Host range of mink cell focus-inducing viruses

The species host range of the recombinant, mink cell focus-inducing (MCF) class of murine retroviruses was determined in vitro and compared to the host range properties of xenotropic and amphotropic murine viruses. In contrast to xenotropic and amphotropic viruses, MCF viruses were restricted in the number of mammalian species they would infect. Cell lines from mouse, rat, mink, ferret, and cat were susceptible to MCF infection and certain virus isolates could infect rabbit cells, but cells from Chinese hamster, buffalo, bat, dog, monkey, and human were resistant to infection by most MCF viruses. The resistance of some of the latter cells was abrogated by phenotypic mixing with xenotropic virus, which demonstrated that MCF species host range was mediated by virus envelope-cell surface interaction. The host range uniformity of the various MCF isolates and the unique species distribution of sensitivity are consistent with the conclusion from other evidence that the MCF viruses comprise a class distinct from xenotropic and amphotropic viruses.     

Role of endogenous murine leukemia virus in immunologically triggered lymphoreticular tumors II. Isolation of B-tropic mink cell focus-inducing (MCF) murine leukemia virus

Previous work has shown that (BALB/c x A)F, (CAF₁) mice with an immunological disorder, the graft-versus-host reaction (GVHR), express an oncogenic murine leukemia virus (MuLV) which appears to be responsible for the subsequent development of lymphoreticular tumors. A mink cell focus-inducing (MCF) virus has been isolated from a reticulum cell neoplasm induced in a BALB/c mouse by serially passaged B-tropic MuLV originating in a CAF, GVHR mouse. The cloned MCF virus has a dual host range. It grows both in mouse cells where it is XC negative, B tropic, and in mink lung cells where it induces characteristic foci. It is partially neutralized by xenotropic MuLV antiserum and it partially competes in a homologous ecotropic gp70 radioimmunocompetition assay. Both ecotropic and xenotropic MuLV interfere with its infectivity. In these biological properties, the GVHR MCF virus closely resembles recombinant AKR MCF virus. We discuss the possible origin and significance of a presumptive recombinant MuLV in a low virus model where lymphoreticular tumors are triggered by an immunological disturbance.     

Use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses

A rapid and sensitive focal immunofluorescence assay (FIA) using monoclonal antibodies or heterologous antisera was employed for detection and biological cloning of viruses capable of inducing viral antigens on cell surfaces. The FIA was performed directly on a variety of live cells in tissue culture dishes and was used successfully with C-type murine leukemia viruses (MuLV) of different tropism including ecotropic, xenotropic, amphotropic, and dual-tropic recombinant mink cell focus-inducing (MCF) viruses. With the FIA, we were able to titrate and distinguish ecotropic Friend-MuLV and Friend-MCF viruses present in mixtures. Dual-tropic MCF viruses could be specifically detected directly in mouse cells by using MCF-specific monoclonal antibodies. These antibodies replaced the requirement for production of typical MCF cytopathic effect in mink cells for MCF virus detection, and also allowed efficient titration in mouse cells of MCF virions pseudotyped with ecotropic envelope proteins. Furthermore, by picking foci of fluorescent cells and using their cell-free viral progeny, MCF viruses were cloned from complex pseudotypic mixtures. This allowed the cloning of viruses present at low frequency in heterogeneous mixtures obtained from leukemic tissues.     

Different murine cell lines manifest unique patterns of interference to superinfection by murine leukemia viruses

