Thymic epithelial genotype influences the production of recombinant leukemogenic retroviruses in mice

By using T1 oligonucleotide fingerprinting and mapping techniques, we analyzed the genomic structure of retroviruses produced by thymocytes and splenocytes of reciprocal bone marrow-and thymus-grafted chimeras. We found that the genetic factor(s) derived from NZB mice that suppresses the development of thymic leukemia in (AKR X NZB)F1 mice also prevents the formation of recombinant leukemogenic viruses and the expression of preleukemic changes in the (AKR X NZB)F1 thymocytes. The NZB mouse gene or genes appeared to exert this suppressive effect by acting on the thymic reticuloepithelial cells and not on the thymic lymphocytes of (AKR X NZB)F1 hybrids. Prospective studies with thymic epithelial grafts from young mice showed that the AKR thymic epithelium could mediate the formation and expression of leukemogenic recombinant viruses and preleukemic changes in thymocytes that lead to the development of thymic leukemia, whereas the (AKR X NZB)F1 thymic epithelium was deficient in this regard. Our results also confirmed a previous observation that during in vivo generation of recombinant leukemogenic viruses, the acquisition of polytropic virus-related sequences in the 3' portion of the p15E gene and the U3 region and in the 5' part of the gp70 gene can occur independently.     

Thymic epithelium is programmed to induce preleukemic changes in retrovirus expression and thymocyte differentiation in leukemia susceptible mice: studies on bone marrow and thymic chimeras

In the leukemia-prone AKR thymus, ecotropic and xenotropic-related viruses are expressed that generate leukemogenic recombinant viruses before the onset of leukemia. We have shown previously that (AKR X NZB)F1 hybrid mice do not develop leukemia because they severely restrict the expression of these retroviruses in their thymuses. The thymic microenvironment of the (AKR X NZB)F1 mice appeared to be of particular importance in determining this restriction, which was specified by an NZB-derived genetic influence. In the present study we analyze reciprocal thymus graft and irradiation bone marrow chimeras to establish that this influence is exerted by thymic reticuloepithelial cells. Prospective studies with thymic epithelial grafts from young mice show that the AKR thymic epithelium can simultaneously induce the amplified expression of retroviral genes, and changes in patterns of thymocyte differentiation that precede the development of leukemia, whereas the (AKR X NZB)F1 thymic epithelium is deficient in this regard.     

Fv-1 regulation of lymphoma development and of thymic ecotropic and xenotropic MuLV expression in mice of the AKR/J x RF/J cross.

The incidence of spontaneous thymic lymphoma has been studied in crosses between AKR/J and RF/J mice. AKR mice develop a high incidence of this disease. RF mice transmit a marked resistance to development of the disease to F1 hybrid mice of the AKR x RF cross. This resistance is associated with a reduction of endogenous ecotropic and xenotropic MuLV expression in the prelymphomatous thymus. The RF gene governing the coordinate suppression of these three phenotypes has been mapped to the Fv-1 locus. These results indicate that the particular Fv-1 allele of AKR mice provides a permissive genetic background for endogenous ecotropic and xenotropic MuLV expression and that these viral activities may be etiologically involved in the development of spontaneous thymic lymphoma in the mouse.     

In vitro studies of the mechanism of leukemogenesis. II. Characterization of endogenous murine leukemia viruses isolated from AKR thymic epithelial reticulum cell lines

