Murine xenotropic type C viruses I. Distribution and further characterization of the virus in NZB mice.

The xenotropic mouse type C virus, originally detected in cultured embryo cells from New Zealand Black (NZB) mice, has been recovered from over 50 adult NZB animals and 15 NZB embryos. Its presence is best detected by measuring its ability to rescue a murine sarcoma virus (MSV) genome from a non-virus-producing MSV-transformed rat cell. The virus can serve as a helper for replication of MSV. It has a distinct type-specific coat and is a prototype for a third serotype of mouse type C viruses, NZB. The xenotropic virus may have an evolutionary role since it has a wide host range, including the ability to infect avian cells. It is produced spontaneously by all cells cultivated from NZB tissues and accounts for the high concentration of viral antigens associated with NZB tissues. The extent of virus production is similar in both male and female mice. All cell clones established from embryos also produce the virus. A variability in the intracellular regulation of virus replication is suggested since tissue cells from the same animal differ quantitatively in their ability to produce xenotropic viruses. Since enhanced spontaneous virus production is associated with cells from NZB mice, the virus may play a role in the autoimmune disease of this mouse strain.     

Genetics of xenotropic virus expression in mice. I. Evidence for a single locus regulating spontaneous production of infectious virus in crosses involving NZB/B1NJ and 129/J strains of mice.

The extent of infectious xenotropic virus expression in homogenized splenic tissues from the high-virus-expressing NZB/BINJ mice and the non-virus-expressing 129/J mice and their crosses has been examined. The data suggest that a single autosomal "dominant-like" gene controls the spontaneous production and release of infectious xenotropic virus in NZB mice. Analysis of infectious virus production in second-backcross families [(F1 X 129) X 129] confirmed this conclusion. Variations in the amount of X-tropic virus released were evident in all genetic crosses. Virus titers (expressed as focus-forming units per milliliter) of supernatant fluid ranged from high levels in the NZB mice to somewhat lower levels in crosses involving the 129 mice. In the absence of a definite pattern in the titers observed in the genetic crosses studied, the term dominant-like is proposed for the single gene regulating the expression of X-tropic virus in NZB mice.     

Envelope and long terminal repeat sequences of a cloned infectious NZB xenotropic murine leukemia virus

An infectious NZB xenotropic murine leukemia virus (MuLV) provirus (NZB was molecularly cloned from the Hirt supernatant of NZB-IU-6-infected mink cells, and the nucleotide sequence of its env gene and long terminal repeat (LTR) was determined. The partial nucleotide sequence previously reported for the env gene of NFS-Th-1 xenotropic proviral DNA (Repaske, et al., J. Virol. 46:204-211, 1983) is identical to that of the infectious NZB xenotropic MuLV DNA reported here. Alignment of nucleotide or deduced amino acid sequences, or both, of xenotropic, mink cell focus-forming, and ecotropic MuLV proviral DNAs in the env region identified sequence differences among the three host range classes of C-type MuLVs. Major differences were confined to the 5' half of env; a high degree of homology was found among the three classes of MuLVs in the 3' half of env. Alignment of the nucleotide sequence of the LTR of NZB xenotropic MuLV with those of the LTRs of NFS-Th-1 xenotropic, mink cell focus-forming, and ecotropic MuLVs revealed extensive homology between the LTRs of xenotropic and MCF247 MuLVs. An inserted 6-base-pair repeat 5' to the TATA box was a unique feature of both NZB and NFS-Th-1 xenotropic LTRs.     

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.     

Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.     

Laboratory and wild-derived mice with multiple loci for production of xenotropic murine leukemia virus.

Mendelian segregation analysis was used to define genetic loci for the induction of infectious xenotropic murine leukemia virus in several laboratory and wild-derived mice. MA/My mice contain two loci for xenotropic virus inducibility, one of which, Bxv -1, is the only induction locus carried by five other inbred strains. The second, novel MA/My locus, designated Mxv -1, is unlinked to Bxv -1 and shows a lower efficiency of virus induction. The NZB mouse carries two induction loci; both are distinct from Bxv -1 since neither is linked to the Pep-3 locus on chromosome 1. Finally, one partially inbred strain derived from the wild Japanese mouse, Mus musculus molossinus, carries multiple (at least three) unlinked loci for induction of xenotropic virus. Although it is probable that inbred strains inherited xenotropic virus inducibility from Japanese mice, our data suggest that none of the induction loci carried by this particular M. m. molossinus strain are allelic with Bxv -1.     

Susceptibility of wild mouse cells to exogenous infection with xenotropic leukemia viruses: control by a single dominant locus on chromosome 1.

