B-tropic Murine Leukemia Virus Research Articles

The small-molecule 3G11 inhibits HIV-1 reverse transcription.

The small-molecule 6-(tert-butyl)-4-phenyl-4-(trifluoromethyl)-1H,3H-1,3,5-triazin-2-one (3G11) inhibits HIV-1 replication in the human T cell line MT-2. Here, we showed that 3G11 specifically and potently blocks HIV-1 infection. By contrast, 3G11 did not block other retroviruses such as HIV-2, simian immunodeficiency virus (SIVmac ), bovine immunodeficiency virus, feline immunodeficiency virus, equine infectious anemia virus, N-tropic murine leukemia virus, B-tropic murine leukemia virus, and Moloney murine leukemia virus. Analysis of DNA metabolism by real-time PCR revealed that 3G11 blocks the formation of HIV-1 late reverse transcripts during infection prior to the first-strand transfer step. In agreement, an in vitro assay revealed that 3G11 blocks the enzymatic activity of HIV-1 reverse transcriptase as strong as nevirapine. Docking of 3G11 to the HIV-1 reverse transcriptase enzyme suggested a direct interaction between residue L100 and 3G11. In agreement, an HIV-1 virus bearing the reverse transcriptase change L100I renders HIV-1 resistant to 3G11, which suggested that the reverse transcriptase enzyme is the viral determinant for HIV-1 sensitivity to 3G11. Although NMR experiments revealed that 3G11 binds to the HIV-1 capsid, functional experiments suggested that capsid is not the viral determinant for sensitivity to 3G11. Overall, we described a novel non-nucleoside reverse transcription inhibitor that blocks HIV-1 infection.

Contribution of SAM and HD domains to retroviral restriction mediated by human SAMHD1

The human SAMHD1 protein is a novel retroviral restriction factor expressed in myeloid cells. Previous work has correlated the deoxynucleotide triphosphohydrolase activity of SAMHD1 with its ability to block HIV-1 and SIVmac infection. SAMHD1 is comprised of the sterile alpha motif (SAM) and histidine–aspartic (HD) domains; however the contribution of these domains to retroviral restriction is not understood. Mutagenesis and deletion studies revealed that expression of the sole HD domain of SAMHD1 is sufficient to achieve potent restriction of HIV-1 and SIVmac. We demonstrated that the HD domain of SAMHD1 is essential for the ability of SAMHD1 to oligomerize by using a biochemical assay. In agreement with previous observations, we mapped the RNA-binding ability of SAMHD1 to the HD domain. We also demonstrated a direct interaction of SAMHD1 with RNA by using enzymatically-active purified SAMHD1 protein from insect cells. Interestingly, we showed that double-stranded RNA inhibits the enzymatic activity of SAMHD1 in vitro suggesting the possibility that RNA from a pathogen might modulate the enzymatic activity of SAMHD1 in cells. By contrast, we found that the SAM domain is dispensable for retroviral restriction, oligomerization and RNA binding. Finally we tested the ability of SAMHD1 to block the infection of retroviruses other than HIV-1 and SIVmac. These results showed that SAMHD1 blocks infection of HIV-2, feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Equine infectious anemia virus (EIAV), N-tropic murine leukemia virus (N-MLV), and B-tropic murine leukemia virus (B-MLV).

