T1 oligonucleotide maps of moloney and HIX murine leukemia viruses

HIX virus shares approximately 80% of the large RNase T1-resistant oligonucleotides derived from its genome with Moloney IC murine leukemia virus. In addition, each virus possesses oligonucleotides not found in the other. The oligonucleotides unique to either HIX or Moloney (that could be ordered) were found to lie clustered in corresponding regions in the 3' halves of the HIX and Moloney IC T1 oligonucleotide maps. A second clonal isolate of Moloney virus, Moloney clone-1, was found to differ from the Moloney IC isolate by about 5–10% of its large RNase Tl-resistant oligonucleotides. HIX virus was more closely related to Moloney IC than to Moloney clone-1 virus. These results provide good evidence to support the proposal that HIX virus arose by recombination involving Moloney IC and an as yet unidentified xenotropic virus.     

Chimeras between SRS and Moloney murine leukemia viruses reveal novel determinants in disease specificity and MCF recombinant formation

SRS 19-6 MuLV is a murine retrovirus originally isolated in mainland China. A noteworthy feature of this virus (referred to as SRS MuLV here) induces tumors of multiple hematopoietic lineages, including myeloid, erythroid, T-lymphoid and B-lymphoid. To identify the determinants of disease specificity, chimeras between SRS and Moloney MuLV (M-MuLV) were generated by molecular cloning, and the pathogenic properties of the chimeras were investigated. The results indicated that, while the M-MuLV LTR can confer lymphoid specificity to SRS MuLV, the SRS LTR by itself was not sufficient to confer multiple lineage tumorigenesis to M-MuLV; additional sequences in gag or pol were also required. Thus, a secondary determinant for myeloid/erythroid leukemia in SRS MuLV is located in gag-pol. In these chimeras, an independent determinant for T-lymphoma was found in M-MuLV gag-pol. It was also interesting that insertion of M-MuLV env into SRS MuLV decreased the rate of leukemogenicity, while insertion of SRS env into M-MuLV (SEM) accelerated leukemogenesis. The enhanced pathogenicity of SEM was found to correlate with earlier formation of MCF recombinants. The basis for the accelerated MCF recombinant formation was investigated. The endogenous polytropic MuLV env sequences contributing to several SEM MCF recombinants were identified, and the cross-over points were identified. While no obvious differences in the relative homologies between SRS MuLV env and polytropic env vs. M-MuLV and polytropic envs suggested a reason for the more rapid MCF recombinant formation, an overlapping but different set of polytropic env proviruses were found to participate in MCF formation for M-MuLV vs. SEM. Thus, the mechanisms for MCF formation appear to differ for M-MuLV and SEM.     

Different recombinant murine leukemia viruses use different cell surface receptors

Retroviruses can be grouped by viral interference measurements into classes which use common cell surface receptors. We previously tested a large number of isolates of mink cell focus-inducing (MCF) murine leukemia viruses (MuLVs), and reported that all of them share a distinct receptor on NIH/3T3 cells (A. Rein, Virology 120, 251, 1982). We now extend this generalization to several additional recombinant isolates, including two (SL3-2 and GPA-V2) which would not be considered MCFs on the basis of host-range data. We note the superiority of interference tests, based on positive, unambiguous data, over host-range tests for virus classification. We also show that in contrast to the MCFs, which are all derived from ecotropic MuLVs, a recombinant derived from wild mouse amphotropic MuLV (S. Rasheed et al., Int. J. Cancer 29, 345, 1982) uses a unique receptor on NIH/3T3 cells. This suggests that (a) mouse cells contain more than one type of endogenous env sequence; and (b) there is some specificity in the generation of recombinants, since ecotropic MuLVs appear to give rise only to MCFs, while amphotropic MuLV has generated a distinct type of recombinant. It also represents a second case (in addition to the MCFs) in which an env gene recombinant is more pathogenic than its exogenous parent. We also show that xenotropic MuLV does not interfere with MCFs in NZB mouse cells; thus, despite the close homology between MCF and xenotropic env sequences, the gp70 of xenotropic MuLV appears to have no detectable affinity for the MCF receptor.     