Thymic epithelial reticulum (TER) cell lines were established from thymuses of a young healthy AKR mouse (A2T), a preleukemic AKR mouse (A6T), and two lymphoma-bearing AKR/Ms mice (ASLT-1 and ASLT-2). Numerous type-C virus particles with occasional budding forms were observed in all cell lines. Expression of XC-detectable, N-tropic, ecotropic virus was observed in every cell line, whereas the presence of xenotropic and mink cell focus-inducing (MCF) viruses could be detected only in TER cells derived from preleukemic and leukemic mice. Expression of xenotropic virus in various cells of newborn and young AKR mice could readily be induced by IUdR treatment, whereas MCF virus was never detected in these cells, with the exception of the A2T cell line after more than 20 passages, in which MCF virus with dual-tropic infectivity emerged in addition to ecotropic and xenotropic viruses. These spontaneous and induced MCF viruses were purified, and their virological properties were characterized. The cloned MCF viruses (MCFs AT1, AT2, AT3, and AT4-IU) showed dual tropism and produced cytopathic effect-like foci in mink lung cells. Preinfection with either ecotropic or xenotropic virus interfered with the infectivity of MCF viruses. Spontaneous leukemogenesis in AKR mice was accelerated by the inoculation of MCF viruses. These findings indicate that TER cells could serve as the host cells for the genetic recombination of the endogenous MuLV; the recombinant MuLV, MCF virus, appears to be most closely associated with leukemogenesis in AKR mice.     

Emv-13 (Akv-3): a noninducible endogenous ecotropic provirus of AKR/J mice

All AKR/J mice carry at least three endogenous ecotropic viral loci which have been designated Emv-11 (Akv-1), Emv-13 (Akv-3), and Emv-14 (Akv-4) (Jenkins et al., J. Virol. 43:26-36, 1982.) Using two independent AKR/J-derived sets of recombinant inbred mouse strains, AKXL (AKR/J x C57L/J) and AKXD (AKR/J x DBA/2J), as well as the HP/EiTy strain (an Emv-13-carrying inbred strain partially related to AKR/J mice) (Taylor et al., J. Virol. 23:106-109, 1977), we have examined the association of these endogenous viral loci with virus expression. Strains which transmit Emv-11 or Emv-14 or both were found to produce virus spontaneously, whereas strains that transmit Emv-13 alone were negative for virus expression. Restriction endonuclease digestion and hybridization with an ecotropic virus-specific hybridization probe of DNAs from strains which transmit only Emv-13 yielded enzyme cleavage patterns identical to those observed with DNAs from strains transmitting Emv-11 or Emv-14 or both. These findings indicate the absence of any gross rearrangement of Emv-13 proviral sequences. Cell cultures derived from recombinant inbred strains that carry only Emv-13 failed to express detectable infectious virus, viral proteins, or cytoplasmic ecotropic virus-specific RNA even after treatment with 5-iodo-2-deoxyuridine or 5-azacytidine, an inhibitor of DNA methylation. Our results indicate that a mechanism(s) other than methylation of Emv-13 proviral DNA is responsible for inhibition of Emv-13 expression.     

Oncogenicity of AKR endogenous leukemia viruses.

Four biologically distinct groups of endogenous murine leukemia virus (MuLV) have been isolated from AKR mice. These viruses included (i) ecotopic XC+ MuLV that occur in high titer in normal tissues and serum of AKR mice throughout their life span, (ii) ecotropic XC- MuLV that are produced in high titers by leukemia cells, (iii) xenotropic MuLV that are readily demonstrable only in aged mice, and (iv) polytropic MuLV thatarise in the thymuses of aged mice as a consequence of genetic recombination between ecotropic and xenotropic MuLV. Virus of each of these biological classes were assayed in AKR mice for their ability to accelerate the occurrence of spontaneous leukemia. Certain isolates of ecotropic XC- MuLV and polytropic MuLV were found to have high oncogenic activity. These viruses induced 100% leukemias within 90 days of inoculation. In contrast, ecotropic XC+ MuLV that were obtained from AKR embryo fibroblasts and xenotropic MuLV that were obtained from the lymphoid tissues of aged AKR mice did not demonstrate oncogenic activity. These findings demonstrate fundamental differences between XC- and XC+ ecotropic MuLV that are found in leukemic and normal tissues, respectively. Furthermore, these findings point to the role of ecotropic XC- and polytropic MuLV in the spontaneous leukemogenesis of AKR mice.     

Differential expression of murine leukemia virus loci in chemically induced hybrid cells.