Interference to superinfection by murine leukemia viruses (MuLV) was analyzed in cells chronically infected with other MuLVs. A new sensitive focal immunofluorescence assay employing monoclonal antibodies was used to detect foci of virus infection in live cell monolayers. Monoclonal antibodies were chosen which reacted with the challenge virus but not with the interfering virus. The results obtained confirmed some of the findings of previous workers using Moloney sarcoma virus pseudotypes as challenge viruses on mouse and nonmouse cells. In addition, SC-1 mouse cells nonproductively infected with defective spleen focus-forming virus were found to be resistant to superinfection by recombinant dual-tropic viruses. Furthermore, results indicated that interference patterns between some pairs of viruses differed in different cell types. Thus, xenotropic MuLV blocked superinfection by recombinant dual-tropic viruses in SC-1 feral mouse cells, but not in two lines of NZB mouse cells. Also, in a Mus dunii tail fibroblast cell line some unique patterns of interference were observed. One ecotropic MuLV blocked infection by two xenotropic viruses and three recombinant dual-tropic viruses. Two other ecotropic viruses blocked infection by only one of the two xenotropic viruses tested. These two ecotropic viruses also differed from each other in their ability to block the three recombinant viruses. In addition, two strains of amphotropic MuLV also differed in their interference capacity. As expected, strain 1504A did not block any viruses tested, whereas strain 4070A surprisingly blocked one xenotropic and one ecotropic MuLV. The lack of homogeneity in interference patterns seen in the Mus dunii cells suggested either that a large number of heterogeneous virus receptors were present on this cell line or that interference in these cells might operate through a mechanism other than blocking of virus receptors by the envelope protein of the interfering virus.     

Detection and quantitation of phenotypically mixed viruses: Mixing of ecotropic and xenotropic murine leukemia viruses

Methods were devised for detection and titration of phenotypically mixed particles in harvests of mixed infections by ecotropic and xenotropic murine leukemia viruses. Such harvests contain, in addition to parental-type virions, particles which induce ecotropic virus infection of heterologous cells and particles which induce xenotropic virus infection of mouse cells. The phenotypically mixed particles show neutralization and interference specificities corresponding to the host range determinant. These findings, in conjunction with the relative proportions of infectious particles in mixed infection harvests, are compatible with the hypothesis that the restriction of mink cells for infection by ecotropic viruses is fully circumvented by phenotypic mixing, while the resistance of mouse cells to xenotropic virus infection is at two levels, one of which cannot be overcome by phenotypic mixing.     

Pseudotypes of vesicular stomatitis virus with envelope antigens provided by murine mammary tumor virus.

Infection of two mouse mammary carcinoma cell lines with vesicular stomatitis virus (VSV) resulted in the formation of at least two types of particles containing the VSV genome but expressing different envelope characteristics (VSV pseudotypes). One of these VSV pseudotypes was infectious for a cell line derived from normal mouse mammary epithelial cells and mouse embryo cells but noninfectious for 3T3 cells, mink lung cells, and Vero cells. If mouse mammary tumor cells were treated with dexamethason some days prior to infection with VSV, the titer of this pseudotype was significantly increased. In contrast, the second pseudotype was infectious for mink cells, but not for the other cell lines tested, and the titer of this second pseudotype was unaffected by the presence of dexamethasone. The first pseudotype was found to be almost completely neutralized by anti-murine mammary tumor virus (MuMTV) serum whereas the second pseudotype was only partially neutralized at a higher antiserum concentration. Neither pseudotype showed the neutralization, host range, or interference properties of either ecotropic or xenotropic murine C-type viruses. These results suggest that the first pseudotype is VSV(MuMTV). The other pseudotype is less well defined but conceivably may represent a xenotropic MuMTV. In the course of these studies, a filterable agent was observed in GR mammary carcinoma cultures that reactivated the infectivity of VSV neutralized by antiserum. This agent was transmissible to mink cells.     

Effect of murine host genotype on MCF virus expression, latency, and leukemia cell type of leukemias induced by Friend murine leukemia helper virus

