Herpesvirus saimiri strains from three DNA subgroups have different oncogenic potentials in New Zealand white rabbits

Herpesvirus saimiri is a primate tumor virus that induces acute T-cell lymphomas in New World monkeys. Strains of this virus have been previously classified into three groups on the basis of extreme DNA variability of the rightmost region of unique L-DNA. To compare the oncogenic potentials of various strains, we inoculated New Zealand White rabbits with viruses representing groups A, B, and C of herpesvirus saimiri. The results showed that a group C strain were highly oncogenic in New Zealand White rabbits; however, group A or B viruses were not oncogenic in these rabbits. Analysis of DNAs of tumor tissues and lymphoid cell lines established from tumors showed that the viral genome exists in circular episomal form. To identify which part of the genome of the group C strain is responsible for the highly oncogenic phenotype, group B-C recombinant strains were constructed by an efficient drug selection technique. Two group B recombinant strains in which the right-end 9.2 kilobase pairs of unique DNA is replaced by group C virus DNA were oncogenic in rabbits, indicating that the rightmost sequences contribute to the oncogenic properties of the group C strain. Oncogenicity of herpesvirus saimiri has been traditionally evaluated in New World monkeys; infection of rabbits with group C strain 484-77 offers a much more accessible animal model to study the mechanism of oncogenicity of this virus.     

Centrosome abnormalities, genomic instability and carcinogenic progression

Centrosome abnormalities are a frequent finding in various malignant tumors. Since centrosomes form the poles of the mitotic spindle, these abnormalities have been implicated in chromosome missegregation and the generation of aneuploid cells which is commonly found in many human neoplasms. It is a matter of debate, however, whether centrosome alterations can drive cells into aneuploidy or simply reflect loss of genomic integrity by other mechanisms. Since these two models have fundamentally different implications for the diagnostic and prognostic value of centrosome abnormalities, we will discuss the relevance of abnormal centrosomes in the context of different oncogenic events as exemplified by high-risk human papillomavirus-associated carcinogenesis.     

Are type C viruses transforming agents?

Type C RNA viruses have been considered oncogenic because they are found associated with animal tumors and can induce cancers in several animal species. Those viruses that rapidly cause cancer appear to contain an oncogenic gene which resembles genetic sequences present in normal cells. This gene codes for a transforming protein which may be a normal cellular enzyme or a slightly altered cellular product. Its mechanism for transforming a cell is not yet known. Other oncogenic viruses, such as the chronic leukemia viruses, may not produce an oncogenic protein but may affect, by other means, specific target cells so they become malignant. Recent evidence now suggests that the majority of endogenous type C viruses are not transforming agents but inherited in the host to function in other biologic processes. These viruses do not contain transduced cellular genes which are responsible for cancer. Their role probably depends on their expression of other gene products which aid in normal development. These observations suggest that the ultimate control of human cancer may result from the identification of the oncogenic cellular-like genes transduced by some type C viruses even if a true human oncogenic virus is not isolated.     

Development of cellular anti-tumor immunity in chickens bearing tumors induced by Rous sarcoma virus.

The technique of peripheral lymphocyte stimulation in response to antigen was used to demonstrate the existence of cell-mediated anti-tumor immunity in chickens bearing tumors induced by avian sarcoma viruses. The expression of this anti-tumor response against virus-containing transformed cell culture supernatant fluids and cell extracts varied among animals in terms of time after inoculation with the oncogenic agent. Animals whose tumors had completely regressed rapidly lost the ability to mount such continuing immunity. In addition, we found evidence that some normal animals express endogenous levels of natural immunity against neoplasms induced by these viruses and/or against the viruses themselves.     

GNAQQ209L expression initiated in multipotent neural crest cells drives aggressive melanoma of the central nervous system

Primary leptomeningeal melanocytic neoplasms represent a spectrum of rare tumors originating from melanocytes of the leptomeninges, which are the inner two membranes that protect the central nervous system. Like other non‐epithelial melanocytic lesions, they bear frequent oncogenic mutations in the heterotrimeric G protein alpha subunits, GNAQ or GNA11. In this study, we used Plp1‐creERT to force the expression of oncogenic GNAQ^Q209L in the multipotent neural crest cells of the ventro‐medial developmental pathway, beginning prior to melanocyte cell differentiation. We found that this produces leptomeningeal melanocytic neoplasms, including cranial melanocytomas, spinal melanocytomas, and spinal melanomas, in addition to blue nevus‐like lesions in the dermis. GNAQ^Q209L drove different phenotypes depending upon when during embryogenesis (E9.5, E10.5, or E11.5) it was induced by tamoxifen and which Cre driver (Plp1‐creERT, Tyr‐creERT², or Mitf‐cre) was used. Given these differences, we propose that melanocytes go through temporary phases where they become sensitive to the oncogenic effects of GNAQ^Q209L. R26‐fs‐GNAQ^Q209L; Plp1‐creERT mice will be useful for defining biomarkers for potentially aggressive leptomeningeal melanocytomas and for developing new therapeutics for advanced disease.     

