Feline Leukaemia Viral Antigens and Antisera to the Group Specific Antigens of the Murine Leukaemia Viruses

GEERING et al.¹ reported that feline leukaemia viruses shared one of the group specific antigens of the murine leukaemia viruses, gs-3, as detected by immunoprecipitation in agar gels with broadly reactive rat antisera to the group specific antigens of the murine leukaemia viruses (MuLV). Subsequently, they found that this shared group specific antigen was also present in the hamster and rat C-type viruses². Work by Schafer³ and our own immunodiffusion⁴ and complement fixation studies have confirmed the immunological reactivity between the feline leukaemia viral antigens and broad-reacting murine leukaemia group specific antisera. We have now applied this interspecies immunological reaction between the murine and feline C-type viruses to quantitative studies of the feline leukaemia viruses. Broad-reactive murine leukaemia-sarcoma group specific antisera prepared in rats by the induction of murine sarcoma virus (MSV) tumours^{5, 6} were found to be as useful and nearly as sensitive as a feline leukaemia-sarcoma specific, group specific antiserum for the in vitro detection and assay of the noncytopathogenic feline leukaemia virus (FeLV).     

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.     

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.     

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.     

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.)     

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.     

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.     

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.     

Feline Leukemia Virus-B Envelope Together With its GlycoGag and Human Immunodeficiency Virus-1 Nef Mediate Resistance to Feline SERINC5

Human SERINC5 (SER5) protein is a recently described restriction factor against human immunodeficiency virus-1 (HIV-1), which is antagonized by HIV-1 Nef protein. Other retroviral accessory proteins such as the glycosylated Gag (glycoGag) from the murine leukemia virus (MLV) can also antagonize SER5. In addition, some viruses escape SER5 restriction by expressing a SER5-insensitive envelope (Env) glycoprotein. Here, we studied the activity of human and feline SER5 on HIV-1 and on the two pathogenic retroviruses in cats, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV). HIV-1 in absence of Nef is restricted by SER5 from domestic cats and protected by its Nef protein. The sensitivity of feline retroviruses FIV and FeLV to human and feline SER5 is considerably different: FIV is sensitive to feline and human SER5 and lacks an obvious mechanism to counteract SER5 activity, while FeLV is relatively resistant to SER5 inhibition. We speculated that similar to MLV, FeLV-A or FeLV-B express glycoGag proteins and investigated their function against human and feline SER5 in wild type and envelope deficient virus variants. We found that the endogenous FeLV recombinant virus, FeLV-B but not wild type exogenous FeLV-A envelope mediates a strong resistance against human and feline SER5. GlycoGag has an additional but moderate role to enhance viral infectivity in the presence of SER5 that seems to be dependent on the FeLV envelope. These findings may explain, why in vivo FeLV-B has a selective advantage and causes higher FeLV levels in infected cats compared to infections of FeLV-A only.     

A comparison of the enzymatic responses of the DNA polymerases from four RNA tumor viruses.

DNA polymerases from avian, feline, murine and simian RNA tumor viruses exhibit substantial differences in optimal assay conditions and vary widely in their template-primer preferences. Avian DNA polymerase utilizes both natural and synthetic template-primers efficiently in the presence of Mg⁺⁺ as well as Mn⁺⁺. By contrast, the mammalian viral DNA polymerases are much more responsive to poly(A)·oligo(dT) than to other template-primers, and exhibit up to 20-fold greater activity with Mn⁺⁺ than with Mg⁺⁺. In addition, simian sarcoma virus DNA polymerase shows no detectable response to poly(C)·oligo(dG) over a wide variety of conditions stimulatory to the other viral enzymes.     

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.     