Leukemias induced by neonatal inoculations of several mouse strains with different strains of Friend murine leukemia helper virus (F-MuLV) were followed for time of disease onset, cytochemical analysis of predominant cell types in leukemic organs, and expression of infectious mink cell focus-inducing (MCF) viruses detected by mink cell foci or MCF-specific monoclonal antibodies. Most BALB.B and IRW mice had a rapidly appearing, severe anemia and hepatosplenomegaly consisting of erythroid cells. MCF viruses were usually isolated from enlarged spleens of IRW mice. In contrast, C57BL/10 mice had a lower incidence of disease and much slower course. Splenomegaly and lymphadenopathy with mild anemia were seen, and the predominant cell types were either myeloid (chloroleukemia) or lymphoid. MCF viruses were never isolated from this mouse strain. (C57BL/10 X IRW)F1 mice were intermediate in latency, but all mice had disease by 8 months. Myeloid, lymphoid, and some mixed leukemias with an erythroid component were observed, but in no case did we see the severe anemia or pure erythroid involvement typical of IRW and BALB.B mice. MCF viruses were, however, isolated from 22% of these mice regardless of leukemia cell type. DBA/2 mice had a disease pattern similar to the (C57BL/10 X IRW)F1 mice, and MCF viruses were isolated from three of six mice tested. Inoculation of IRW mice with the low virulence B3 strain of F-MuLV produced disease with a longer latency than F-MuLV 57, but similar cell types were transformed by both viruses. In vitro cell lines were derived from 14 mice, and most were tumorigenic in vivo. Three lines released infectious MCF virus, and three others expressed MCF-specific cell surface antigens but did not release virus. Eight lines expressed no MCF infectious virus or viral antigens. Several lines released infectious xenotropic viruses and/or expressed xenotropic MuLV cell surface antigens recognized by monoclonal antibodies reactive with xenotropic viruses. The lack of MCF expression in many primary leukemic tissues as well as in in vitro derived leukemia cell lines of C57BL/10 and (B10 X IRW)F1 mice suggested that MCF virus generation and expression may not be required for leukemogenesis in some mouse strains or in some hemopoietic lineages.     

Characterization of an amphotropic murine C-type virus that is NB-tropic.

Cocultivation of SIPC-2 myeloma cells with clone A₃₁, BALB/3T3 cells results in the continuous production of a C-type virus by the A₃₁ cells that has been designated S/A-1. S/A-1 virus possessed the essential properties of a murine retrovirus, including a buoyant density of 1.16 g/cm³, C-type morphology, high molecular weight (70 S) RNA, and an RNA-dependent DNA polymerase. After cloning by three endpoint dilution passages in A₃₁, and SIRC cells, the virus was shown to possess ecotropic as well as xenotropic activity. For example, the S/A-1 virus producibility infected mouse cells, including BALB/3T3, NIH/3T3, SC-1, 3T3 FL, and F₁ cells as well as non-mouse cells, including normal rat kidney, rabbit corneal (SIRC), Chinese hamster peritoneal, mink lung, Pekin duck embryo, human rhabdomyosarcoma, and guinea pig embryo cells. The S/A-1 virus infected A₃₁, cells do not induce XC-syncytia and the virus does not induce foci in mink lung cells. Thus, the data suggest that it is an amphotropic virus whose ecotropism is different (i.e., NB-tropic) from the previously reported amphotropic viruses (i.e., N-tropic). In addition, S/A-1 virus is different from the mink cell focus-inducing (MCF) viruses (J. W. Hartley, N. K. Wolford, L. J. Old, and W. P. Rowe 1977, Proc. Nat. Acad. Sci. USA74, 789–792) in terms of its lack of focus-inducing activity on mink lung cells, B-tropism, and ability to replicate in human rhabdomyosarcoma and F₁ mouse cells. The structural proteins of cloned S/A-1 virus were compared to those of 1504-A virus, a previously described amphotropic virus isolated from feral mice (J. W. Hartley and W. P. Rowe 1976, J. Virol.19 19–26; S. Rasheed, M. B. Gardner, and E. Chan 1976, J. Virol.19, 13–18), by electrophoresis on a linear 7–20% acrylamide slab gel and staining with Coomassie blue. S/A-1 differed from 1504-A in terms of (a) the mobility of a protein with a molecular weight of approximately 12,000 daltons (pl5) and (b) the fact that it has a “double” p30.     

