely in their cleavage pattern. The HindII cleavage pattern demonstrated the identity of the large DNA isolate with BPV 2 of Lancaster et al. (personal communication). The other apparently new type of bovine papilloma virus, which was detected twice thus far, is tentatively designated as BPV 3.

Analysis of early and late Epstein-Barr virus associated polypeptides by immunoprecipitation.

Abstract The Epstein-Barr virus-producing cell lines P3HR-1 and B95-8 and the nonproducer cell lines Raji clone No. 7 and NC37 were induced to viral antigen synthesis by the tumor promoter TPA and then analyzed by immunoprecipitation with human sera for early and late virus-associated polypeptides. After labeling of producer cells for a 4-day period with [ 35 S]methionine, two polypeptides with molecular weights of 140,000 and 150,000 were identified reacting predominately with virus capsid antigen (VCA + ) sera. Analysis of purified Epstein-Barr virus demonstrated that the 140,000 polypeptide presumably represents an envelope protein while the 150,000 polypeptide is a nucleocapsid protein. In 4-hr radioactively labeled producer cells an additional polypeptide with a molecular weight of 130,000 was found to be immunoreactive with VCA + sera. Immunoprecipitation of [ 35 S]methionine-labeled cell extracts from nonproducer cells resulted in the specific precipitation of two polypeptides with molecular weights of 85,000 and 35,000 which most likely represent early EBV-associated proteins. Producer cells exhibit three additional apparently early EBV-associated polypeptides with molecular weights of 120,000, 18,000, and 16,000. None of these polypeptides could be detected in EBV genome-negative Ramos cells after TPA treatment.

Identification of a gene function of herpes simplex virus type 1 essential for amplification of simian virus 40 DNA sequences in transformed hamster cells.

Infection with herpes simplex viruses (HSV) lead to a significant increase of the simian virus 40 (SV40) DNA content in the SV40-transformed hamster cell lines CO631 and Elona. Analysis of this gene-amplifying activity revealed (i) that it cosedimented with infectious herpesvirions in sucrose density gradients, (ii) that it was abolished by anti-HSV antibodies or (iii) by antiviral drugs acting on the HSV-induced DNA polymerase; and analysis of temperature-sensitive mutants showed that this DNA polymerase was an essential component of HSV-induced, gene-amplifying activity in SV40-transformed hamster cells.

Localization of viral DNA-replication in sections of human warts by nucleic acid hybridization with complementary RNA of human papilloma virus type 1

SummaryUsing complementary RNA of human papilloma virus type 1 (HPV 1) and in situ hybridization techniques the localization of viral DNA replication was studied in sections of 38 human virus acanthomata from 31 different patients. In five cases significant labeling was detected by autoradiography. Labeling started always in the first or the second suprabasal cell layer and was strongly limited to the nuclei. A remarkable early beginning of the vacuolated process seemed to be correlated with the visible DNA replication. No labeling could be detected in the basal cell layer. This suggests that these cells contain at the most only a small number of viral genomes. Our findings represent only the situation in human warts definitely caused by HPV 1. We are not able to say, wether our negative hybridization results in the remaining warts indicate either the scarcity of viral genomes within these sections or their infection by a different agent.ZusammenfassungUm eine Aussage über die Lokalisation der Virusreplikation in menschlichen Virusakanthomen zu gewinnen, unterzogen wir Gefrierschnitte von 38 Warzen, die wir bei 31 verschiedenen Patienten entfernt hatten, einer insitu-Hybridisierung mit komplementärer RNA des Virustypen HPV 1. In fünf Fällen zeigte sich autoradiographisch eine signifikante Markierung. Diese begann immer in der ersten oder zweiten suprabasalen Zellschicht und war streng auf die Zellkerne beschränkt. Auffallend war eine deutliche Korrelation zwischen dem Ort dieser Markierung und dem ebenda einsetzenden Beginn der Zellvakuolisierung. Die Basalzellschicht war frei von markierten Zellen. Dies läßt vermuten, daß die Zellkerne hier nur eine sehr geringe Anzahl viraler Genome enthalten. Unsere Befunde können nur auf Warzen bezogen werden, die definitiv von dem Virustypen HPV 1 hervorgerufen wurden. Ob die übrigen unserer Warzen, die einen negativen Hybridisierungsbefund zeigten, lediglich eine unter unserer Nachweismöglichkeit liegende Virusgenomanzahl enthielten oder ob sie durch einen anderen Virustypen hervorgerufen wurden, läßt sich nicht sagen.