Inhibitor of the DNA-dependent DNA Polymerase of Some RNA Tumour Viruses in Feline Sera

AN inhibitor of the RNA-dependent DNA polymerases^1,2 of mammalian C-type viruses was found in sera from rats bearing transplantable tumours, induced by murine C-type RNA tumour viruses^3,4. Partially purified polymerases of murine leukaemia virus³ and feline leukaemia virus (FeLV)⁴ were shown to be antigenic in rabbits and a rat, respectively. We have detected an inhibitor of the DNA-dependent DNA polymerase^5,6 of feline and murine C-type viruses in the sera of cats inoculated in utero and/or postnatally with the Gardner-Arnstein strain of feline sarcoma virus (FSV)⁷ and in the sera of cats bearing spontaneous sarcomas, lymphomas or carcinomas.     

C-Type Virus associated with Gibbon Lymphosarcoma

C-TYPE viruses have been established as the causal agents of leukaemia in murine and feline species and have been characterized^1,2. C-type virus is also probably associated with fibrosarcoma in non-human primates^3–6. To determine whether viruses with identical characteristics are associated with other neoplasms in simian species, we looked for C-type viruses in cases of leukaemia. A gibbon (Hylobates lar) with a disseminated tumour (later confirmed as lymphosarcoma) was made available to the Comparative Oncology Laboratory by Dr Malcolm Jones of the University of California, San Francisco Medical Center. The principal sites of involvement (lymph node, liver and bone marrow) were extensively overrun with massive neoplastic cells, which were predominantly prolymphocytic forms. Electron microscopy revealed C-type particles identical to those observed in vitro in sections from lymph nodes, liver, spleen and bone marrow.     

Cat Interferon inhibits Feline Leukaemia Virus Infection in Cell Culture

TRANSMISSION of feline leukaemia can be accomplished with tissue extracts from cases which occur naturally¹. Virus particles which are morphologically indistinguishable from the murine and avian C-type viruses are present in cats with the transmitted disease². Feline leukaemia virus (FeLV) replicates in cat cell cultures³ and infected cells are demonstrable by the indirect immunofiuorescent antibody test which detects FeLV group-specific antigen as granular punctate fluorescence in the cytoplasm of acetone fixed cells⁴; this method allows easy quantitation of the antiviral effect of interferon. We report the production and assay of feline interferon using the fluorescent antibody test with FeLV infected cat cell cultures.     

Retention of Antigen Specificity of Murine Sarcoma Viruses grown in Hamster Cells <Emphasis Type="Italic">in vitro</Emphasis>

TUMOURS can be induced in hamsters by the various strains of murine sarcoma virus (MSV)^1–6. Tumours differ, however, in the antigens which are expressed. Whereas the cell line HT-1, derived from early passages of a hamster tumour induced by the Moloney strain of MSV (M-MSV), contains no trace of infectious virus or virion antigen^2,7, tumours induced by the Harvey (H), Kirsten (Ki) and later passages of the M-MSV-(GLV) viruses have yielded sarcoma viruses with a hamster-specific host range^3–6,8 which do not share envelope^4–6,9 or group specific¹⁰ antigens with murine viruses. The HT-1 cell does retain the MSV genome which can be rescued by murine leukaemia viruses². Such rescued viruses are termed pseudo-types and contain the envelope and group-specific antigens of the rescuing virus. The virus preparation from tumours induced by M-MSV(GLV) differed from the other hamster-specific viruses in that a non-sarcomagenic C-type virus could be isolated from cultures infected beyond the cell transformation end point⁶. This virus was also hamster-specific in host range and antigenic properties and specifically interfered with cell transformation by the various hamster-specific virus strains⁹. This virus also shared an ether-stable virion-antigen with a C-type virus found in a lymphoma which occurred spontaneously in a hamster¹⁰. This shared antigen seems to be the principal structural polypeptide of hamster C-type viruses and is structurally similar but antigenically distinct from its mouse homologue (unpublished work of S. O., C. Foreman, G. K. and R. V. G.). These findings led us to propose that the hamster-specific non-sarcomagenic C-type virus was a hamster leukaemia virus (in the generic but not necessarily the pathological sense) and the virus is therefore designated HaLV^9,10. The hamster-specific sarcoma viruses are considered to be pseudotypes of MSV rescued in vivo by HaLV and are abbreviated accordingly; for example, M-MSV(HaLV) represents the hamster-specific sarcoma virus rescued from M-MSV induced tumours. This is plausible because HaLV is able to rescue the MSV genome from HT-1 cells⁶. (This change in the nomenclature has been made in order to reflect the antigenic composition of the hamster-specific virus more accurately. In addition, to indicate the virus rescued from M-MSV(GLV)-induced hamster tumours, a terminal G is added after the parentheses. This has been done only to distinguish it from the virus obtained from M-MSV induced hamster tumours, for there is no evidence of residual activity from GLV.)     