Characterization of inducible type-C RNA viruses of mouse strains from different geographic areas

The distribution of endogenous type-C RNA viruses was studied in inbred strains of mice and some subspecies of Mus musculus of different geographical origins. The following groups of inducible viruses were characterized by their host range and immunological properties: (1) viruses indistinguishable from one of the three prototype viruses endogenous to BALB/c mice; (2) viruses coding for proteins immunologically related to different prototype endogenous viruses; (3) viruses whose p12 structural proteins were immunologically indistinguishable from that of BALB:virus-2, but whose p30 major structural proteins and envelope glycoproteins differed immunologically from those of previously characterized endogenous viruses. These findings suggest that endogenous viruses have undergone numerous genetic interactions during the process of evolution leading to inducible viruses of present day mouse strains. A class of xenotropic virus spontaneously released by NZB mice is endogenous to but not inducible from embryo cells of other previously studied mouse strains. Viruses which could not be distinguished from the NZB xenotropic virus by host range analysis or radioimmunological techniques were chemically inducible from embryo cells of several mouse strains originating in Asia and Europe. These results indicate that the biological regulatory mechanisms that affect expression of this virus have evolved differently in such strains from control mechanisms that developed in standard inbred strains.     

Phenotypic mixing between murine leukemia viruses: characteristics of ecotropic virus infection of heterologous cells.

Ecotropic murine leukemia viruses (MuLV) were capable of infecting mink, rabbit, duck, guinea pig, human, and cat cells, but not chicken cells, when phenotypically mixed with xenotropic MuLV. Mink cell cultures inoculated with the phenotypically mixed pools showed XC plaques with one-hit dose response kinetics; these plaques represented colonies derived from outgrowth of single infected cells rather than spreading areas of infection, which showed two-hit kinetics. Clonal lines of mink and rabbit cells chronically infected with N-tropic ecotropic (AKR-L1), B-tropic ecotropic (WN1802B), and NB-tropic ecotrpic (Friend) MuLV were established. Production of virus by these xenogenic cell lines was generally much lower than by mouse cells, as was the number of cells with gs-antigen detected by immunofluorescence. However, essentially all cells registered as virus-producers on infectious center plating. Treatment with IUdR enhanced virus production by 10-fold, and immunofluorescence staining as well, in the three infected mink cell lines. The viruses retained their ecotropic and Fv-1-determined host range characteristics through more than 6 to 12 months of chronic virus production in the heterologous cells.     

Endogenous type C RNA virus of mink (Mustela vison).

A type C RNA virus was isolated from mink lung cell line (American Type Culture Collection No. CCL 64) which had been cocultivated with 5-bromodeoxyuridine (BUDR)-treated mouse spleen cells. The virus has type C RNA virus morphology as demonstrated by electron microscopy. The complement fixation and immunofluorescent tests performed with mouse anti-p30 antisera show a distinctive difference between mink and mouse type C viruses. Complement fixation tests also indicate that mink type C virus is antigenically different from rat, feline leukemia, feline endogenous (RD-114), baboon, and woolly monkey type C viruses. The virus propagates in cells of mouse, rat, cat, sheep, dog, and human origin, but not in bovine (MDBK) or simian (BSC-1) cells. The infection of rabbit (SIRC) cells and cells of virus origin (mink lung) was followed by delayed and low-titer polymerase release in tissue culture media. The virus sediments in sucrose density gradients as a broad band of densities, 1.13–1.17 g/ml, and contains 70 and 4S RNA. The protein profile is similar to that observed in other mammalian type C viruses. The DNA complementary to the poly(A)-containing virion RNA hybridized to a high degree (72%) with the RNA from virus-producing mink lung cells but not with the RNA from mouse cell lines or uninfected mink lung cell line. The nucleotide sequences homologous to mink viral cDNA were found in mink cell DNA from both virus-producing and nonproducing cells, but not in the DNA of mouse, rat, or feline origin. The virus here described therefore represents an endogenous mink type C virus.     

Conversion of restrictive mouse cells to permissiveness during sequential and mixed double infection by murine leukemia viruses.