Role of Human TRIM5α in Intrinsic Immunity

Human immunodeficiency virus (HIV) has a very narrow host range. HIV type 1 (HIV-1) does not infect Old World monkeys, such as the rhesus monkey (Rh). Rh TRIM5α was identified as a factor that confers resistance, intrinsic immunity, to HIV-1 infection. Unfortunately, human TRIM5α is almost powerless to restrict HIV-1. However, human TRIM5α potently restricts N-tropic murine leukemia viruses (MLV) but not B-tropic MLV, indicating that human TRIM5α represents the restriction factor previously designated as Ref1. African green monkey TRIM5α represents another restriction factor previously designated as Lv1, which restricts both HIV-1 and simian immunodeficiency virus isolated from macaque (SIVmac) infection. TRIM5 is a member of the tripartite motif family containing RING, B-box2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase, and TRIM5α is thought to degrade viral core via ubiquitin–proteasome-dependent and -independent pathways. The alpha isoform of TRIM5 has an additional C-terminal PRYSPRY domain, which is a determinant of species-specific retrovirus restriction by TRIM5α. On the other hand, the target regions of viral capsid protein (CA) are scattered on the surface of core. A single amino acid difference in the surface-exposed loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA affects viral sensitivity to human TRIM5α and was also shown to be associated with viral load in West African HIV-2 patients, indicating that human TRIM5α is a critical modulator of HIV-2 replication in vivo. Interestingly, L6/7 of CA corresponds to the MLV determinant of sensitivity to mouse factor Fv1, which potently restricts N-tropic MLV. In addition, human genetic polymorphisms also affect antiviral activity of human TRIM5α. Recently, human TRIM5α was shown to activate signaling pathways that lead to activation of NF-κB and AP-1 by interacting with TAK1 complex. TRIM5α is thus involved in control of viral infection in multiple ways.

A human TRIM5α B30.2/SPRY domain mutant gains the ability to restrict and prematurely uncoat B-tropic murine leukemia virus

Human TRIM5α restricts N-tropic murine leukemia virus (N-MLV) but not B-tropic MLV (B-MLV) infection. Here we study B30.2/SPRY domain mutants of human TRIM5α that acquire the ability to inhibit B-MLV infection prior to reverse transcription without losing the ability to restrict N-MLV infection. Remarkably, these mutants gain the ability to decrease the amount of particulate B-MLV capsids in the cytosol of infected cells. In addition, these mutants gain the ability to restrict SIVmac and HIV-2 infection. B-MLV and SIVmac infections were blocked by the mutant TRIM5α proteins prior to reverse transcription. Thus, the range of retroviruses restricted by human TRIM5α can be increased by changes in the B30.2/SPRY domain, which also result in the ability to cause premature uncoating of the restricted retroviral capsid.

Complex determinants within the Moloney murine leukemia virus capsid modulate susceptibility of the virus to Fv1 and Ref1-mediated restriction

Two of the most well-known genetic mechanisms in mammalian cells which control the susceptibility of cells to productive infection by retroviruses and lentiviruses rely on the cellular Fv1 and Ref1 restriction factors, which act, after viral entry, to prevent productive infection through their interactions with viral capsid (CA) sequences. While previous studies of Fv1 restriction involving N- and B-tropic murine leukemia viruses (MLVs) had demonstrated that the identity of a single amino acid residue at CA110 (arginine vs. glutamic acid) determines whether the resulting virus is N (arg) or B-tropic (glu), analogous studies of dual-tropic MLVs, such as Moloney MLV (Mo-MLV), have shown that additional residues other than CA110 are also involved in the specification of dual-tropic host range. Here we have further studied the CA determinants of Mo-MLV host range, with an emphasis on identifying additional CA residues and unique combinations of CA residues which differentially influence the ability of the resulting virus to infect murine and human cells. First, we show that CA82, a residue previously identified to affect the pattern of Fv1 restriction of different MLV viruses in murine cells, is a particularly strong potentiator of B-tropism in an Mo-MLV background carrying a glutamic acid residue at CA110 (A110E substitution), and that interestingly, different residues at CA82 lead to distinct patterns of restriction in human but not in murine cells. We also identify another CA residue, CA214, as a similarly potent potentiator of B-tropism, in the context of the A110E substitution. While another substitution at CA110, A110R, leads to strong potentiation of N-tropism in murine cells, in the absence of additional mutations, we found that A110R alone was not sufficient to confer appreciable restriction in Ref1-expressing cells, despite the fact that authentic N-MLV shows strong restriction in those cells. In conjunction with the A110R substitution, substitutions at CA82, but not at CA214, do lead to significant restriction in human cells, thus demonstrating a distinction between the interactions between those two determinants of restriction and CA110. Finally, using cell lines engineered to express the TRIM5α hu gene product, recently identified as the Ref1 restriction factor, and RNAi technology to knock-down expression of TRIM5α hu in human cells, we directly demonstrate that the unique patterns of restriction observed in human cells with the different mutants are consistent with a TRIM5α hu-mediated restriction. These studies shed further light on the complex determinants within the viral CA gene product which control the susceptibility of murine and human cells to retroviral infection.