The time course of murine leukemia virus production after chemical induction was determined in hamster-mouse somatic cell hybrids containing the xenotropic murine leukemia virus induction locus Bxv-1 or the ecotropic locus Akv-2. By using these hybrids, induction could be studied in the absence of secondary virus spread because xenotropic viruses cannot infect hybrid cells and ecotropic viruses cannot infect hybrids which have lost mouse chromosome 5. After induction, hybrids with Bxv-1 produced only a transient burst of virus, whereas those with Akv-2 continued to produce virus for periods in excess of 3 months. The presence or absence of other mouse chromosomes in the hybrid lines did not alter these induction patterns. Thus, endogenous murine leukemia virus loci differ in their response to induction, and both inducibility and the kinetics of virus expression are controlled at or near these proviral loci.     

Natural immunity in mice to the envelope glycoprotein of endogenous ecotropic type C viruses: neutralization of virus infectivity.

The ability of naturally immune mouse sera to neutralize ecotropic AKR murine leukemia virus (MuLV) was examined by using unfrozen virus preparations harvested for 1 h. In this assay several mouse sera significantly and consistently neutralized MuLV infectivity. The ability of these sera to neutralize was correlated with the presence of antibodies against MuLV detectable in a radioimmune precipitation assay using radioactively labeled intact virions. This neutralization was specific, in that either N- or B-tropic viruses, but not Friend MuLV, were neutralized. In addition, neutralization could be abrogated with purified AKR MuLV gp71 at concentrations that do not interfere with virus infectivity but could not be abrogated with Rauscher MuLV gp71. Neutralizing activity could be removed by absorption with intact AKR MuLV, but not by absorption with Friend MuLV, a BALB/c xenotropic virus, or with NZB xenotropic virus. All the neutralizing activity of (B6C3)F1 mouse sera was associated with the immunoglobulin G fraction.     

Fv-1 determinants in xenotropic murine leukemia viruses studied with biological assay systems: Isolation of xenotropic virus with N-tropic Fv-1 activity in the cryptic form.

By a biological assay system using phenotypically mixed ecotropic and xenotropic murine leukemia viruses, we investigated whether in the virions of a xenotropic virus there is N- or B-tropic Fv-1 determinant in active form. The existence of N-tropic Fv-1 determinant was demonstrated in SL-XT-1 xenotropic virus isolated from the spleen of a 3-month-old SL mouse, and the N-tropic Fv-1 tropism was confirmed by analysis of the phenotypically mixed viruses harvested from clonal SC-1 cells doubly infected with the SL-XT-1 and B-tropic ecotropic viruses. However, neither N- nor B-tropic Fv-1 determinant was demonstrated in any xenotropic viruses isolated from embryo cells of BALB/c, NZB, or DBA/2 mice, or Cas E #1-IU, and xenotropic-like virus isolated from a wild mouse.     

Comparison of the restriction endonuclease maps of unintegrated proviral DNAs from four xenotropic murine leukemia viruses.

From purified linear and superhelical DNAs, the restriction endonuclease maps of four xenotropic murine leukemia virus DNAs from NFS, NZB, BALB/c, and AKR mice were determined with ten restriction endonucleases. Each xenotropic proviral DNA was found to be a unique restriction endonuclease map, with differences in the gag, pol, env, and terminal repeated sequence regions. However, type-specific SacI and EcoRI sites in the env region were identical in all four xenotropic murine leukemia virus DNAs and were not found in ecotropic murine leukemia virus DNA. Comparison of the xenotropic murine leukemia virus DNA maps with maps of ecotropic murine leukemia virus DNA showed that the pol and terminal repeated sequence regions were highly conserved. Other similarities in ecotropic and some xenotropic viral DNAs suggest common origins.     

RNase T1-resistant oligonucleotides of Akv-1 and Akv-2 type C viruses of AKR mice.