Conversion from restrictive (two-hit) to permissive (one-hit) kinetics was observed when N- or B-tropic murine leukemia viruses were titrated on mouse embryo fibroblasts of nonpermissive Fv-1 genotype that had previously been nonproductively infected with the same virus at an average multiplicity of one. The effect was transient, disappearing within about 24 hr after the first infection, and was abrogated by exposure of the first virus preparation to increasing doses of ultraviolet light, with a D₃₇ inactivation dose of 2550 ergs/mm², about three times that for inactivation of viral replication. Prior infection of nonpermissive mouse cells with the NZB xenotropic virus did not alter their restrictive response to later infection with ecotropic viruses. Low multiplicity infection of $\text{NIH}\text{3T3}$ cells with a B-tropic virus, followed by treatment with iodoeoxyuridine, failed to induce productive infection by endogenous or exogenous virus. Cells of F₁ hybrid Fv-1 genotype, which are restrictive for both N- and B-tropic viruses, were converted to permissiveness for virus of either tropism after low multiplicity infection with virus of the opposite tropism. No evidence of NB-tropic recombinant progeny could be detected under these experimental conditions. The implications of these experimental observations with respect to the mechanism of restriction governed by the Fv-1 gene are discussed.     

Genetic relationship of wild mouse amphotropic virus to murine ecotropic and xenotropic viruses.

The genomic relationship between amphotropic and ecotropic wild mouse (Mus musculus) leukemia viruses (MuLV) and between these viruses and the ecotropic and xenotropic MuLVs of inbred mice was determined by DNA-RNA hybridization using viral complementary DNA and polyA-containing 70S viral RNA. The results indicate that (a) the genomes of individual cloned amphotropic and ecotropic wild mouse virus isolates are closely related and (b) these nucleotide sequences are also related to, yet distinguishable from, the genomes of prototype ecotropic (Rauscher, Gross-AKR) and xenotropic (AT-124, NZB, AKR, Balb/c) MuLVs of inbred mice.     

Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: phosphorylation of protein synthesis initiation factor elF-2 and ribosome-associated protein P1.

Mice of different inbred strains varied in their immune response to endogenous leukemia viruses (MuLV). The ability of mice to produce antibodies against the gp70 proteins of xenotropic and ecotropic MuLV was to some extent dependent upon the level to which these mice naturally expressed their endogenous leukemia viruses. Three patterns of response were observed upon immunization with AKR leukemia cells: (1) Mice of low leukemic strains, which did not contain inducible ecotropic or xenotropic MuLV, produced antibodies that reacted with the gp70 proteins of both xenotropic and ecotropic MuLV; (2) mice of low leukemic strains, which contained low levels of ecotropic and inducible xenotropic MuLV, produced antibodies that reacted primarily with the gp70 proteins of ecotropic MuLV; and, (3) mice of high leukemic strains, which contained high levels of ecotropic and inducible xenotropic MuLV, failed to produce antibodies that reacted with the gp70 proteins of either xenotropic or ecotropic MuLV. Antibodies induced by immunization with leukemia cells reacted with type-specific antigenic determinants of the gp70 protein; by absorption analysis distinctive antigenic determinants could be identified on the gp70 proteins of BALB xenotropic, NZB xenotropic, and AKR ecotropic MuLV.     

Isolation of a fibroblast nonproducer cell line containing the Friend strain of the spleen focus-forming virus.

A BALB/c spleen homogenate containing a mixture of B-tropic type-C helper virus and the Friend strain of spleen focus-forming virus (SFFV) was found by end point titration to contain equivalent biologic titers of helper virus and SFFV. By infection of BALB/c 3T3 cells with an appropriate dilution of this homogenate, we have isolated two single cell clones which contain SFFV free of replicating type-C helper virus. When these SFFV-containing nonproducer cells were superinfected with a cloned stock of the Friend strain of helper type-C virus,.a virus mixture was obtained which produced abundant splenic foci within 9 days in young adult BALB/c mice. The nonproducer cells containing the SFFV genome are similar in morphology to uninfected BALB/c 3T3 cells, do not release virus particles, and' are virus negative by electron microscopy. Thus, the Friend strain of SFFV has been obtained free of replicating helper virus. The results suggest that SFFV represents a class of replication-defective RNA type-C viruses which are unable to transform mouse fibroblasts but which, like mammalian RNA-containing sarcoma viruses, are associated with a rapid malignant disease in mice.     