Modulation of specific T cell responses by concurrent infection with Leishmania major and LP-BM5 murine leukemia viruses

C57BL/6 mice infected with a murine leukemia virus (MuLV) mixturedesignated LP-BM5 develop an immunodeficiency syndrome termedMAIDS, characterized by a variety of T and B cell abnormalities,including elevated levels of IgE, suggesting that IL-4 expressionis increased in these animals. It has been suggested that theimmunodeficiency associated with MAIDS is caused by a conversionof immune responses normally characterized by T_h1 developmenttowards a T_h2- dominated response. Mice of the same strain,infected with Leishmania major, mount a protective T_h1 responsewith the induction of high levels of IFN- and undetectable IL-4.We therefore infected mice with L. major at differing time pointsbefore and after virus infection and assessed the effects onT cell responsiveness, cytokine production and survival to L.major, as well as the effect on MAIDS-associated pathology.We have also immunized C57BL/6 mice with trinitrophenol-keyholelimpet haemocyanln (TNP-KLH), which leads to a predominantlyT_h2 response, and compared the effects of MAIDS on the responseto TNP-KLH with the effect of MAIDS on L. major infection. Ourresults show that significant immunodeficiency with regard toinfection by L. major is only apparent after 8 weeks of LP-BM5MuLV infection, by which time T and B cell defects are welladvanced. Further, we have found that the strongly polarizedT_h1 response stimulated by L. major infection can modulate theeffect of MAIDS on T cells, leading to the survival of antigen-specificT cells. Our results suggest that the impairment of immune responsesto either TNP–KLH or L. major is due not to an alterationof the balance of T_h1/T_h2 subsets but to a general loss of reactivityin antigen-specific CD4⁺ cells. However, prior activation ofT_h1 but not T_h2 cells can inhibit the development of lymphoproliferationand immunodeficiency caused by MAIDS.     

XC cell fusion by murine leukemia viruses: Fusion from without

Concentrated murine leukemia virus (MuLV) or MuLV producing cells induce XC cell fusion within an hour leading to syncytia formation. While MuLV inactivated by UV irradiation,-propiolactone or hydroxylamine treatment still caused cell fusion, Bromelin- or trypsin treated MuLV was no longer able to fuse XC cells. Though sonicated MuLV induced no XC cell fusion, it interfered with cell fusion as caused by untreated MuLV. XC cells infected by diluted MuLV of a titer lower than 1×10⁵ PFU/ml formed no syncytia although they produced MuLV. The call fusion mechanism is discussed.     

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.     

Multiparameter analyses of spontaneous nonthymic lymphomas occurring in NFS/N mice congenic for ecotropic murine leukemia viruses

Mouse strains congenic for ecotropic retrovirus genes have a much higher frequency of spontaneous lymphomas than the background NFS/N strain. In this study, most of these lymphomas have been identified as B-cell in origin by morphologic features, identification of immunoglobulin class, and cell-surface antigens. The classification suggested by Pattengale and Taylor proved to be applicable to the lymphomas studied. Most were of large follicular center cells and are considered typical of the type formerly designated as "reticulum cell sarcoma, type B." Many lymphomas contained a large proportion of nonneoplastic cells which partially obscured their neoplastic component. The role of ecotropic murine leukemia viruses as etiologic agents for B-cell lymphomas remains equivocal. However, because the only difference between the NFS/N and congenic mice is the expression of viruses in the latter, it appears that these viruses are somehow involved in induction of B-cell lymphomas.     