Restriction of multiple divergent retroviruses by Lv1 and Ref1.

The mouse gene Fv1 encodes a saturable restriction factor that selectively blocks infection by N-tropic or B-tropic murine leukemia virus (MLV) strains. Despite the absence of an Fv1 gene, a similar activity is present in humans that blocks N-MLV infection (Ref1). Moreover, some non-human primate cell lines express a potentially related inhibitor of HIV-1 and/or SIVmac infection (Lv1). Here, we examine the spectrum of retrovirus-restricting activities expressed by human and African green monkey cell lines. Human cells restrict N-MLV and equine infectious anemia virus (EIAV), but not HIV-1, HIV-2, SIVmac or SIVagm, whilst AGM cells restrict N-MLV, EIAV, HIV-1, HIV-2 and SIVmac. Remarkably, in each example examined, restriction of infection by a given retrovirus can be abrogated at least partially by saturation with another retrovirus, provided that it is also restricted but regardless of whether it is closely related. These data suggest that restriction factors in human and non-human primate cells are able to recognize and block infection by multiple, widely divergent retroviruses and that the factors themselves may be related.

One base change is sufficient for host range conversion of murine leukemia virus from B to NB tropism.

Comparison of the whole p30 (CA) sequence between B-tropic WN1802B murine leukemia virus and its NB-tropic derivative revealed a single base change at the 313th nucleotide. We constructed hybrid proviruses differing only at the 313th nucleotide of p30 along the whole genome, and examined the host range of the produced viruses. The single point mutation in p30 was found sufficient for the B to NB tropism conversion.

Host range conversion of murine leukemia virus resulting from recombination with endogenous virus

Ecotropic murine leukemia viruses (MuLVs) are classified into B-N-, or NB-tropic MuLV by their host range determined by the Fv-1 gene product. B-tropic MuLV is restricted in N-type mouse cells (Fv-1 n/n) and N-tropic MuLV is restricted in B-type mouse cells (FV-1 b/b). Although forced passages in a restrictive host grant a wider host range (NB-tropism), we show here a host range conversion from B to N tropism. The conversion was most likely a result of recombination between the exogenously infected B-tropic MuLV and an endogenously expressed N-tropic MuLV in a C57BL/6 mouse cell line, YH-7.

Transplacental transmission of a leukemogenic murine leukemia virus

Recombinant inbred BXH-2 mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life and have a high incidence of spontaneous myeloid leukemia. These traits are not characteristic of the progenitor strains (C57BL/6J and C3H/HeJ) or of 11 other recombinant inbred BXH strains. Genetic analysis has shown that the virus is not transmitted through the germ line, suggesting that the virus is passed from one generation to the next by horizontal transmission. An additional ecotropic proviral locus was detected in some mice of this strain after several generations of inbreeding. We show that BXH ecotropic virus was transmitted to other strains when fostered on viremic BXH-2 mice and that these mice go on to develop tumors of hematopoietic origin. Our earlier finding that virus is expressed early in gestation suggested that the ecotropic MuLV is also transmitted in utero. In order to determine the stage at which the ecotropic MuLV is transmitted in utero, preimplantation stage embryos were transferred to the uteri of recipient ecotropic virus-negative mice. These mice were found to be negative for the presence of the ecotropic MuLV, suggesting that transplacental transmission of the ecotropic virus readily occurs in BXH-2 mice. Although other viruses, including human lentiviruses, are transmitted across the placental barrier, transplacental transmission of MuLV is a rare event. Thus, the BXH-2 mouse strain may contribute to our understanding of the mechanism of transplacental transmission and pathogenesis and offers a potential new model for use in drug therapy of exogenously transmitted viruses related to lentiviruses.