We used two-dimensional gel electrophoresis to obtain fingerprints of RNase T1-resistant oligonucleotides derived from the genomes of Akv-1 and Akv-2 type C viruses of AKR mice. The fingerprints of these two viruses look identical. The products of pancreatic RNase digestion of corresponding oligonucleotides of the two viruses were indistinguishable. These observations are consistent with, but not proof of, the possible identity of the genomes of the Akv-1 and Akv-2 viruses and, thus, of the viral genetic material believed to comprise the Akv-1 and Akv-2 loci of AKR mice.     

Chromosomal assignment of two endogenous ecotropic murine leukemia virus proviruses of the AKR/J mouse strain.

The AKR/J mouse strain is genetically fixed for three different ecotropic murine leukemia virus genomes, designated Akv-1, Akv-3, and Akv-4 (Emv-11, Emv-13, and Emv-14). With recombinant inbred strains and crosses with linkage-testing stocks, Akv-3 and Akv-4 were placed on the mouse chromosome map. Akv-3, which encodes a replication-defective provirus, maps near the agouti coat color locus, a, on chromosome 2. Akv-4, which is replication competent, maps near the neurological mutant gene locus trembler, Tr, on chromosome 11. Akv-1 and Akv-2 (Emv-12), an ecotropic provirus carried by AKR/N but not AKR/J, have previously been mapped to chromosome 7 and 16, respectively. Thus, the four Akv proviruses mapped to date are on four different chromosomes. Akv-3 is the second ecotropic murine leukemia virus provirus to be mapped near the agouti locus. The results are discussed in relation to possible nonrandomness of viral integration.     

Evidence for a B lymphocyte defect underlying the anti-X anti-erythrocyte autoantibody response of NZB mice.

The autoimmune hemolytic anemia of NZB mice is pathogenetically mediated by a genetically prescribed anti-erythrocyte autoantibody response directed to the X erythrocyte autoantigen. The cellular locus of the immunoregulatory defect underlying the anti-X response was explored by adoptively transferring bone marrow cells (BMC) from NZB mice to lethally irradiated histocompatible recipients. Before adoptive transfer, BMC from donor mice were assayed for antigen-binding lymphocytes with receptors for the X autoantigen (X-ABL) by immunocytoadherence assays and for anti-X autoantibody-secreting cells (X-PFC) by plaque-forming cell assays. Twelve weeks after adoptive transfer, splenic lymphocytes from recipient mice were assayed for X-PFC and humoral anti-X autoantibody by Coombs' tests. Transfer of 15 to 30 x 10(6) BMC containing 6 to 12 x 10(3) X-ABL but no X-PFC from 6- to 8-week-old NZB mice to lethally irradiated BALB/c, B10.D2, C57BL/Ks, and DBA/2 mice produced X-PFC in 70% of the recipients. Development of X-PFC was not simply dependent upon available X-ABL since transfer of 15-30 x 10(6) BMC, containing comparable numbers of X-ABL, from BALB/c, B10.D2, C57BL/Ks, or DBA/2 mice to NZB or syngeneic recipients did not produce X-PFC. Transfer of BMC from NZB mice to BALB/c, B10.D2, and DBA/2 mice with weekly administrations of AKR anti-theta antiserum had no effect on the development of X-PFC; Tlymphocyte ablation was evidenced by the absence of theta+ spleen cells. These results suggest that the pathogenetic anti-X response is not genetically prescribed at the level of macrophages, humoral factors, or T cells, but rather appears to be a phenotypic expression of a primary B lymphocyte defect permitting or promoting differentiation of NZB X-ABL.     

Differentiated mouse epithelial cell line with hepatocyte characteristics

An epithelial cell line, 3105, with an unusual growth pattern has been derived from the liver of an (NZB x NZW)F1 mouse. When confluent, it forms a monolayer of closely packed cells interspersed with holes that do not fill in during cultivation. By electron microscopy, the line has tight and intermediate junctions as well as desmosomes typical of epithelial cells. It produces several enzymes normally present in liver including hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, glucose-6-phosphatase, and alkaline phosphatase; has cytochromes P-450 and b5; and spontaneously release xenotropic but not ecotropic endogenous mouse type C viruses. Inoculation of the cell line into athymic nude mice gives rise to benign cysts in 2-3 months. This mouse epithelial line with hepatocyte characteristics should be helpful to investigators as a cell model of normal liver cell differentiation.     