Although xenotropic murine leukemia viruses cannot productively infect cells of laboratory mice, cells from various wild-derived mice can support replication of these viruses. Although the virus-sensitive wild mice generally lack all or most of the xenotropic proviral genes characteristic of inbred strains, susceptibility to exogenous infection is unrelated to inheritance of these sequences. Instead, susceptibility is controlled by a single dominant gene, designated Sxv, which maps to chromosome 1. Sxv is closely linked to, but distinct from Bxv-1, the major locus for induction of xenotropic murine leukemia viruses in laboratory mice. Genetic experiments designed to characterize Sxv show that this gene also controls sensitivity to a wild mouse virus with the interference properties of mink cell focus-forming murine leukemia viruses, and that Sxv-mediated susceptibility to xenotropic murine leukemia viruses is restricted by the mink cell focus-forming virus resistance gene Rmcf. These data, together with genetic mapping of the mink cell focus-forming virus cell surface receptor locus to this same region of chromosome 1, suggest that Sxv may encode a wild mouse variant of the mink cell focus-forming virus receptor that allows penetration by xenotropic murine leukemia viruses.     

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.     

Endogenous xenotropic murine leukemia virus-related sequences map to chromosomal regions encoding mouse lymphocyte antigens.

DNAs of all inbred mouse strains contain multiple copies (18 to 28 copies per haploid mouse genome) of endogenous xenotropic murine leukemia virus-related sequences detectable by Southern analysis with a xenotropic murine leukemia virus env gene-specific probe. After PvuII digestion, we identified a subset of xenotropic murine leukemia virus-related sequences that are well resolved by agarose gel electrophoresis and can be mapped to specific chromosomes by using recombinant inbred mouse strains. Interestingly, three of six xenotropic proviral loci that we mapped were integrated near genes encoding mouse lymphocyte antigens (Ly-m22, chromosome 1; Ly-m6, chromosome 2; and Ly-m10, chromosome 19) and a fourth xenotropic proviral locus mapped near a gene on chromosome 4 that has a major influence on xenotropic virus cell surface antigen levels. These studies indicate that xenotropic proviral loci are located on many different mouse chromosomes and may be useful markers for molecularly cloning and characterizing regions of the mouse genome important in lymphocyte development.     

Nucleotide sequence of the env-specific segment of NFS-Th-1 xenotropic murine leukemia virus.

The sequence of 863 contiguous nucleotides encompassing portions of the pol and env genes of NFS-Th-1 xenotropic proviral DNA was determined. This region of the xenotropic murine leukemia virus genome contains and env-specific segment that hybridizes exclusively to xenotropic and mink cell focus-forming but not to ecotropic proviral DNAs (C. E. Buckler et al., J. Virol. 41:228-236, 1982). The unique xenotropic env segment contained several characteristic deletions and insertions relative to the analogous region in AKR and Moloney ecotropic murine leukemia viruses. Portions of an endogenous env segment cloned from a BALB/c mouse embryo gene library that had a restriction map and hybridization properties typical of xenotropic viruses (A. S. Khan et al., J. Virol. 44:625-636, 1982) were also sequenced. The sequence of the endogenous env gene was very similar to the comparable region of the NFS-Th-1 xenotropic virus containing the characteristic deletions and insertions previously observed and could represent a segment of an endogenous xenotropic provirus.     

Nonecotropic murine leukemia viruses in BALB/c and NFS/N mice: characterization of the BALB/c Bxv-1 provirus and the single NFS endogenous xenotrope

We used hybridization probes that react specifically with xenotropic and mink cell focus-forming virus envelope sequences to characterize the nonecotropic proviruses of BALB/c and NFS/N mice. Analysis of somatic cell hybrids with different BALB/c chromosomes showed that the 9 xenotropic and more than 20 MCF virus-related proviral sequences in this mouse were present on more than nine BALB/c chromosomes. Multiple copies were found on chromosomes 1, 4, 7, 12, and probably 11, and the copies found on a single chromosome were not identical by restriction enzyme mapping. We also identified and characterized the proviral sequences that give rise to infectious xenotropic virus in both BALB/c and NFS/N mice. BALB/c contains the major locus for induction of infectious virus in inbred mice, Bxv-1, which is on chromosome 1. We showed that this locus contains a single xenotropic provirus on an 18-kilobase HindIII fragment. Restriction enzyme analysis of a hybrid cell DNA that contains only the Bxv-1 xenotropic provirus showed that the Bxv-1 provirus contains restriction enzyme sites characteristic of the infectious virus induced from BALB/c fibroblasts. The Bxv-1 provirus and its flanking sequences also contain the same restriction sites as the provirus thought to contribute U3 long terminal repeat sequences to leukemogenic (class I) AKR MCF viruses. Analysis of cell hybrids made with the nonvirus-inducible strain NFS/N showed that the single xenotropic virus env gene of NFS mice, here termed Nfxv-1, is not on chromosome 1. Unlike that of Bxv-1, the restriction map of Nfxv-1 does not resemble that of any known infectious xenotropic virus including xenotropic viruses isolated from NFS mice. These data suggest that Bxv-1, but not Nfxv-1, is a full-length xenotropic provirus that can be transcribed directly to produce infectious virus.     