Sequence analysis of Gardner-Arnstein feline leukaemia virus envelope gene reveals common structural properties of mammalian retroviral envelope genes

We have sequenced the envelope (env) gene and most of the adjacent 3' long terminal repeat (LTR) of Gardner-Arnstein feline leukaemia virus subtype B. The LTR of this virus contains, at corresponding positions, all signal sequence elements known from other retroviral LTRs. The deduced amino acid sequence of the longest open reading frame was compared with env polypeptide sequences of several murine leukaemia viruses. This allowed us to predict the positions of both p12/15env and gp70 polypeptides as well as a hydrophobic leader polypeptide. The env polypeptides of the different viruses show long stretches of homology and similar hydrophilicity profiles in the p12env region and in the carboxy-terminal half of gp70 (constant region). The most extensive variations are confined to certain parts of the amino-terminal half of gp70 (differential region). In this region, however, feline leukaemia virus and murine mink cell focus forming viruses are still closely related. A correspondingly spaced pattern of identical, short amino acid sequences appears in three different parts of the env polyprotein, suggesting its evolution from a primordial env-related precursor by tandem duplications.     

Template specificities of the RNA dependent DNA polymerase of different murine C-type virus particles.

The crude RNA dependent DNA polymerase of seven different C-type viruses (AMV, Kirsten-MSV produced by NRK or $\text{NIH}\text{3T3}$ cells, Moloney-MuLV, Kirsten-MuLV, the murine myeloma associated virus (MuMAV) from FLOPC-1 and MOPC-21) was analyzed for their ability to utilize four different synthetic $\text{RNA}\text{DNA}$ hybrids or three different $\text{DNA}\text{DNA}$ duplexes as templates. The polymerases from AMV and murine sarcoma or leukemia viruses were distinctly different in their template stimulated activities and the two MuMAV polymerases were different from all of the other enzymes. MuMAV RDDPs were not stimulated by any of the synthetic $\text{RNA}\text{DNA}$ hybrid templates to the same level as the enzymes of the other C-type viruses and their ability to distinguish between templates was also different.     

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.     

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.     

Streptovaricins inhibit Focus Formation by MSV (MLV) Complex

We recently reported that the streptovaricins inhibit the reaction by which DNA is transcribed from the RNA template resident in murine leukaemia virions (MLV)¹. The reports^{2, 3} which first indicated that this DNA polymerase is present in oncogenic RNA viruses have been confirmed and extended^4–8. The enzyme provides a mechanism whereby an RNA virus can insert stable genetic information into a host chromosome. Gallo and co-workers described an RNA dependent DNA polymerase in lymphoblastic leukaemic cells which was inhibited by N-demethylrifampicin⁹ and this antibiotic, together with a number of other rifamycin derivatives, also inhibited the oncogenic viral DNA polymerase¹⁰. Like the streptovaricins, the rifamycins are ansa macrolide antibiotics (Fig. 1).     

Feline cytotoxic immune mechanisms against virus-associated leukemia and fibrosarcoma

Humoral and cellular cytotoxic immune mechanisms of cats were compared against feline leukemia virus (FeLV)- and feline sarcoma virus (FeSV)-transformed cells. The groups of animals studied were nonexposed control cats; FeLV-infected immune or viremic tumor-bearing cats; FeSV-inoculated tumor progressor or regressor cats, and cats immunized with FeSV-transformed autochthonous fibroblasts (ATF). Sera containing complement-dependent antibodies (CDA), which lysed FeLV-producer lymphoma lines, had no cytotoxic effects when tested against FeLV-producer FeSV-transformed fibroblasts. Sera with lytic CDA activity were also tested for antibody-dependent cellular cytotoxic (ADCC) effects with peripheral blood lymphocytes (PBL) from nonimmune cats. No ADCC activity was detected against either lymphoid or fibroblast target lines. To demonstrate that cat PBL contained ADCC effector cells, antibody-coated murine target cells were employed and positive results obtained. Natural killer (NK) assays were performed using PBL from normal and tumor-bearing cats. Cytotoxic effects were only detectable to FeLV-producer lymphomas, and comparable levels of NK activity were found in normal and lymphoid tumor-bearing animals. In cats immunized with ATF, a population of effector cells was found in peripheral blood which had functional characteristics of cytotoxic T lymphocytes (CTL). The killing of ATF by CTL-like cells was not inhibited by FeLV/FeSV immune sera or by sera from autochthonous immune cats. The comparative importance of humoral and cellular cytotoxic mechanisms against FeLV- and FeSV-induced tumors is discussed.