Janet S. Butel

Construction and characterization of an SV40 mutant defective in nuclear transport of T antigen

An SV40-adenovirus 7 hybrid virus, PARA(cT), has been described that is defective for the nuclear transport of SV40 large tumor antigen. An SV40(cT) mutant was constructed using SV40 early and late region DNA fragments derived from PARA(cT) and wild-type SV40 respectively. The SV40(cT)-3 construct is defective for viral replication, but can be propagated in COS-1 cells. T antigen induced by SV40(cT)-3 is localized in the cytoplasm of infected cells. The cT mutation also inhibits the transport of wild-type T antigen; COS-1 cells lose their constitutive expression of nuclear T antigen after infection with SV40(cT)-3. Sequence analysis revealed that the cT mutation results in the replacement of a positively charged lysine in wild-type T antigen with a neutral asparagine at amino acid number 128, demonstrating that the alteration of a single amino acid is sufficient to abolish nuclear transport. Implications of the cT mutation on possible mechanisms for the transport of proteins to the nucleus are discussed.

DNA Sequences Similar to Those of Simian Virus 40 in Ependymomas and Choroid Plexus Tumors of Childhood

BACKGROUND Ependymomas and papillomas of the choroid plexus occur in early childhood. The ubiquitous human polyomaviruses, BK virus and JC virus, have been associated with the induction of these neoplasms in animal models. A related monkey polyomavirus, simian virus 40 (SV40), is highly tumorigenic in rodents and also induces choroid plexus papillomas. METHODS We tested the possibility that polyomaviruses were associated with these tumors in humans. Tumors from 31 children--20 with choroid plexus neoplasms and 11 with ependymomas--were evaluated for the presence of polyomavirus T-antigen gene sequences by means of amplification with the polymerase chain reaction. RESULTS Ten of the 20 choroid plexus tumors and 10 of the 11 ependymomas contained amplification products that preferentially hybridized to probes specific for SV40 viral DNA rather than BK or JC viral DNA. In two specimens, DNA sequencing demonstrated that the amplified sequence was identical to the sequence of that region of the SV40 gene. In three other specimens, amplification with SV40-specific primers revealed a 574-bp segment of the SV40 viral gene. In 7 of 11 tumors examined by immunohistochemical staining, viral T antigen was expressed in the nuclei of the neoplastic cells. CONCLUSIONS Half of the choroid plexus tumors and most of the ependymomas that we studied contained and expressed a segment of T-antigen gene related to SV40. These results suggest that SV40 or a closely related virus may have an etiologic role in the development of these neoplasms during childhood, as in animal models.

Association between simian virus 40 and non-Hodgkin lymphoma

BACKGROUND Non-Hodgkin lymphoma has increased in frequency over the past 30 years, and is a common cancer in HIV-1-infected patients. Although no definite risk factors have emerged, a viral cause has been postulated. Polyomaviruses are known to infect human beings and to induce tumours in laboratory animals. We aimed to identify which one of the three polyomaviruses able to infect human beings (simian virus 40 [SV40], JC virus, and BK virus) was associated with non-Hodgkin lymphoma. METHODS We analysed systemic non-Hodgkin lymphoma from 76 HIV-1-infected and 78 HIV-1-uninfected patients, and non-malignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumours; 54 colon and breast carcinoma samples served as cancer controls. We used PCR followed by Southern blot hybridisation and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. FINDINGS Polyomavirus T antigen sequences, all of which were SV40-specific, were detected in 64 (42%) of 154 non-Hodgkin lymphomas, none of 186 non-malignant lymphoid samples, and none of 54 control cancers. This difference was similar for HIV-1-infected patients and HIV-1-uninfected patients alike. Few tumours were positive for both SV40 and Epstein-Barr virus. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B-cell and follicular-type lymphomas. INTERPRETATION SV40 is significantly associated with some types of non-Hodgkin lymphoma. These results add lymphomas to the types of human cancers associated with SV40.

SV40 and human tumours: myth, association or causality?

An increasing number of scientific reports have described evidence for a polyomavirus, simian virus 40, in a highly select group of human tumours. How did a simian virus infect humans and is the virus a passenger in tumours or is it important in their pathogenesis?

Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene

The role of the hepatitis B virus (HBV) X protein in liver tumorigenesis is unresolved. Transgenic mice harboring the X gene (nt 1376–1840 under the control of the human α‐1‐antitrypsin regulatory elements) (ATX mice) display only minor histopathologic alterations of the liver. To determine if ATX mice are more susceptible to the effects of hepatocarcinogens, 12‐ to 15‐d‐old male ATX and control littermate mice were injected with a single dose (2 μg/g body weight) of diethylnitrosamine (DEN). The animals were killed 6–10 mo after exposure and were analyzed for histological changes in the liver. One hundred percent of the DEN‐treated ATX mice developed abnormal liver lesions. When their liver tissues were compared by stereological analysis with those of non‐transgenic animals, the ATX mice had a relative twofold increase in the total number of focal lesions and a twofold increase in the incidence of hepatocellular carcinoma. Elevated levels of X protein and p53 protein were not detected in carcinogen‐induced nodules or tumors. These results are consistent with a model in which the expression of the HBV X protein potentiates the induction of DEN‐mediated liver disease.

