A unique and important set of recently recognized genes of the mouse are those that are the genomes of endogenous retroviruses (i.e., the RNA viruses that replicate by transcribing the genome into a DNA copy that integrates into cellular DNA). During the course of evolution of many vertebrate, DNA copies of retrovirus genomes have become established as germ line chromosomal elements. In the mouse there are at least two distinct classes of retrovirus in the germ line, i.e., the B-type, or mammary tumor viruses, and the C-type, or leukemia viruses; it is the latter that will be discussed here.
C-type viruses of mice, or murine leukemia viruses (MuLV), fall into three major categories: the ecotropic, xenotropic, and amphotropic classes. Ecotropic viruses are capable of infecting mouse cells but not heterologous species cells; xenotropic viruses affect only heterologous cells; while amphitropic viruses infect both categories. These viruses are closely related to one another in that the virion structural proteins p30 and reverse transcriptase are the same. The viruses differ, however, in the envelope glycoproteins, which confer the type-specific interference, neutralization, and host range phenotypes.
The ecotropic viruses are typified by the classical AKR MuLV; comparable strains are found in other high lymphoma strains of mice, such as C58, PL/J, and C3H/Fg. The most familiar example of a xenotropic MuLV is the endogenous virus of NZB mice, long thought to be an noninfectious MuLV, but shown by Levy and Pincus to be infectious for a variety of non-murine cells. Amphotropic viruses to date are known to occur only in certain populations of wild Mus musculus.
The three classes of MuLV show quite distinct patterns in their natural occurrence. Ecotropic viruses are carried by some strains of mice, but are not found in others. Among those strains that do carry the ecotropic viruses there are marked differences in the level of expression. The high lymphoma strains show titers of ecotropic MuLV throughout life, with virus appearing in the tissues at almost the time of birth. The virus can be induced from embryonic cells of such mice in tissue culture by treatment with the thymidine analogues 5-IUdR and 5-BUdR, and tissue culture cloning studies show that all cells possess the inducible viral genome.
Other strains of mice, referred to as low-ecotropic-virus yielders, on occasion yield the same MuLV as found in high virus mice, but it only appears late in life and in low titer. Strains with this pattern include BALB/c, C57BL, C57BR, DBA/2, and many others. Other strains, including C57L, NZB, 129, and NIH swiss and its inbred derivatives, NFS/N, have never yielded ecotropic MuLV, either in vivo or in vitro. Nucleic acid hybridization studies with viral cDNA probes have shown that the complete set of genome sequences of ecotropic MuLV is present in the cellular DNA of high- and low-virus mice, but a portion of the sequences, amounting to about 20% of the genome, is not present in DNA of the virus-negative strains.
The high-ecotropic virus phenotype shows classical Mendelian heredity patterns, and a number of the loci for ecotropic virus inducibility have been mapped. The mapped loci include two on chromosome 7, the Akv-1 and Fgv-1 loci, present in AKR/J and C3H/FgLw, respectively; they occupy non-allelic sites, Akv-1 being 12 map units from Gpi-1, and Fgv-1 being very close to Hbb. Two as yet unnamed loci in C57BL/10 and its H-2 congenic derivative B10.BR have also been mapped; one is on chromosome 8, linked to Es-1, and the other is on chromosome 11, linked to Es-3.
Strains of mice that are high for expression of ecotropic MuLV generally carry multiple loci for virus induction. AKR carries two such loci, C3H/Fg carries three, and C58 carries three or possibly four. The multiple loci are probably the result rather than the cause of the high virus phenotypes, since hybrids carrying only one virus-inducing locus per cell usually show as high virus expression as do the inbred strains with homozygosity at multiple loci. Indeed, we have seen on several occasions appearance of new loci for virus induction in congenic mice carrying the Akv-1 locus.
While much mapping of the ecotropic genomes in the various strains of mice remains to be done, it seems very likely that they are not at allelic sites in different strains of laboratory mice. Rather the loci must be thought of as being strain specific.