Retrovirus-induced murine acquired immunodeficiency syndrome: natural history of infection and differing susceptibility of inbred mouse strains

C57BL mice (Fv-1b) develop a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia virus (MuLV), a derivative of Duplan-Laterjet virus which contains B-tropic ecotropic and mink cell focus-inducing MuLVs and a putative defective genome which may be the proximal cause of disease. The stages of development of this disease were defined for C57BL mice on the basis of lymphadenopathy and splenomegaly; histopathological changes consistent with B-cell activation; and alterations in expression of cell surface antigens affected by proliferation of T cells, B cells, and macrophages. By using this disease profile as a standard, the response of adult mice of various inbred strains and selected F1 hybrids was compared. We show that although the strains which are highly sensitive are of the Fv-1b genotype (i.e., permissive for B-tropic MuLVs), certain Fv-1b strains, e.g., BALB/c and A/J, are resistant to murine acquired immunodeficiency syndrome, whereas certain Fv-1n strains (permissive for N-tropic MuLVs but restrictive for B-tropic MuLVs), notably P/N, BDP, and AKR, show moderate sensitivity and (C57BL/6 x CBA/N)F1 mice (Fv-1n/b and thus dually restrictive) are of relatively high susceptibility. The results of virus recovery tests suggest that apparently anomalous sensitivity, based on predicted Fv-1 restriction, may reflect MuLV induction and/or mutation to provide a helper virus for which the host is permissive.

Fv-1 N- and B-tropism-specific sequences in murine leukemia virus and related endogenous proviral genomes.

Oligonucleotide probes specific for the Fv-1 N- and B-tropic host range determinants of the gag p30-coding sequence were used to analyze DNA clones of various murine leukemia virus (MuLV) and endogenous MuLV-related proviral genomes and chromosomal DNA from four mouse strains. The group of DNA clones consisted of ecotropic MuLVs of known Fv-1 host range, somatically acquired ecotropic MuLV proviruses, xenotropic MuLV isolates, and endogenous nonecotropic MuLV-related proviral sequences from mouse chromosomal DNA. As expected, the prototype N-tropism determinant is carried by N-tropic viruses of several different origins. All seven endogenous nonecotropic MuLV-related proviral sequence clones derived from RFM/Un mouse chromosomal DNA, although not recognized by the N probe, showed positive hybridization with the prototype B-tropism-specific probe. The two xenotropic MuLV clones derived from infectious virus (one of BALB:virus-2 and one of AKR xenotropic virus) failed to hybridize with the N- and B-tropic oligonucleotide probes tested and with one probe specific for NB-tropic Moloney MuLV. One of two endogenous xenotropic class proviruses derived from HRS/J mouse chromosomal DNA (J. P. Stoye and J. M. Coffin, J. Virol. 61:2659-2669, 1987) also failed to hybridize to the N- and B-tropic probes, whereas the other hybridized to the B-tropic probe. In addition, analysis of mouse chromosomal DNA from four strains indicates that hybridization with the N-tropic probe correlates with the presence or absence of endogenous ecotropic MuLV provirus, whereas the B-tropic probe detects abundant copies of endogenous nonecotropic MuLV-related proviral sequences. These results suggest that the B-tropism determinant in B-tropic ecotropic MuLV may arise from recombination between N-tropic ecotropic MuLV and members of the abundant endogenous nonecotropic MuLV-related classes including a subset of endogenous xenotropic proviruses.

The tandem direct repeats within the long terminal repeat of murine leukemia viruses are the primary determinant of their leukemogenic potential.