Cellular changes in the thymuses of preleukemic AKR mice: correlation with changes in the expression of murine leukemia viruses.

Thymus cells of preleukemic and leukemic AKR mice express on their cell surface elevated levels of antigens associated with the murine leukemia virus (MuLV) proteins gp70 and p30. The gp70 antigenicity is contained in a 70,000 dalton polypeptide that corresponds to the viral envelope protein, while the p30 antigenicity is contained in two polypeptides of 85,000 and 95,000 daltons that correspond to glycosylated forms of the polyprotein product of the gag gene.The expression of these viral coded proteins on the cell surface of thymocytes varies both quantitatively with the age of the mouse and qualitatively with the cellular populations that express these antigens. Four discrete stages in the leukemic pathway can be identified. First, low numbers of cells from the thymuses of young (2 month old) AKR mice express p30 (<0.25%) and gp70 (2–7%) antigens. Expression of gp70 antigen is restricted to large cells in the subcapsular region of the thymus. Second, thymuses of 6 month old AKR mice show a selective depletion of cortical thymocytes with a concomitant increase in the medullary region of the thymus. Thymus cells of these mice contain elevated numbers of cells that express an increased concentration of p30 and gp70 antigens. Viral antigens are found on the surface of all large cells of the subcapsular region of the thymus, and in variable numbers (2–85%) of small cells of the cortical and medullary regions. Third, the thymuses of some 8 month old AKR mice demonstrate selective hypertrophy of a single thymic lobe. The enlarged lobe contains a population of cells that are intermediate in size between the small cortical cells and leukemic blast cells. This new cell population expresses elevated levels of p30 and gp70 viral antigens. These cells, which are not leukemic (since transfer of high numbers of these cells to syngeneic hosts does not induce transplantable disease), may represent preleukemic thymocytes. Fourth, thymuses of mice with overt leukemia contain primarily leukemic blast cells. These cells express extremely high levels of viral antigens on their cell surfaces, and upon transfer of these cells to syngeneic hosts, they rapidly induce transplantable leukemias.The increased expression of viral antigens on the surface of thymus cells is correlated with an increased production of infectious ecotropic and xenotropic MuLV in the thymus. During aging, the percentage of cells producing ecotropic MuLV increases 10-fold, while the percentage of cells producing xenotropic MuLV increases 100 fold.     

Tryptic peptide analysis of avian oncovirus gag and pol gene products.

Tryptic digests of the internal proteins p30, p15, p12, and p10 of mouse xenotropic, ecotropic, and amphotropic type C viruses were subjected to cation-exchange chromatography. Analysis of these maps revealed that the p30 proteins from representative isolates of all three viral subgroups were distinguishable. The p15 proteins were all unique. The p12 proteins of NZB xenotropic and wild-mouse amphotropic viruses were not identical and yielded peptide maps remarkably different from that of the ecotropic virus. The p10 proteins of xenotropic and ecotropic viruses were identical and were dissimilar to that of the wild-mouse amphotropic virus.     

Maternally transmitted resistance to lymphoma development in mice of reciprocal crosses of the RF/J and AKR/J strains

Females but not males of the low-lymphoma RF/J strain transmit a non-Mendelian factor which suppresses the development of lymphoma in F1 crosses with mice of the high-lymphoma AKR/J strain. Suppression of lymphoma was also evident in the first backcross generation to the parental AKR strain, but only when (RF female x AKR male)F1 mice had been the female parent. This "maternal resistance factor" was transmitted independently of the dominant, lymphoma-suppressing Fv-1n allele transmitted by both males and females of the RF strain, but the suppressive capacities of the two factors appeared to be additive. In this cross, F1 progeny of RF females also showed marked suppression of ecotropic murine leukemia virus expression by comparison with mice of the reciprocal F1 cross, but this suppression of virus expression was not detected in the lymphoma-suppressed AKR backcross population. The observation of lymphoma suppression in the absence of ectropic virus suppression in mice of the (RF X AKR)F1 female x AKR male backross generation indicates a qualitative or quantitative difference in the determination of these two effects.     