Expression of viral envelope glycoprotein and transformation genes in cells transformed by a defective Kirsten murine sarcoma virus.

Concurrent expression of transformation properties and virion envelope glycoproteins has been observed as a property of several clones of normal rat kidney cells transformed by, but not producing, Kirsten murine sarcoma virus. The present studies were carried out to determine whether a genetic linkage exists between the viral sarcoma and envelope genes in these cells. Several alternative models for the possible structure and origin of the sarcoma and envelope genes were considered. One possibility, that the viral envelope gene was derived from an endogenous rat virus, was studied by characterization of the interference properties of the transformed cells. The sarcoma virus genome of envelope-positive clones was efficiently rescued by woolly monkey and murine xenotropic but not by murine ecotropic viruses. Thus, the interference properties of cells producing the envelope glycoprotein are analogous to those of a cell producing murine ecotropic virus, indicating that the envelope was of murine viral origin. In these experiments it was also found that sarcoma viruses rescued from envelope-positive cells upon superinfection with primate and xenotropic murine viruses could transform host cells for both xenotropic and ecotropic viruses, indicating that these superinfecting viruses became phenotypically mixed with the ecotropic envelope expressed in transformed, envelope-positive cells. Possible linkage between the envelope and transformation genes was analyzed by the frequency of concurrent rescue of sarcoma and envelope genes. Transfer of the Kirsten sarcoma viral genome to uninfected cells upon rescue by superinfection with woolly monkey virus showed a high frequency of apparent segregation of the transformation and envelope genes [from 29 to 57% for (KSV env⁺)NRK-6]. The model supported by the present data is that the transformed, envelope-positive cells were infected with a virus which contained both the envelope and the sarcoma genes.     

Polymorphism in the major core protein (p30) of murine leukemia viruses as identified by mouse antisera.

Antisera prepared in C57BL/6 mice against the AKR K36 leukemia cells detect] group-specific and class-specific antigenic determinants on the major core protein (p30) of murine leukemia virus (MuLV). Sera that contain a predominance of antibodies against class-specific antigens react preferentially with the p30 proteins of ecotropic MuLV; these sera do not react with the p30 proteins of xenotropic MuLV. The use of MuLV class-specific antisera in immunological assays enables the assignment of p30 antigens in viruses and tissues to ecotropic or xenotropic origins.     

Pathogenesis of the slow disease of the central nervous system associated with wild mouse virus. III. Role of input virus and MCF recombinants in disease

Inbred mice bearing the FV-1n marker when inoculated at birth with an ecotropic murine leukemia virus (WM 1504-E) obtained from wild mice develop a progressive central nervous system (CNS) disease manifested by hindlimb paralysis and incoordination that begins by 6 to 7 weeks of age. Studies of infected SWR/J mice at 3 days to 4 months of age indicated the following: (1) Expression of MuLV gp69/70 and p30 antigens in CNS rises beginning as early as 3 days after inoculation and increases with time. (2) Neuronal damage is evident by Day 7, and neuronal lesions develop in all mice by Day 14. (3) WM 1504-E virus can be isolated from CNS tissue by 48 hr after initiating infection. (4) Upon passage into susceptible newborn mice, the WM 1504-E isolates cause neuronal disease. (5) "Dual-tropic" mink cell focus forming (MCF) -like virus is found in splenic but not CNS tissues by 8 weeks after initiating infection. (6) MCF viruses that arise by env gene recombination between WM 1504-E and endogenous xenotropic viruses do not cause CNS disease upon inoculation into susceptible newborn mice. Similarly inoculated WM 1504-amphotropic virus (WM 1504-A) does not cause CNS disease (7). Results in SWR/J mice can be duplicated in C3H/St and C57Br/cdj mice. These observations define the wild mouse ecotropic virus as the sole infectious agent responsible and sufficient for the development of this neurologic disease. Evidently, the disease from this "slow virus infection" begins early in life shortly after introduction of the infectious agent, but becomes clinically apparent only as neuronal destruction accumulates over the lifetime of the host.     

