By in situ hybridization analyses, we have assessed the presence of ecotropic MuLV RNA in the brains of C58 mice as a function of age. Only a few ecotropic MuLV-positive cells were observed in weanling mice, but the number of positive cells in the brain increased progressively with increasing age of the mice. Throughout the lives of the mice, the ecotropic MuLV RNA-positive cells were primarily located in well-defined white-matter tracts of the brain (commissura anterior, corpus callosum, fimbria hippocampi, optical tract, and striatum) and of the spinal cord. Cells of the subventricular zone also expressed ecotropic MuLV RNA, and in older mice a small number of positive cells were present in the grey matter. Infection of endogenous ecotropic MuLV provirus-less CE/J mice in utero with ecotropic MuLV clone AKR-623 resulted in the extensive infection of brain cells. The regional distribution of ecotropic MuLV RNA-containing cells was the same as observed in the brains of C58 mice, in which cells became infected by endogenously activated virus, but the number of positive cells was higher.
The chromosomal DNA of all vertebrate species investi-gated contains numerous endogenous retroviral proviruses and proviruslike elements (6). The origin of these endogenous se-quences is uncertain, and little is known about their potential effects on the normal development and physiology of their hosts or their causation of disease. The human genome carries over 1,000 retroviruslike elements (6, 12, 19, 29). Mouse chro-mosomes also carry a diverse group of retroviruses (6, 27). Four groups of endogenous murine leukemia viruses (MuLVs) are found in mice: ecotropic, xenotropic, polytropic, and mod-ified polytropic MuLVs. Some of these proviruses can become transcriptionally activated, resulting in the formation of in-fectious virions that can play a critical role in pathogenesis (27). For example, expression and replication of endogenous N-tropic, ecotropic MuLV in mouse strains that are
permis-sive for their efficient replication (Fv-1n/n[16]), such as AKR
and C58, almost invariably result in the development of onco-genic recombinant viruses (mink cell focus-forming viruses) which induce thymic tumor formation (10, 28). Additionally, replication of endogenous murine retroviruses may have other detrimental effects on their host, including proviral insertional mutagenesis or aberrant activation of cellular genes (27). Fi-nally, endogenous N-tropic, ecotropic MuLVs are associated with a unique paralytic disease of AKR and C58 mice, age-dependent poliomyelitis (23, 24). In these mice, endogenous N-tropic, ecotropic MuLV proviruses are activated during em-bryogenesis and infect cells in the developing spinal cord (1, 2, 26). Expression of ecotropic MuLV in spinal cord glial cells renders anterior horn neurons susceptible to cytocidal infec-tion by an entirely different virus, lactate dehydrogenase-ele-vating virus (LDV) (1, 2, 7, 24), via an unknown mechanism.
Destruction of the motor neurons by LDV infection results in paralysis.
Paralytic disease is also caused in mice by infection of fetuses and newborns with the neuropathogenic ecotropic MuLV Cas-Br-E (17, 25) or neuropathogenic variants of Friend and Molo-ney MuLVs (30). Anterior horn neuron destruction in this case seems mediated indirectly by infection of glial cells with the MuLVs (11). Infection of glial cells by these viruses has been documented to occur in both the spinal cord and the brain. As the neuropathogenic ecotropic MuLVs and the en-dogenous ecotropic MuLVs of C58 and AKR mice are closely related, infect some of the same cell types in the mouse spinal cord, and are involved in similar neuropathogenic diseases, it seemed likely that ecotropic MuLVs generated from endoge-nous proviruses also infect cells in the brains of C58 and AKR mice and perhaps could affect central nervous system (CNS) functions or give rise to variants that cause CNS disease in old age.
Accordingly, we examined in detail, by in situ hybridization, the expression of ecotropic MuLV proviruses in the brains of C58 and DBA2/J mice as well as CE/J mice that were infected in utero with ecotropic MuLV. Our results reveal a highly specific pattern of ecotropic MuLV provirus expression in cer-tain regions of the brains of these mice which increases greatly with increase in the age of C58 mice.
C58/M mice of various ages, bred in the animal facility of the Department of Microbiology, University of Minnesota, were anesthetized and perfused with phosphate-buffered saline. Brains were removed from a minimum of three mice of each age, fixed by immersion in neutral formalin for 4 h, and then
placed in ethanol at 48C for at least 4 h. The tissues were
embedded in Amerffin (American Scientific Products,
Minne-apolis, Minn.). Sections 8mm thick were cut and floated on a
drop of 3% (vol/vol) Elmer’s white glue on a slide pretreated with Denhardt’s medium and then subjected to acetylation. The slides were dried, deparaffinized, pretreated, and
hybrid-* Corresponding author. Mailing address: Department of Microbi-ology, University of Minnesota, Box 196 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455.