A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development

Although alterations in the p53 tumor suppressor gene are detected frequently in human breast cancers, mammary tumors are observed infrequently in p53null mice. This has led to the suggestion that absence of p53 alone is not sufficient for induction of mammary tumors. However, early death of p53null mice from thymic lymphomas may obscure tumor phenotypes that would develop later. Therefore, p53null mammary epithelium was transplanted into cleared mammary fat pads of wild type p53 BALB/c hosts to allow long-term analysis of mammary tumor phenotypes. Five treatments were compared for their effects on tumor incidence in hosts bearing transplants of p53null and p53wt mammary epithelium. The treatment groups were: (1) untreated; (2) continuous hormone stimulation with pituitary isografts; (3) multiple pregnancies; (4) DMBA alone; and (5) DMBA+pituitary isografts. The tumor incidences in p53null vs p53wt mammary transplants for each treatment group were 62% vs 0%, 100% vs 0%, 68% vs 0%, 60% vs 4% and 91% vs 14%, respectively. The mammary tumors that developed in the p53null mammary epithelium were all adenocarcinomas and were frequently aneuploid. These data demonstrate that the absence of p53 is sufficient to cause development of mammary tumors and that hormonal stimulation enhances the tumorigenicity of p53null mammary epithelium to a greater extent than DMBA exposure alone. This model provides an in situ approach to examine the molecular basis for the role of p53 in the regulation of mammary tumorigenesis.

The History of Tumor Virology

In the century since its inception, the field of tumor virology has provided groundbreaking insights into the causes of human cancer. Peyton Rous founded this scientific field in 1911 by discovering an avian virus that induced tumors in chickens; however, it took 40 years for the scientific community to comprehend the effect of this seminal finding. Later identification of mammalian tumor viruses in the 1930s by Richard Shope and John Bittner, and in the 1950s by Ludwik Gross, sparked the first intense interest in tumor virology by suggesting the possibility of a similar causal role for viruses in human cancers. This change in attitude opened the door in the 1960s and 1970s for the discovery of the first human tumor viruses--EBV, hepatitis B virus, and the papillomaviruses. Such knowledge proved instrumental to the development of the first cancer vaccines against cancers having an infectious etiology. Tumor virologists additionally recognized that viruses could serve as powerful discovery tools, leading to revolutionary breakthroughs in the 1970s and 1980s that included the concept of the oncogene, the identification of the p53 tumor suppressor, and the function of the retinoblastoma tumor suppressor. The subsequent availability of more advanced molecular technologies paved the way in the 1980s and 1990s for the identification of additional human tumor viruses--human T-cell leukemia virus type 1, hepatitis C virus, and Kaposis sarcoma virus. In fact, current estimates suggest that viruses are involved in 15% to 20% of human cancers worldwide. Thus, viruses not only have been shown to represent etiologic agents for many human cancers but have also served as tools to reveal mechanisms that are involved in all human malignancies. This rich history promises that tumor virology will continue to contribute to our understanding of cancer and to the development of new therapeutic and preventive measures for this disease in the 21st century.

The Dynamics of Herpesvirus and Polyomavirus Reactivation and Shedding in Healthy Adults: A 14-Month Longitudinal Study

Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

Oncogenicity and cell transformation by papovavirus SV40: the role of the viral genome.

Publisher Summary This chapter evaluates the available knowledge of simian virus 40 (SV40) oncogenesis and assesses the implications for carcinogenesis by DNA-tumor viruses as a whole. It summarizes different tests that can be employed to detect virus-induced changes in SV40-transformed cells. Inoculation of such cells into susceptible hosts usually results in the production of tumors, the ultimate criterion necessary to establish that malignant transformation has indeed occurred. Fusion or cocultivation of the transformed cell with normal susceptible cells may sometimes succeed in the rescue of infectious virus. The optimum conditions required to elicit the production of virus from a majority of the transformed cells have not yet been established. Transformed cells are usually immune to superinfection by the transforming virus. Nucleic acid hybridization experiments indicate that multiple copies of the SV40 genome is present, probably integrated into the host cell chromosome. Virus-specific mRNA is present in the transformed cells. The extent of the transcription of the viral genome seems to vary from one transformed cell line to the next. No relationships have been established between the extent of transcription in a given transformed cell line and the number and/or magnitude of virus-specific changes of that cell line. A series of in vitro tests may detect a variety of surface changes on the transformed cells. These tests include immunofluorescence, agglutination, cytotoxicity, colony inhibition, and mixed hemadsorption.

SV40 DNA IN HUMAN OSTEOSARCOMAS SHOWS SEQUENCE VARIATION AMONG T-ANTIGEN GENES

Authentic simian virus 40 (SV40) has been detected in association with human choroid plexus and ependymoma tumors, and SV40‐like DNA sequences have been found in some human osteosarcomas. We report here an analysis of human osteosarcoma samples for the presence of SV40 DNA using PCR and primers directed at 4 distinct sites of the SV40 genome, coupled with sequence analysis. Authentic SV40 DNA sequences were detected in 5 of 10 osteosarcoma tumor samples. The SV40 regulatory region in each case was identical and of archetypal length (non‐duplicated enhancer), as is usually found in natural isolates of SV40 from monkeys and in human brain tumors. A section of the gene that encodes a viral late gene product (VP1) was detected in 5 of 10 tumors and had an exact match with the known sequence of SV40. Two separated segments of the large T‐antigen (T‐ag) gene were found in the same 5 tumors. Analysis of the DNA sequences encoding the T‐ag carboxy terminus revealed sequence variation among the tumors, as observed previously in viral DNA associated with human brain tumors. There does not appear to be a preferential association of a T‐ag variable domain sequence with a given tumor type. No sequences from the regulatory region of human polyomaviruses JCV and BKV were detected in the bone tumors. We also noted less efficient recovery of SV40 DNA from tumor samples fixed in paraffin as compared to frozen tumors. Our results confirm the presence of SV40 DNA in human bone tumors and, based on the sequence variation observed for the carboxy terminus of the T‐ag gene, suggest that there is not a specific SV40 strain associated with human osteosarcomas. Int. J. Cancer 72:791–800, 1997.