Complementarity between RNA dimerization elements favors formation of functional heterozygous murine leukemia viruses

The cis-elements that direct packaging and dimerization of retroviral RNAs overlap, and it has been suggested that dimerization is required for RNA packaging. This also implies that heterodimerization would be necessary for co-packaging and recombination. Moreover, co-packaging of distinct RNAs may be reduced if incapable of heterodimerizing. In this study, we have designed a novel two-vector rescue system in which co-packaging and interstrand transfer are necessary for transduction. Thus, the rescue titer is a measure of the ability of a given vector combination to co-package and subsequently generate a provirus. In the current MLV-based set-up, we explored Akv- and MLV-like-endogenous virus (MLEV)-derived vectors with modulated dimerization signals. Results show that rescue is influenced by competition at the level of RNA packaging, as well as complementarity between dimerization elements. Altogether, the results support the hypothesis that complementarity between dimerization elements may favor co-packaging of distinct retroviral RNAs.     

An immunological focus assay for murine leukemia viruses on viable attached cell lines

An assay is described for the detection and isolation of murine leukemia virus (MuLV)-infected cells in viable monolayers. The procedure utilizes antisera or monoclonal antibodies which specifically bind cell surface viral antigens of infected cells. Bound antibodies are subsequently detected by binding with ¹²⁵I-labeled Staphylococcus aureus protein A followed by autoradiography of the tissue culture vessel. Focal areas of infection can be identified from the autoradiograph and infected cells can subsequently be isolated and subcultured as MuLV-producing cell lines. With appropriate antibodies the procedure should be useful for the direct isolation of minor components, including mutants, in complex virus mixtures.     

In vitro properties of murine autoreactive T cell clones with specificity for erythrocytes

A panel of IL-2 dependent T cell clones was generated from the lymph nodes of Balb/c mice immunized with RRBC which carry both foreign determinants specific for RRBC and crossreactive determinants shared with MRBC. From this panel, two clones designated L1R1 41 and L2R1 10 were successfully maintained in long-term culture for over six months and possessed essentially similar properties. They were slow growing and secreted a late-acting B cell growth factor but neither BSF-1 (Il-4) nor gamma interferon. Although Thy-1+ they were negative for other differentiation antigens including Ly-1, Ly-2 and L3T4. Both clones underwent antigen dependent proliferation in vitro. At a T cell:irradiated spleen cell ratio of 1:10 they responded to RRBC and MRBC equally well whereas at a ratio of 10:1 only an anti-self response was obtained. These results illustrate how antigen presentation may influence the capacity of the immune system to discriminate between self and non-self.     

Pathogenicity of BALB/c-derived N-tropic murine leukemia viruses

N-tropic murine leukemia viruses have been observed in connection with radiation-induced osteosarcomagenesis in BALB/c mice. We have investigated the bone disease-inducing potential of molecularly cloned, BALB/c-derived N-tropic viruses in the random-bred NMRI mouse strain. The germ-line virus and an exogenous virus isolate were found to induce high incidences of osteopetrosis and lymphomas and a lower incidence of osteomas. Two viruses derived from somatically acquired proviruses of independent radiation-induced osteosarcomas induced lower incidence of osteopetrosis and lymphomas. Nucleotide sequence analysis of the long terminal repeat regions and RNase T1 fingerprint analysis revealed only few differences between the isolates. The possible involvement of N-tropic murine leukemia viruses in radiation-induced osteosarcomagenesis in the BALB/c mouse strain is discussed.     

Clonal specificity analysis of mitogen-activated murine T lymphoblasts

We have determined the frequencies and specificities of MHC-reactive and MHC-restricted cytotoxic T lymphocyte precursors (CTL-p) in mitogen (ConA)-activated splenocytes of normal unprimed mice. The limiting dilution (LD) system supported the growth of one out of three Lyt2+ T cell blasts. The generated CTL-populations lysed blast cell targets specifically as determined by split well analyses. MHC-gene product expression was necessary for lysis to occur, since MHC-negative F9 teratocarcinoma cells were not lysed. The frequency determinations and split well analyses revealed: 1) equally high numbers (approximately 1/100) of CTL-p that generated specific allo-MHC or self-MHC reactive CTL populations, 2) high frequencies of CTL-p which recognized hapten (TNP) or minor H (MH)-antigens in the context of self MHC or allo-MHC determinants. The results are discussed with respect to antigen, restriction and receptor specificities of mitogen-activated unprimed T cell blasts.     

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.