Analysis of integrated proviral DNA sequences with an octadecanucleotide probe designed for specific identification of spleen focus-forming virus in the mouse genome.

The Friend spleen focus-forming virus (SFFV) is an envelope gene recombinant between the ecotropic Friend murine leukemia virus and the endogenous xenotropic mink cell focus-forming retroviral sequences. We synthesized an octadecanucleotide complementary to the 3' end of the SFFV env gene designed for discriminating the SFFV proviruses from the xenotropic mink cell focus-forming virus and ecotropic exogenous or endogenous viral sequences. Under appropriate hybridization conditions this probe allowed the identification, in addition to few endogenous DNA fragments, of multiple SFFV proviruses integrated in the genome of Friend malignant cells. Therefore this probe should be of interest in further characterizing the SFFV integration sites and possibly the SFFV ancestor gene.     

Isolation of infectious xenotropic mouse type C virus by transfection of a heterologous cell with DNA from a transformed mouse cell

An endogenous xenotropic type C virus has been isolated from a Kirsten sarcoma virus-transformed BALB/c mouse cell line by transfection of a mink fibroblast cell with the DNA from the transformed cells. The results indicate that transfection may be used as a technique to isolate this endogenous type C virus without the need to chemically induce the cell line containing the provirus prior to attempting to isolate the virus.     

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.     

Leukemogenicity and cell transformation mechanisms in vitro by Gross murine leukemia virus: analysis of virus subpopulations.

The leukemogenic activity of Gross murine leukemia virus adapted to rats was tested in W/Fu rats and NIH/Swiss mice. All animals infected with this virus developed thymic and nonthymic T-cell leukemia with a short latency period. It was observed that cell-free extracts from thymic lymphoma tissue of mice and rats, induced by either Gross murine leukemia virus or Gross murine leukemia virus adapted to rats, consisted of both small-plaque-forming and large-plaque-forming viruses, as determined by the XC plaque test. MCF-type virus was found in these virus complexes. Transformed cell foci were induced in SC-1 cell layers by double infection of the cloned MCF-type virus and an ecotropic virus. SC-1 cells containing transformed cell foci were shown to be tumorigenic upon inoculation into nude mice. The formation of transformed cell foci in mink lung cells was also observed after double infection with the cloned MCF-type virus and a xenotropic virus. The possible mechanism of leukemogenesis by endogenous viruses is discussed.     

Mechanism of restriction of ecotropic and xenotropic murine leukemia viruses and formation of pseudotypes between the two viruses.

Ecotropic and xenotropic murine leukemia viruses (MuLV's) constitute separate interference groups; within each group there is cross-interference, but between the groups there is no detectable interference. Interference is manifest against pseudotypes in which the vesicular stomatitis virus genome is contained within the coat of one of the murine leukemia viruses. The pseudotypes display the cell specificity of the leukemia viruses: pseudotypes with an ecotropic MuLV coat infect mouse cells but not rabbit or mink cells; pseudotypes with a xenotropic MuLV coat infect rabbit or mink cells well but mouse cells very poorly. Efficient pseudotype formation also occurs between the two MuLV classes, and both the interference patterns and the cell specificity of these pseudotypes are entirely determined by their envelope. Using these pseudotypes, ecotropic MuLV infection could be established in xenogeneic cells, and the resulting progeny could be scored by using a conventional XC cell assay. Also, xenotropic MuLV infection could be established in a mouse cell, showing that no absolute intracellular barrier against xenotropic virus growth exists in murine cells. The major barriers against both xenotropic and ecotropic MuLV therefore are cell surface barriers. Xenogeneic cells probably lack receptors for ecotropic MuLV, but murine cells may either lack receptors for xenotropic MuLV or have receptors that are blocked by endogenous expression of the glycoprotein of endogenous xenotropic MuLV.     

RNA metabolism of murine leukemia virus II. Endogenous virus-specific RNA in the uninfected BALB/c cell line JLS-V9.

Type C virus-specific RNA sequences of BALB/c endogenous virus were detected in JLS-V9 cells (an uninfected BALB/c derived line) by annealing cell RNA with 3-H-labeled virus-specific DNA. Endogenous viruses used in preparing the 3-H-labeled DNA (mostly xenotropic) was prepared from JLS-V9 cells induced to produce virus with iododeoxyuridine. In whole-cell extracts, two virus-specific RNA species, 38S and 27S, were detected. No 60 to 70S virus-specific RNA was found. The same two species of virus-specific RNA were observed in isolated cytoplasmic RNA and in cytoplasmic RNA selected for polyadenylic acid-containing species by binding and elution from oligo(dT) cellulose. Very little, if any, of the virus-specific RNA was active as messenger RNA on polyribosomes. No virus-specific RNA transcribed from genes coding for the BALB/c endogenous N-tropic virus was detected, since 3-H-labeled DNA prepared from endogenous N-tropic virus did not hybridize measurably with JLS-V9 RNA.     

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.