To map the viral sequences encoding the leukemogenic determinant(s) of nondefective murine leukemia viruses (MuLVs), we constructed chimeric viral genomes in vitro between cloned viral DNAs from the highly leukemogenic Gross passage A (Gross A) MuLV and from the related nonleukemogenic BALB/c N-tropic MuLV. Infectious chimeric MuLVs, recovered from murine cells microinjected with these DNAs, were inoculated into newborn mice to test the leukemogenic potential of these viruses. We found that the U3 long terminal repeat region from Gross A genomes was sufficient to confer an intermediate leukemogenic potential to chimeric MuLVs. Sequencing data indicated that the U3 tandem direct repeat was responsible for this effect. Adding most of the Gross A p15E-coding sequences to the Gross A U3 long terminal repeat enhanced the leukemogenic potential of chimeric viruses significantly. Adding a larger 3'-end env region (all p15E-coding sequences and 345 base pairs of the carboxy terminus of gp70) to the Gross A U3 long terminal repeat restored the full leukemogenic potential of Gross A MuLV. Chimeric viruses harboring only the Gross A 3'-end env region were, however, nonleukemogenic. Similar chimeric MuLVs, constructed with genomes from the parental weakly leukemogenic BALB/c B-tropic MuLVs and nonleukemogenic BALB/c N-tropic MuLVs, were also studied. Our data indicate that the U3 tandem direct repeat sequences appear to be necessary and sufficient to confer some leukemogenic potential to MuLV. However, env 3'-end sequences, mostly the p15E-encoding sequences, are required for the expression of fully leukemic phenotypes.

Physical mapping of the Fv-1 tropism host range determinant of BALB/c murine leukemia viruses.

The murine leukemia viruses (MuLVs) have different host ranges and were originally designated N-tropic and B-tropic if they replicated preferentially in vitro on NIH and BALB/c fibroblasts, respectively. It was later found that N-tropic MuLVs were in fact restricted in BALB/c cells, that B-tropic MuLVs were restricted in NIH cells, and that both viruses were restricted in (BALB X NIH) F1 cells. A single gene, Fv-1, with two alleles, Fv-1b and Fv-1n, determines this dominant restriction. A virus-encoded protein seems to carry the viral host range determinant which is recognized by the Fv-1 gene product. To map the viral DNA sequences encoding this determinant, we constructed viral DNA recombinants in vitro between the cloned infectious viral DNA genomes from BALB/c N-tropic and B-tropic MuLVs. Infectious recombinant MuLVs were recovered by microinjecting these recombinant DNAs into murine Fv-1- SC-1 cells and were subsequently tested in vitro for their host ranges (N- or B-tropic). We found that a short 302-base pair 5'-end fragment was necessary and sufficient to confer a specific host range to a recombinant. Our sequencing data revealed that this fragment codes for amino acid sequences in gag p30. They also showed that only two consecutive amino acid differences, Gln-ArgN- and Thr-GluB-, in p30 are responsible for the N- and B-tropic host ranges of the BALB/c MuLVs, respectively. Therefore, it appears that the Fv-1b and Fv-1n gene products can discriminate between these two p30 amino acid sequences.

Nucleotide sequences of gag-pol regions that determine the Fv-1 host range property of BALB/c N-tropic and B-tropic murine leukemia viruses.

Previously, in vitro recombinant DNA studies demonstrated that genetic determinants of N-tropism and B-tropism, or Fv-1-related host range properties of murine leukemia viruses, were located in a BamHI-HindIII DNA segment derived from the 5' portion of the cloned viral genome. We sequenced this segment and its immediate 5' region from cloned DNA of two BALB/c mouse C-type viruses (WN1802N and WN1802B) and found base differences at 12 positions out of the otherwise identical 1,390-base-pair sequences. Analysis of the most likely reading frame showed that 6 of the 12 base differences would result in four encoded amino acid changes, three of which occur at positions 109 (glutamine in WN1802N versus threonine in WN1802B), 110 (arginine in WN1802N versus glutamic acid in WN1802B), and 159 (glutamic acid in WN1802N versus glycine in WN1802B) of the p30 protein. The remaining one is located at position 36 (threonine in WN1802N versus isoleucine in WN1802B) of the viral polymerase protein. Significant conformational alteration of the p30 protein could be predicted from these amino acid changes.