Serological Identification of Hamster Oncornaviruses

CATS, mice and chickens have indigenous oncornaviruses (oncogenic RNA viruses) which induce leukaemias and sarcomas^1,2. Mouse sarcoma virus (MuSV), like avian sarcoma virus, can induce sarcomas in the hamster^3,4 but some of these MuSV hamster sarcomas release virus that differs both antigenically and with regard to its host range from the original⁵—it can be neutralized by antisera prepared against isolates of virus released from MuSV-transformed cells but not by antisera against murine leukaemia virus (MuLV) and it is sarcomagenic in hamsters but not in mice. Such a virus could be: (a) an indigenous hamster sarcoma virus “activated” by the inoculation of MuSV; (b) an MuSV genome that has acquired a new viral envelope from an indigenous hamster leukaemia virus (HaLV) during its sojourn in hamsters; or (c) a recombinant between HaLV and the sarcomagenic portion of the MuSV genome. In fact, it is known that the hamster possesses a virus (HaLV) which is morphologically similar to MuLV^6,7. This virus lacks⁸ the group-specific (gs) internal MuLV-gs1 antigen characteristic of MuLV^9,10 although it does have the gs antigen (MuLV-gs3) which is common to all mammalian leukaemia viruses investigated so far⁸.     

Increased Transformation Efficiency of Simian Virus 40 in Rat Embryo Cells infected with Rauscher Leukaemia Virus

SIMIAN virus 40 (SV40) is oncogenic for hamsters¹ and Mastomys². In vitro studies have shown that SV40 is capable of transforming cells derived from hamster³, mouse⁴, rabbit⁴, pig⁴, cow⁵, monkey⁶, human⁷, guinea-pig⁸ and rat⁹. We have established and studied continuous lines of uninfected and Rauscher leukaemia virus (RLV) infected rat embryo (RE) cells¹⁰. Rat embryo cells exposed to RLV have produced infectious virus and complement-fixing (CF) antigen characteristic of the murine leukaemia-sarcoma virus complex for more than 18 months. This communication describes increased transformation efficiency of SV40 in RLV infected RE cells (RLV-RE) compared with uninfected RE cells.     

Inhibition of Leukaemia Virus Replication by Polyadenylic Acid

A PREVIOUS communication from this laboratory demonstrated that the DNA polymerase of the Rauscher leukaemia virus is strongly inhibited in vitro by unprimed, single stranded polyribonucleotides¹ as a result of competition between the polymers and the active template for the same enzyme binding site. This inhibition was apparently specific, since partially purified preparations of DNA polymerase from Escherichia coli and BALB/c mouse embryos were not inhibited in the same conditions. We attempted to determine therefore whether single stranded polyribonucleotides would have any effect on the activities of oncogenic RNA viruses in cultured cells.     

Hormone-activated Expression of the C-type RNA Tumour Virus Genome

THE concept of vertical transmission of specific viral information, particularly that possibly associated with the induction of malignancy in mice has been postulated. Moreover, it has been hypothesized that this genetic information may be expressed either in the form of whole virus in certain selected laboratory animal strains or as the operon involved in regulating cellular replication (oncogene)¹. To detect this proposed genetically transmitted message, one uses a group specific antisera against the C-type RNA tumour viruses having as one of its components, gs-3, first described by Gerring et al.². A similar group specific antigen has subsequently been reported by Schafer³, Gilden⁴ and Sarma et al.⁵ and designated “interspec”, meaning that the antigen is common to the internal components of the C-type RNA virion of several mammalian species.     