That these virus-inducing loci indeed represent viral genomes has been shown clearly by the nucleic acid hybridization studies of Chattopadhyay et al. ( 1, 2). When cellular DNA of various mouse strains is tested for the ability to hybridize with single stranded radiolabeled DNA probes prepared for ecotropic MuLV, and the number of copies of the virus-related sequences is determined from the kinetics of the reaction, a generally excellent correlation is found between the biologic and biochemical observations. The quantitative hybridization analyses show that there are two major populations of viral sequences in the cell DNA. One set, which corresponds to the genes for the MuLV group-specific proteins is present at a frequency of roughly 50 copies per haploid genome. A second set of sequences, corresponding to the type-specific (presumably envelope) genes, is present in a small number of copies, ranging from 1 to 3 or 4 copies per haploid genome in the ecotropic virus-positive strains, and being absent in the ecotropic virus-negative strains. The strains of mice that show multiple loci on Mendelian segregation analysis show multiple copies by hybridization kinetics, while those that are low in virus or are high-virus strains with a single locus, such as PL and RF, show a lower copy number by hybridization. In segregating crosses of hybrids carrying the Akv-1 locus on the ecotropic virus negative NIH Swiss background, the presence of the type-specific nucleic acid sequences segregated with virus inducibility and with chromosome 7 markers, establishing that viral DNA sequences are indeed present at this locus.
The pattern of xenotropic virus distribution among mouse strains is somewhat different. While there are marked differences in expression of xenotropic virus between mice, it seems probable that all strains of mice have the capacity to produce the virus. NZB mice regularly show high levels of spontaneous expression, while some other strains manifest lower levels of spontaneous virus production or are IUdR-inducible in tissue culture. In still other strains, xenotropic virus has been detected only on rare occasions, and it is not inducible from their cells in culture; strain 129 has not yielded virus, but it expresses MuLV glycoproteins with xenotropic antigenic specificity. As with the ecotropic virus, in the mouse strain with high level expression of xenotropic virus (NZB), Mendelian segregation analysis shows that there are multiple loci for virus induction, while in the strains with lower inducibility levels there is only a single locus. Nucleic acid hybridization studies have shown that the complete genome of xenotropic viruses is present in the DNA of all mouse strains tested, and in multiple copies, ranging from 2 to as many as 8 per haploid genome. In this case, the number of copies does not correlate with the biologic activity. Whether the multiple copies are clustered or scattered throughout the genome is not yet known. The mapping studies to date have succeeded in locating loci for induction of xenotropic virus in three cases, C57BL/10, BALB/c and the LP (loop tail) linkage testing stock; in all three instances the locus is on chromosome 1, 10 to 20 map units from Dip-1. Whether the three loci are allelic has yet to be established.
The amphotropic MuLV strains show a third pattern of natural history. As mentioned, these viruses have been detected only in certain colonies of feral mice, and have never been detected in laboratory strains. In their occurrence in wild mice, transmission seems to be primarily maternal, with infection by transuterine or milk-borne routes; genetic transmission has not yet been clearly established. However, the complete set of viral genome sequences is present in the cellular DNA of all mice, both feral and laboratory; and, again, the subset of viral sequences that corresponds to type-specific virion components is present in multiple copies, differing in number in the inbred strains. It seems quite possible that the amphotropic genomes are not present in the chromosomal DNA as complete linear copies, but they may require genetic recombination between products of separated loci before virus of this type is formed.
Indeed, there is much mounting evidence that new viruses are being generated constantly within the lifetime of high-virus mice. A new class of viruses called MCF (for "mink cell focus inducing") strains, since they induce foci of cell damage or transformation in a tissue culture line of mink lung cells, emerges in high lymphoma strains prior to the time of onset of lymphomas. These viruses represent unique genetic recombinants between ecotropic virus and some as yet unidentified class of endogenous MuLV sequences, perhaps representing xenotropic genomes. Each of these viruses appears to be a unique recombinant in the envelope gene region and as such does not represent the product of a single chromosomal locus, but is rather the outcome of an interaction between endogenous viruses coded by two (or more?) separate loci.
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