Analysis of neoplasms induced by Cas-Br-M MuLV tumor extracts

Cas-Br-M is an ecotropic murine leukemia virus isolated from wild mice that induces a wide spectrum of hematopoietic neoplasms, including T and B cell lymphomas, myelogenous leukemias, and erythroleukemias. The purpose of this study was to determine if the induction of neoplasms belonging to multiple lineages was due to the ecotropic virus itself or to the generation of cell lineage-specific recombinant viruses. The results demonstrate that in some instances (two of 12 tumor extracts tested), recombinant viruses can be recovered from primary Cas-Br-M-induced tumors that will induce lymphomas of single lineages in mice inoculated as newborns. One of these viruses is a recombinant mink cell focus-forming virus that induces T cell lymphomas, and the other is a replication-defective, fibroblast-transforming virus that induces early B lineage lymphomas in mice. Histologic and flow microfluorometric cell surface antigen analyses of primary and in vitro adapted tumors are presented in support of a modified scheme of hematopoietic cell development.     

New molecular mechanisms of virus-mediated carcinogenesis: oncogenic transformation of cells by retroviral structural protein Envelope

RNA tumor viruses as classified in Retroviruses have been isolated and identified to induce tumors in a variety of animals including chickens, mice, and rats, or even in human in the last 100 years, since the first one has been reported in 1908. The RNA tumor viruses have been historically classified into two groups, acute transforming RNA tumor viruses and nonacute RNA tumor viruses. Acute transforming RNA tumor viruses are basically replication-defective and rapidly induce tumors by expressing the viral oncogenes captured from cellular genome in host cells. The first oncogene derived from Rous sarcoma virus was the src non-receptor tyrosine kinase, which has been identified to play the significant roles for signal transduction. On the other hand, nonacute RNA tumor viruses, which consist of only gag, pro, pol, and env regions but do not carry oncogenes, are replication-competent and could activate the cellular proto-oncogenes by inserting the viral long terminal repeat close to the proto-oncogenes to induce tumors with a long incubation period, as is termed a promoter insertion. These molecular mechanisms have been thought to induce tumors. However, very recently several reports have described that the retroviral structural protein Envelope could directly induce tumors in vivo and transform cells in vitro. These are very unusual examples of native retroviral structural proteins with transformation potential. In this review we look back over the history of oncogenic retrovirus research and summarize recent progress for our understanding of the molecular mechanisms of oncogenic transformation by retrovirus Envelope proteins.     

Virus-associated human tumors: cervical carcinomas and papilloma viruses

The latest experimental data on the role of viruses in the origin of human tumors are discussed. This group of viruses consists of T-cell leukemia virus type 1 (HTLV 1), herpes viruses (HHV 8 and Epstein-Barr virus), hepatitis B virus, and human papilloma viruses. The most typical feature of this group of viruses is a very long latent period from the initial infection to the development of the disease that varies between 10 and 40 years. The mechanism of malignant cell conversion is specific for each viral type but is mainly associated with a disruption of functions of cellular genes participating in the control of cell division and proliferation. It can be a direct inactivation of tumor suppressor genes by their interaction with viral gene products (papilloma viruses), or a trans-activation of cellular genes modulating cell proliferation by viral gene products (hepatitis B virus and HTLV 1). Viruses play an initiative role and additional genetic changes in the genome of infected cells are necessary for complete expression of the oncogenic potential of the viral genes. Only these cells will give rise to a monoclonal cell population with uncontrolled proliferation. New approaches for the creation of vaccines against cancers associated with hepatitis B virus and papilloma viruses (hepatocellular carcinomas and cervical tumors, respectively) are in progress. These vaccines have been found to be effective in prevention of the disease in the experimental models and are now beginning to be used for human vaccination.     

Molecular diversity among five different endogenous primate retroviruses.