Inactivation of DNA Polymerases of Murine Leukaemia Viruses by Calcium Elenolate

THE monoterpene elenolic acid can be isolated, after mild acid hydrolysis, from aqueous extracts of the olive plant (Olea europa)^1,2. The hydrolysate yields crystalline calcium elenolate [(C₁₁H₁₃O₆)₂Ca] which is virucidal in vitro for a number of both DNA and RNA viruses³. It also reduces virus yields from hamsters infected with parainfluenza 3 virus⁴ and has minimal toxicity when administered to animals⁵. I report the inhibition of RNA-dependent DNA polymerases of murine leukaemia viruses by calcium elenolate.     

Rat C-Type Virus induced in Rat Sarcoma Cells by 5-Bromodeoxyuridine

HALOGENATED derivatives of uridine are highly effective inducers of latent C-type RNA viruses^1,2 and have been successfully used to induce viruses identical to, or similar to, the C-type RNA tumour viruses in mouse, rat and human cells^3–6. In previous experiments we used 5-bromodeoxyuridine (BrUdR) for induction of focus-forming virus in non-productive rat cells that have been transformed by mouse sarcoma virus². We describe here the induction of a C-type RNA virus in the cells of the rat tumour cell line XC, which contains the Rous sarcoma virus genome⁷. The induced virus possesses the group specific (gs) antigens of rat C-type viruses but not those of chicken C-type viruses.     

Morphological Transformation of Rat Embryo Cells by the Combined Action of 3-Methylcholanthrene and Rauscher Leukaemia Virus

RAT embryo cells infected with either CF-1 or Rauscher C-type RNA murine leukaemia virus, when treated with diethylnitrosamine (DENA), undergo morphological transformation and become aneuploid¹. Untreated cells and cells treated with either virus or chemical alone do not transform. We describe here a similar effect of 3-methylcholanthrene (3 MC) on rat cells infected with Rauscher leukaemia virus.     

Expression of a New Complement-fixing Antigen reactive with Murine Sarcoma Virus Rat Antiserum in Rat Cells transformed by Polyoma Virus

WE reported accelerated transformation by DNA viruses (SV40 and polyoma) in rat embryo (RE) cells chronically infected with a C-type RNA virus^1,2. Recently we found in RE cells transformed by polyoma virus a new complement-fixing (CF) antigen detectable by rat antisera having broad reactivity with the various intraspecies and interspecies antigens of the RNA tumour viruses^3–8; this antigen, however, was distinct from the murine intraspecies and interspecies group-specific (gs) antigens both immunologically and by virtue of other properties. It is also distinct from the polyoma virion (capsid) and tumour (“T”) antigens.     

Coexistence of Intraspecies and Interspecies Specific Antigenic Determinants on the Major Structural Polypeptide of Mammalian C-type Viruses

THE report of a shared viral antigen (termed gs-3) among mammalian C-type viruses from four species¹, extending an earlier report of cross reactivity between mouse and cat viral antigens², has far reaching implications in the search for human cancer viruses or their gene products. The report is confirmed both by the data presented here and also by the data obtained by another laboratory³. Our gel diffusion assays using various selected sera against mouse, hamster and cat crude and purified C-type viral antigens indicate that the cross reactive antigenic determinants are specifically present on the major structural polypeptide of C-type viruses. The polypeptide also carries species specific determinants. These conclusions are drawn from complement fixation and gel diffusion tests using six types of antisera (either individual sera or pools) prepared as described in Table 1.     

Transformation and Productive Infection of Human Osteosarcoma Cells by a Feline Sarcoma Virus

FELINE sarcoma virus (FSV) transforms human embryo cells in vitro¹; it therefore seemed interesting to determine whether this virus could transform human osteosarcoma cells. Defective Moloney sarcoma virus genome can be rescued from non-producer hamster tumour cells by feline leukaemia virus (FeLV)² and because FSV stocks also contain excess FeLV (ref. 1 and unpublished observations of R. V. G.), it was hoped that human osteosarcoma cells transformed by FSV and co-infected with FeLV might yield a human sarcoma virus.     

Induction of Group-specific Interspecies Antibody in a Cat by Immunization with Disrupted Feline Leukaemia Virus

“ALL mice, cats and virtually all chickens seem to be completely refractory to developing antibody to the group-specific, gs, antigens characteristic of the RNA tumour viruses of their own species.”¹ This is explained on the basis of an immune tolerance induced in early embryonic life by the expression of these antigens before the development of immune competence. Avian group-specific (gs) antibody has been demonstrated in the sera of immunized chickens by the immunodiffusion (Ouchterlony)² and complement fixation inhibition³ tests. This report is to record the production of gs antibody in a cat which had been immunized with gs antigen from disrupted feline leukaemia virus (FeLV).     