Reversal of Fv-1 host range by in vitro restriction endonuclease fragment exchange between molecular clones of N-tropic and B-tropic murine leukemia virus genomes

We molecularly cloned unintegrated viral DNA of the BALB/c endogenous N-tropic and B-tropic murine leukemia retroviruses and in vitro passaged N-tropic Gross (passage A) murine leukemia retroviruses. Recombinant genomes were constructed in vitro by exchanging homologous restriction enzyme fragments from N- or B-tropic parents and subsequent recloning. Infectious virus was recovered after transfection of these recombinant genomes into NIH-3T3 cells and cocultivation with the Fv-1 nonrestrictive SC-1 cells. XC plaque assays of recombinant virus progeny on Fv-ln and Fv-lb cells indicated that the Fv-l host range was determined by sequences located between the BamHI site in the p30 region of the gag gene (1.6 kilobase pairs from the left end of the map) and the HindIII site located in the pol gene (2.9 kilobase pairs from the left end of the map).

Thymotropism of murine leukemia virus is conferred by its long terminal repeat.

Several murine leukemia viruses (MuLV) replicate efficiently in the thymus (T+) of the mouse, whereas others are unable to replicate (T-) in this organ. To map the region of the viral genome harboring the sequences responsible for this thymotropic phenotype, we constructed viral DNA recombinants in vitro between cloned infectious viral DNAs from T- BALB/c N-tropic MuLV and from T+ BALB/c B-tropic MuLV or AKR Gross passage A MuLV. (N- and B-tropic refer to the Fv-1 host range of MuLV.) Infectious recombinant MuLVs, recovered from murine cells microinjected with these recombinant DNAs, were injected into newborn mice to test their ability to replicate in the thymus. We found that the long terminal repeat from the T+ BALB/c B-tropic or AKR Gross passage A MuLV genome was sufficient to allow replication of recombinant MuLVs in the thymus. Our sequence data suggested that the U3 tandem direct repeat was responsible for this effect. These results suggest a new role for the U3 long terminal repeat in the replication of MuLV in specific differentiated target cells.

The high leukemogenic potential of Gross passage A murine leukemia virus maps in the region of the genome corresponding to the long terminal repeat and to the 3' end of env.

The Gross passage A murine leukemia virus (MuLV) is a highly leukemogenic, ecotropic fibrotropic retrovirus. Its genome is similar to that of other nonleukemogenic ecotropic fibrotropic MuLVs but differs at the 3' end and in the long terminal repeat. To determine whether these modifications were related to its leukemogenic potential, we constructed a viral DNA recombinant in vitro with cloned infectious DNA from this highly leukemogenic Gross passage A MuLV and from a weakly leukemogenic endogenous BALB/c B-tropic MuLV. Infectious viruses, recovered after microinjection of murine cells with recombinant DNA, were injected into newborn mice. We show here that the Gross passage A 1.35-kilobase-pair KpnI fragment (harboring part of gp70, all of p15E, and the long terminal repeat) is sufficient to confer a high leukemogenic potential to this recombinant.

H-2 control of the cytotoxic antibody response against a newly defined MuLV-related cell-surface antigen: G(B10.A).

H-2-congeneic C57BL mice with milk transmission of B-tropic murine leukemia virus (V+ mice) have a much higher lymphoma incidence than the same strains without milk-transmitted virus (V- mice). Gene(s) within the major histocompatibility complex (H-2) influence virus titers, lymphoma incidence, lymphoma type and the anti-MuLV envelope antibody response. In this paper, we report that the prevalence of cytotoxic antibodies to virus-induced lymphomas is also regulated by the H-2 complex. Milk transmission of MuLV resulted in the formation of cytotoxic antibodies against primary virus-induced C57BL lymphomas. These antibodies detect an antigen that is also present on the RADAI tumor-cell line, and on normal spleen cells of young adult B10.A (H-2a) mice of both V+ and V- sublines, but not on spleen cells of young adult B10 (H-2a) mice of either subline. These cytotoxic antibodies were detected in the sera of B10V+ and B10.A(5R)V+ animals, but not in the sera of B10.AV+ mice. This indicates that the prevalence of these antibodies is controlled by a gene in the K- and/or I-A region of the H-2 complex. The presence of these cytotoxic antibodies in serum is recessively inherited. The specificity of the cytotoxic antibodies was investigated with a standard panel of transplantable tumor-cell lines. Of these, only the RADAI cells expressed the target antigen in direct cytotoxicity tests and by absorption. The ability of B10V+ sera to lyse the B10.AV+ and RADAI tumor cells is ascribed to antibody activity against a new MuLV-related cell-surface protein: G(B10.A). Immunochemical analysis and absorption experiments with different types of purified MuLV and MuLV-infected cell lines indicate that the cytotoxic antibodies belong to low-avidity IgM antibodies that are directed to MuLV.