Genetically transmitted retroviruses of Old and New World monkeys include type C viruses isolated from baboons (M7), macaque (MAC-1), and owl monkeys (OMC-1) and type D viruses from langurs (PO-1-Lu) and squirrel monkeys (SMRV, M534). Each of these isolates is unrelated to the others by nucleic acid hybridization criteria and contains a unique array of virion-associated proteins which can be resolved by agarose gel filtration and polyacrylamide gel electrophoresis under denaturing conditions. The major structural protein of each virus has a distinct primary structure, as determined by two-dimensional tryptic peptide analysis, and is antigenically different from the others. The major virion phosphoproteins of endogenous primate type C viruses (pp15) are also different from those of type D viruses (pp13-pp14). Immunological and structural analyses show that the endogenous langur virus and the horizontally transmitted Mason-Pfizer virus of rhesus monkeys are closely related to one another, consistent with the sequence homology detected in their RNA genomes. Although certain radioimmunoassays detect interspecies antigenic determinants common to either the p30 or gp70 proteins of some of these viruses, no one assay has yet been designed which can detect all groups of endogenous primate retroviridae. The data lead to the conclusion that primates contain a minimum of three different sets of genetically transmitted type C and type D retroviral genes.     

Accelerated development of polyoma tumors and embryonic lethality: different effects of p53 loss on related mouse backgrounds.

Molecular evidence linking polyoma virus to p53 inactivation is thus far lacking, setting this highly oncogenic virus apart from other DNA tumor viruses. As a biological test for interaction, we studied the effects of p53 loss on development of virus-induced tumors. The absence of p53 led to more rapid tumor development on two different mouse backgrounds, indicating synergism between p53 loss and oncogenic pathways controlled directly by the virus. No effects of p53 on tumor type or frequency were noted. Polyoma tumor-derived cells in culture retained p53, and most of these showed induction of p21CIP1/WAF1 in response to DNA damage. These results indicate that p53 functions are not directly and fully impaired by the virus in the intact host. On one mouse background, it was discovered that loss of p53 resulted in complete embryonic lethality prior to 11 days of gestation. This lethality could be rescued by inclusion of gene(s) from a 129/SvJ background.     

Simian Tumour Virus Proliferation in Inoculated <Emphasis Type="Italic">Macaca mulatta</Emphasis>

AN oncorna-type virus (M-PMV) was detected in a spontaneous breast tumour of a female rhesus monkey^1,2. This virus is morphologically and biochemically similar to oncogenic RNA viruses of other species^3–5. Using in vitro tissue culture assays and electron microscopy we have observed proliferation of infectious virus in hyperplastic lymph nodes of M-PMV inoculated infant rhesus monkeys.     

Responsiveness of normal and transformed rat embryo cell cultures to poly I-poly C and interferon

A number of normal rat cell cultures as well as cultures transformed spontaneously, by chemicals, and/or by oncogenic viruses were tested for responsiveness to the interferon inducer polyinosinic·polycytidylic acid, or to exogenous interferon. Responsiveness, or lack thereof, had no correlation with subculture passage number, infection with RNA leukemia virus, morphological transformation by oncogenic RNA or DNA viruses, chemical treatment, or the ability of these cells to produce tumors in isologous host animals. The data indicate that lack of response to interferon or to the inducer is neither a necessary prerequisite nor an absolute result of cellular transformation of rat cells.     

Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.     

Avian reticuloendotheliosis viruses: evolutionary linkage with mammalian type C retroviruses.

Reticuloendotheliosis viruses have been shown to be causative of tumors in a variety of avian species. The major structural protein of these non-genetically transmitted viruses is demonstrated to possess antigenic determinants common to those of all known mammalian type C viruses. These findings establish a mammalian origin for this oncogenic avian retrovirus group. None of the known mammalian type C virus groups demonstrated a closer immunological relationship to avian reticuloendotheliosis viruses. These results suggest that reticuloendotheliosis viruses have been non-genetically transmitted for a long period of evolution or that these viruses may have arisen by relatively recent infection of birds with an as yet undiscovered mammalian type C retrovirus.     

Acquisition of oncogenicity by endogenous mouse type C viruses: effects of variations in env and gag genes.

Several dual-tropic isolates derived from the thymuses of preleukemic or leukemic AKR mice and a more recrnt group of viruses generated by in vitro or in vivo passage of a poorly infectious endogenous virus of C3H mouse cells have been shown to be highly oncogenic. By analysis of the immunological properties of their gag gene-coded structural proteins, each of the AKR-derived isolates and two dual-tropic C3H-derived isolates were found to closely resemble AKR murine leukemia virus. In contrast, gag gene-coded proteins of two other leukemogenic isolates of C3H origin, including one ecotropic and one dual-tropic virus, were indistinguishable from those of Moloney murine leukemia virus. All of the oncogenic isolates, including those of AKR and C3H origin, were found to possess common envelope glycoprotein determinants of a unique class not shared by the nononcogenic ecotropic viruses from which they were derived. These findings support the possibility that oncogenic variants of endogenous ecotropic mouse type C viruses are derived by genetic recombination. This recombinational event appears to involve the acquisition, by different ecotropic viruses, of a common class of endogenous virus-coded envelope glycoprotein determinants which are presumably required, but not necessarily sufficient, for oncogenicity.     