Surface Alterations of Cells Carrying RNA Tumour Virus Genetic Information

CELLS transformed by the DNA tumour viruses, polyoma virus and SV40, are agglutinated by lectins such as wheat germ agglutinin¹, concanavalin A (Con A)² and soybean agglutinin³. Agglutination in these cases presumably reflects changes in the cell surface related to the transformed properties of the cell; studies with a temperature-dependent mutant of polyoma virus has shown that cell surface changes are controlled by viral genes⁴. Here we describe experiments in which we investigated the agglutinability of cells transformed by RNA tumour viruses. One recent report had suggested that cells transformed by RNA tumour viruses were not specifically agglutinated⁵, whereas a second more recent report claimed the specific agglutination of cells transformed by RSV⁶. We find that transformed rat, mouse and cat cells that replicate the sarcoma-leukaemia virus complex of murine (MSV) and feline (FeSV) origin are strongly agglutinated by Con A, but mouse and human cells that replicate the murine and feline leukaemia virus components alone are not agglutinated. The ability to agglutinate is rapidly acquired by normal mouse cells on infection with the murine sarcoma virus at a rate that parallels virus replication. In contrast to the results obtained with cells producing virus, non-virus-producing transformed hamster and mouse cells that synthesize virus-specific RNA are either not agglutinated or are agglutinated to a lesser degree. These results suggest that the cell surface alterations responsible for agglutination are not necessarily associated with the transformed state of the cell, but rather with the possession of sarcoma virus-specific information.     

Expression of oncornavirus antigens in peripheral leucocytes of patients with chronic myeloid leukaemia.

Peripheral leucocytes from 16 patients with chronic myeloid leukaemia (CML) were examined for the presence of oncornavirus p30 antigens by indirect cytoplasmic immunofluorescence. The leucocytes of 12 patients who could be kept in balance by chemotherapy proved to be negative or contained the p30 antigen of mammalian endogenous oncornaviruses as the only viral antigen. In the leucocytes of four patients being in blastoid crisis, an antigen related to the p30 antigen of mammalian leukaemia-sarcoma viruses was detected. In five of six patients decrease in sensitivity to chemotherapy, or blastoid crisis, was preceded by expression of leukaemia-sarcoma virus p30 antigen(s). Leucocytes from 15 CML patients kept in balance by chemotherapy and those from seven being in blastoid crisis, were examined by indirect membrane immunofluorescence for the presence of antigen(s) related to the gp70 antigen of the simian and murine leukaemia-sarcoma virus. All tests proved to be negative.     

Transfer of Immunity to Tumour Isografts by the Systemic Administration of Xenogeneic “Immune” RNA

SPECIFIC immunoreactivity can be conferred on lymphoid cells by incubation with RNA-rich extracts prepared from lymphoid tissues exposed to specific antigens in vivo¹ and in vitro^2,3. We have shown transfer of immunity to tumour specific antigens in vivo⁴ and in vitro⁵ by incubation of syngeneic spleen cells in vitro with RNA extracted from the lymphoid tissues of xenogeneic or syngeneic animals immunized with the tumour to be treated. Administration of these spleen cells to normal animals decreased the development and growth of isografts of the same tumour.     

Specific Inhibition of Mammalian Ribonucleic Acid C-Type Virus Deoxyribonucleic Acid Polymerases by Rat Antisera

Inhibition of the ribonucleic acid (RNA)- and deoxyribonucleic acid (DNA)-dependent DNA polymerase activities of mammalian C-type viruses was obtained with sera from rats bearing murine leukemia virus-induced transplant tumors. Polymerase activities of nonmammalian (viper) C-type virus and murine mammary tumor virus were not inhibited by such sera nor by serum from a rat immunized with the DNA polymerase of feline leukemia virus purified by isoelectric focusing. The latter serum appeared to inhibit preferentially the DNA-dependent DNA polymerase activity of mammalian C-type viruses showing no inhibition of RNA-dependent DNA synthesis.