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ized with DNA probes as described by Blum et al. (4). The MuLV-specific probe was a 168-bp SmaI restriction fragment representing part of the env gene. It was prepared by SmaI digestion of the infectious ecotropic MuLV proviral clone AKR-623, provided by Doug Lowy (22); separation of the fragment by gel electrophoresis; and subcloning into pBSII
KS(1) (Stratagene, La Jolla, Calif.). The probe is specific
for ecotropic MuLVs (2, 7). It does not hybridize to RNA from Mus dunni cells infected with xenotropic or polytropic MuLVs.
The DNA was radiolabeled by random priming by using the Random Priming Labeling Kit from Boehringer Mannheim (Indianapolis, Ind.) according to the procedure recommended
by the manufacturer with35S-dATP and35S-dCTP (Amersham
Corp., Arlington Heights, Ill.). The probe was phenol-chloro-form-isoamyl alcohol extracted before use in hybridizations.
After hybridization, the slides were extensively washed (4), autoradiographed, stained with Mayer’s hematoxylin and 0.5% (vol/vol) eosin Y in ethanol (Sigma, St. Louis, Mo.), and ex-amined with a Leitz microscope. A cell was identified as pos-itive if a focus of grains deposited over a cell body was detected in a sequential tissue section. Magnifications given in the figure legends pertain to those of the microscope. Photographic en-largements vary but are the same for all panels in a single figure.
Rowe and Pincus (26), using infectivity assays, demonstrated that activation of endogenous germ line ecotropic MuLVs in AKR mice occurs during embryogenesis, probably in rare cells, and that progeny virus then spreads to other tissues, including those of the CNS. The same is probably the case for mice of all highly leukemic strains, including C58 mice. Previous Northern (RNA) hybridization analyses of total tissue RNA with a probe specific for the env gene of ecotropic MuLV have shown that full-length 8.2-kb MuLV and the 3-kb env mRNA are present in very small amounts in the brains and spinal cords of C58/M mice as early as 1 month after birth but that the levels pro-gressively increase with increased age of the mice (2). These results have been confirmed in the present study (data not shown) and also apply to AKXD-16 mice (1). Furthermore, other results suggested that the ecotropic MuLV RNA is tran-scribed from proviruses that have been acquired by infection during embryogenesis (2).
Results from in situ hybridization show that the ecotropic MuLV RNA-containing cells in the CNS of C58 mice were predominantly located in white-matter tracts both in the spi-nal cord (reference 2 and data not shown) and, as shown here, in the brain. For example, Fig. 1 shows a large number of ecotropic MuLV RNA-positive cells in the commissura anterior of a 12-month-old C58 mouse. The commissura ante-rior functions as one of the interhemispheric commissures that connect the commissural fibers of the anterior olfactory nucleus to the same nucleus in the other hemisphere, as well as to the main and accessory olfactory bulbs (13, 18). In striking contrast to the situation for this white-matter tract, few, if any, positive cells were seen in the adjacent grey matter (basal ganglia; Fig. 1). As a further example, Fig. 2 illustrates commonly observed ecotropic MuLV RNA-ex-pressing cells in the large white-matter interhemispheric com-missure, the corpus callosum. The corpus callosum connects the neocortexes of the hemispheres of the brain (13, 18). Again, few, if any, ecotropic MuLV RNA-positive cells were found in the adjacent grey matter (neocortex and hippocam-pus). Ecotropic MuLV RNA-positive cells were also routinely found in other white-matter tracts, such as the fimbria hip-pocampi (see Fig. 3B) and the tractus opticus, and, less fre-quently, in the medullary layer of the cerebellum (data not shown).
Although the vast majority of ecotropic MuLV RNA-ex-pressing cells were found within white-matter tracts of the CNS, a few MuLV RNA-positive cells were found in the brain grey matter of old C58 mice. For example, a single positive cell was detected in the hippocampal dentate gyrus of an 8-month-old C58 mouse (data not shown). A focus of positive cells in the stria terminalis is presented in Fig. 3A. However, even these positive cells appeared to be juxtaposed with the white-matter striations of this brain region. Generally, no ecotropic MuLV RNA-positive cells were found in the deep grey matter of the neocortex or in the meninges (data not shown). Like-wise, cells of the choroid plexus and the specialized ependymal cells lining the lateral ventricle were devoid of signal, in stark contrast to cells found in the adjacent fimbria hippocampi, which were positive (Fig. 3B).