Genomes of murine leukemia viruses isolated from wild mice.

The genomes of murine leukemia viruses (MuLV) isolated from wild mice have been studied. Detailed restriction endonuclease maps of the 8.8-kilobase (kb) unintegrated linear viral DNAs were derived for five ecotropic and five amphotropic MuLV's from California field mice, for Friend MuLV, and for one ecotropic and one xenotropic MuLV from Mus musculus castaneus. In general, the California MuLV's were similar in their leftward 6 kb (corresponding to the leftward long terminal repeat [LTR], gag, and pol) and rightward 1 kb (7.8 to 8.8 kb, corresponding to p15E and the rightward LTR). For the region spanning 6.0 to 7.7 kb (which includes the sequences that encode gp70) the amphotropic MuLV's shared few enzyme sites with the ecotropic MuLV's, although the California ecotropic MuLV's were highly related to each other in this region, as were the amphotropic MuLV's. Cross-hybridization studies between amphotropic and California ecotropic MuLV DNAs indicated that they were not homologous in the region 6.3 to 7.6 kb; the California ecotropic viral DNAs cross-hybridized in this region to AKR ecotropic MuLV. When the California viral DNAs were compared with AKR ecotropic viral DNA, many differences in enzyme sites were noted throughout the genome. The U3 regions of the wild mouse LTRs showed partial homology to this region in AKR MuLV. The LTR of Moloney MuLV was highly related to that of the California MuLV's, whereas the LTR of Friend MuLV appeared to be a recombinant between the two types of LTRs. The M. musculus castaneus isolates were most closely related to ecotropic and xenotropic MuLV's isolated from inbred mice. One amphotropic MuLV DNA was cloned from supercoiled viral DNA at its unique EcoRI site in pBR322. Viral DNAs with one and two LTRs were isolated. After digestion with EcoRI, DNAs of both types were infectious. It is concluded that ecotropic and amphotropic MuLV's differ primarily in the region which encodes gp70.     

Activation of Nonexpressed Endogenous Ecotropic Murine Leukemia Virus by Transfection of Genomic DNA into Embryo Cells

We studied the infectivity of endogenous ecotropic murine leukemia virus genomes contained in high-molecular-weight DNA prepared from virus-free cells of the AKR-2B line, and from RF, BALB/c, B6, and (BALB/c x B6)F(1) mouse embryo cells. When DNA prepared from virus-free AKR-2B cells was transfected into NIH-3T3 cells, no virus-positive cultures were observed, a result consistent with previous reports. However, when DNAs from virus-free AKR-2B cells or virus-free cells containing the RF/J or BALB/c ecotropic proviruses were transfected into chicken embryo cells that were then cocultivated with SC-1 (mouse) cells, virus-positive cultures were recovered. The specific infectivities of the AKR provirus(es) contained in virus-free cells and the molecularly cloned Akv-1 provirus were similar when chicken embryo cells were used as primary recipients. Virus-positive cultures were also observed when secondary mouse embryo cells were used as recipients for DNA from virus-free AKR-2B and RF/J cells. The transfected chicken embryo-SC-1 cultures produced XC-positive murine leukemia virus that is N-tropic. Virus-positive recipient cultures were observed 10- to 100-fold more frequently when AKR-2B DNA was used than when BALB/c DNA was used as the donor DNA. Our studies indicate that some nonexpressed ecotropic murine leukemia virus proviruses are activated upon transfection into chicken embryo cells. Such studies suggest that there are different factors governing the expression of murine leukemia virus after transfection into established cell lines (NIH-3T3) and into nonestablished secondary cultures (chicken and mouse).