The Lucke Frog Kidney Tumor and its Herpesvirus

Northern leopard frogs are afflicted with a spontaneous malignantneoplasm of the mesonephros. A herpesvirus is invariably associatedwith tumors obtained from frogs hibernating 30 days or longerand in tumors of frogs taken from breeding ponds. Tumors obtainedfrom prehibernating frogs do not have viruses as detected byelectron microscopy but virus particles are found in some tumorswithin 7 days after the onset of hibernation. Tumors of frogsmaintained at warm temperature in the laboratory do not haveviruses and tumors of frogs maintained at cold temperaturesin the laboratory do contain viruses. However, the productionof viruses in the laboratory follows a distinctly slower chronologythan that which occurs in nature. Injection of cell fractionscontaining the herpesvirus into frog embryos induces tumorsnear the time of metamorphosis in many experimental animals.Embryos injected with tumor extracts not containing herpesvirusesdo not develop tumors. Environmental and laboratory observationsare discussed which may relate to natural transmission of thispresumed viral oncogenic agent.     

Analysis of High-Molecular-Weight Ribonucleic Acid Associated with Intracisternal A Particles

Intracisternal A particles, known primarily for their association with various tumors, have been shown to contain high-molecular-weight (HMW) ribonucleic acid (RNA) by velocity centrifugation, using linear glycerol gradients. This HMW RNA is sensitive to ribonuclease digestion and alkali treatment but is resistant to Pronase treatment. By a double-labeling experiment, HMW RNA was shown to be intrinsic to intracisternal A particles and not to have resulted from cytoplasmic polysomal RNA aggregation. By a reconstitution experiment, it was determined that the results were not due to C-type virus contamination. The synthesis of HMW RNA in intracisternal A particles is inhibited by actinomycin D and ethidium bromide. These observations emphasize that there are probably some taxonomic relationships between intracisternal A particles and oncogenic RNA viruses.     

Avian leukosis virus-induced tumors have common proviral integration sites and synthesize discrete new RNAs: oncogenesis by promoter insertion

Unlike other RNA tumor viruses, avian leukosis viruses (which cause lymphomas and occasionally other neoplasms) lack discrete "transforming genes". We have analyzed the virus-related DNA and RNA of avian leukosis virus (ALV)-induced tumors in an attempt to gain insight into the mechanism of ALV oncogenesis. Our results show that viral gene products are not required for maintenance of neoplastic transformation. Primary and metastatic tumors are clonal and thus presumably derived from a single infected cell. Most importantly, tumors from different birds have integration sites in common. Tumor cells synthesize discrete new poly(A) RNAs consisting of viral sequences covalently linked to cellular sequences. These RNA species are expressed at high levels in tumor cells. Our results suggest that in lymphoid tumors, an ALV provirus is integrated adjacent to a specific cellular gene, and the insertion of the viral promoter adjacent to this gene results in its enhanced expression, leading to neoplasia. These results have potentially important implications for the mechanism of non-viral carcinogenesis.     

Production of Virus by Mammalian Cells Transformed by Rous Sarcoma and Murine Sarcoma Viruses

Cultured cells of mammalian tumors induced by ribonucleic acid (RNA)-containing oncogenic viruses were examined for production of virus. The cell lines were established from tumors induced in rats and hamsters with either Rous sarcoma virus (Schmidt-Ruppin or Bryan strains) or murine sarcoma virus (Moloney strain). When culture fluids from each of the cell lines were examined for transforming activity or production of progeny virus, none of the cell lines was found to be infectious. However, electron microscopic examination of the various cell lines revealed the presence of particles in the rat cells transformed by either Rous sarcoma virus or murine sarcoma virus. These particles, morphologically similar to those associated with murine leukemias, were found both in the extracellular fluid concentrates and in whole-cell preparations. In the latter, they were seen budding from the cell membranes or lying in the intercellular spaces. No viruslike particles were seen in preparations from hamster tumors. Exposure of the rat cells to (3)H-uridine resulted in the appearance of labeled particles with densities in sucrose gradients typical of virus (1.16 g/ml.). RNA of high molecular weight was extracted from these particles, and double-labeling experiments showed that this RNA sedimented at the same rate as RNA extracted from Rous sarcoma virus. None of the hamster cell lines gave radioactive peaks in the virus density range, and no extractable high molecular weight RNA was found. These studies suggest that the murine sarcoma virus produces an infection analogous to certain "defective" strains of Rous sarcoma virus, in that particles produced by infected cells have a low efficiency of infection. The control of the host cell over the production and properties of the RNA-containing tumorigenic viruses is discussed.