Naturally occurring leukemia viruses in H-2 congenic C57BL mice III. Characterization of C-type viruses isolated from lymphomas induced by milk transmission of B-ecotropic virus

The prevalence of different host-range classes of murine leukemia virus (MuLV) was studied in C57BL mice with (V+) and without (V-) milk transmission of a naturally occurring B-tropic ecotropic MuLV. Virus isolates were studied with respect to growth properties, XC-plaque formation, antigen profiles of their envelope proteins (gp70 and P15(E)), gp70 tryptic-peptide maps, and their potential to induce lymphomas after inoculation into newborn mice. B-tropic ecotropic MuLV with the capacity to cause plaques in XC cells was isolated from almost all lymphomas of both V+ and V- sublines. The reaction patterns of these ecotropic isolates with monoclonal antibodies reactive with MuLV-env proteins and the tryptic-peptide maps of the gp70 molecule indicate that they are similar to each other and differ only slightly from the ecotropic MuLV in the spleens of young V+ animals, which is identical to the milk-transmitted virus. XC −, B-tropic dualtropic mink cell focus-inducing (MCF) viruses were isolated from the majority of different types of lymphoma (B cell, T cell, or neither B nor T cell derived), but not from the spleens or milk of young V+ or V- animals. The env proteins of the MCF isolates are highly heterogeneous, but most isolates originating from B10.AV+ T-cell lymphomas share MCF-related epitopes in their gp85 envelope precursor with AKR MCF-247 virus. Most MCF viruses isolated from non-T lymphomas do not possess these epitopes. The results indicate that also in this model the generation of dualtropic MCF viruses might be important in lymphoma induction, although only some of the cloned MCF viruses show enhanced oncogenic properties in comparison with ecotropic isolates. A cloned oncogenic MCF virus induced different lymphoma types in C57BL/10 (=B10, H-2 b) and B10.A (H-2 a) mice, similar to what was found earlier with the milk-transmitted virus. Hence, the lymphoma-type differences are not due to differences in the B-tropic ecotropic viruses transmitted through the milk in these strains, but reflect an influence of the H-2 complex on the phenotype of the virus-induced lymphomas.

Ecotropic murine leukemia virus DNA content of normal and lymphomatous tissues of BXH-2 recombinant inbred mice.

BXH-2 recombinant inbred mice spontaneously produce a B-tropic murine leukemia virus (MuLV) beginning early in life and have a high incidence of non-T-cell lymphomas. These traits are not characteristic of the progenitor strains (C57BL/6J and C3H/HeJ) or of 11 other BXH recombinant inbred strains. Since B-tropic virus expression may be causally related to the high incidence of lymphoma in this strain, we have analyzed the ecotropic MuLV DNA content of both normal and lymphomatous tissues of BXH-2 mice. Southern analysis and hybridization with an ecotropic MuLV DNA-specific probe showed that DNA of normal BXH-2 tissues contained both parental N-tropic MuLV proviruses but lacked endogenous B-tropic MuLV DNA sequences. In addition, none of 116 F1 hybrid mice derived from male BXH-2 mice spontaneously produced ecotropic MuLV early in life. These results suggest that the B-tropic virus is horizontally transmitted in BXH-2 mice. Southern analysis of DNA from tumor tissues of 12 BXH-2 mice showed that amplification of ecotropic-specific DNA sequences had occurred in lymphomatous tissues of 3 mice and suggested that these tumors were monoclonal. The number of newly acquired proviruses, which appeared to be structurally nondefective and integrated at different sites, varied from one to three copies. Since lymphomatous tissues from only 3 of 12 mice examined carried additional detectable ecotropic proviruses, these results suggest that amplification of ecotropic MuLV DNA sequences is not required for maintenance of transformation in BXH-2 lymphomas.