The spleen focus-forming virus (SFFV)-specific neoantigen shares cross-reactive determinants with mink cell focus-inducing (MCF) virus gp70.

We recently reported that the spleen focus-forming virus [(SFFV), a replication-defective leukemogenic virus] coded for a cell surface neoantigen which could be detected by lymphocyte-mediated cytolysis. Because the SFFV genome has recently been characterized as a recombinant between both ecotropic and xenotropic viral information, we examined whether the SFFV-specific neoantigen, as defined by in vitro cytolysis, shared determinants with antigens coded for by mink cell focus (MCF)-inducing viruses (also xenotropic/ecotropic recombinants). As detected by direct cytolysis of SFFV- and MCF-infected target cells, the SFFV-specific neoantigen was found to be partially cross-reactive with determinants on the surface of MCF virus-infected cells. The cross-reactive determinants appeared to be coded for by the envelope region of both AKR and Moloney MCF viruses, in that antisera specific for MCF gp 70 were capable of blocking SFFV-specific neoantigen-directed cytolysis. These results provide further evidence in support of the recombinant nature of the SFFV genome and its close relationship to MCF viral genetic information. However, in that the SFFV-specific neoantigen was not totally cross-reactive with MCF gp70, it remains possible that additional antigenic determinants associated with SFFV-encoded transformation may be present.     

Leukemogenic properties of AKR dualtropic (MCF) viruses: amplification of murine leukemia virus-related antigens on thymocytes and acceleration of leukemia development in AKR mice

The late preleukemic period in AKR mice (6–8 months of age) is characterized by amplified expression of murine leukemia virus (MuLV)-related cell surface antigens on thymocytes. Analysis of thymic biopsies of preleukemic AKR mice at 180 days of age showed that antigen amplification was prognostic of spontaneous leukemia development within approximately 100 days. In the present study we have investigated the viral basis of this preleukemic change in AKR thymus. Cloned isolates of ecotropic, xenotropic, and dualtropic MuLV that were derived from preleukemic or leukemic AKR thymus were tested by intrathymic injection of young AKR mice to determine which of the three classes of MuLV could induce antigen amplification, and, subsequently, accelerate leukemia development in the same animal. Only dualtropic MuLV exhibited these two activities in vivo. Considerable heterogeneity was observed among the 13 dualtropic MuLV isolates examined. In vitro three distinct serological phenotypes could be recognized on the basis of expression of MuLV gag gene-coded antigens GCSA and MuLV env gene-coded antigens. G_IX, G_(ERLD), G_(RADAI), G_(ARSL2) on the surface of infected cells. Virus isolates also differed in (i) relative ecotropic-xenotropic host range (dualtropism) as assayed by ratios of infectious titers on mouse and mink cells, (ii) induction of mink cell foci (MCF), and (iii) infectivity for NIH/3T3 cells. In vivo the two activities of antigen amplification and leukemia acceleration could be dissociated and thus represent distinct virus phenotypes. Seven isolates of dualtropic MuLV induced antigen amplification; these viruses encoded G_IX, G_(ERLD), and G_(AKSL2) antigens, were MCF inducing, had SC-1/mink titer ratios ≥ 0.4, and were infectious for NIH/3T3 cells. Only three of these virus isolates (MCF 247, MCF 69L1, and MCF 13) accelerated leukemia development. Analysis of dose-response relationships and kinetics of virus-induced antigen amplification by MCF 69L1 virus implicated virus infection of thymocytes in the preleukemic change. Moreover, the level of MuLV antigen expression on thymocytes at approximately 1 month postinjection of MCF 69L1 virus correlated directly with development of early leukemia. It is apparent that leukemia development in young AKR mice which can be induced experimentally by intrathymic injection of cloned isolates of dualtropic MuLV exhibits the hallmarks of spontaneous disease in this strain and thus can serve as a useful model for study of thymic leukemogenesis in mice.