All hybridizations included negative controls. The ecotropic MuLV-specific probe did not hybridize to sections of CNS
FIG. 1. Distribution of ecotropic MuLV RNA-containing cells in the commissura anterior of the brain of a 12-month-old C58 mouse. The brain was fixed in formalin, embedded in paraffin, and sectioned. Eight-micrometer-thick sections cut sagittally were hybridized with the35S-labeled ecotropic MuLV-specific probe
(168-bp SmaI fragment). The sections were coated with photographic emulsion, exposed, and then stained with hematoxylin and eosin. Positive cells are indicated by arrows. (A) Light-field exposure; (B) dark-field exposure. ca, commissura anterior; BG, basal ganglia. Magnification for both panels,3200 (magnification pertains to the microscope; see text).
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tissue from C58 mice that had been treated with RNases A and
T1(1, 2) or to sections of CNS tissues from CE/J mice, which
are devoid of endogenous ecotropic MuLVs (reference 2 and data not shown). An unrelated cDNA probe, LDV-specific cDNA 4-55, also did not hybridize to the sections of brain tissue from C58 mice (data not shown).
Of special interest was the repeated observation that eco-tropic MuLV RNA-expressing glial cells in white-matter tracts were generally located in foci (Fig. 2 and 3A). Why positive cells are often found in foci is not entirely clear, as white-matter tracts are uniformly composed of the same cell types. The foci of expressing cells may represent the progeny of precursor cells infected with ecotropic MuLV that have pop-ulated the same region of white matter. Alternatively, an in-tracellular trans-acting factor(s) may induce the expression of newly acquired ecotropic MuLV proviruses in specific regions of white matter. The foci probably did not result from contin-uous horizontal spread of the ecotropic MuLV infection, since MuLV provirus integration occurs only in dividing cells (20) and CNS cells in adult mice are thought generally to be mi-totically inactive.
The distribution of ecotropic MuLV RNA-containing cells in brains of 1-month-old C58 mice was comparable to that described for the 8- to 12-month-old mice. However, there were fewer positive cells in the brains of the young mice than there were in the brains of the older mice (data not shown),
as was predicted from the Northern hybridization analyses (1, 2).
Recombinant inbred AKXD-16 mice (obtained from Jack-son Laboratories, Bar Harbor, Maine) carrying the replication-competent endogenous N-tropic MuLV provirus emv-11 of AKR mice (14, 15) demonstrated patterns of ecotropic MuLV expression in the brain similar to those of C58 mice of the same age (data not shown). The distribution of positive cells in white-matter tracts was very similar to that reported for mice infected as fetuses or newborns with neuropathogenic strains of ecotropic MuLV, such as Cas-Br-E (11).
[image:3.612.60.558.73.410.2]Another area where large numbers of ecotropic MuLV RNA-containing cells were consistently found in all C58 or AKXD-16 mice was the subventricular zone (SVZ). Panels A and B of Fig. 4 document expression of ecotropic MuLV RNA in cells in the SVZ immediately adjacent to the lateral ventricle in an 8-month-old C58 mouse. Additionally, many glial cells within the adjacent corpus callosum were also expressing ret-roviral RNA. The presence of ecotropic MuLV RNA-positive cells in the SVZ is of special interest, as the cells populating this region are glial cell precursors and are mitotically active throughout life (13) and thus might be susceptible to infection by ecotropic MuLV (20) in adult mice. Interestingly, we also found large numbers of ecotropic MuLV RNA-expressing cells in the SVZs of 12-month-old DBA/2J mice (Fig. 4C and D). Their density and distribution in the SVZ were similar to those
FIG. 2. Foci of ecotropic MuLV RNA-positive cells (arrows) distributed along the corpus callosum of the sagittally cut brain of a 12-month-old C58 mouse. In situ hybridization was conducted as described in the legend to Fig. 1. (A) Light-field exposure; (B) dark-field exposure. CC, corpus callosum; H, hippocampus; N, neocortex; CPu, caudate putamen; LV, lateral ventricle. Magnification for both panels,3100 (magnification pertains to the microscope; see text).
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of the ecotropic MuLV RNA-containing cells in SVZs of old C58 mice (Fig. 4). In contrast, no positive cells were detected outside the SVZs of old DBA/2J mice, either in white-matter or grey-matter tracts, including the corpus callosum and fim-bria hippocampi (data not shown), which in old C58 mice normally contain large numbers of ecotropic MuLV RNA-positive cells. DBA/2J mice carry only a replication-defective ecotropic MuLV provirus (emv-3) (8, 9). However, with in-creasing age of the mice replication-competent ecotropic MuLVs become generated as a result of back mutation or recombination with other endogenous proviruses (3). These recombinant ecotropic MuLVs replicate primarily in cells of lymphoid tissues (9). Our results suggest that the recombinant virus may also infect mitotically active cells in the SVZ. Fur-thermore, these results indicate that the ecotropic MuLV RNA-containing cells in the SVZs of adult C58 mice are not the source of positive cells found elsewhere in the adult brain.
The ecotropic MuLV RNA-positive cells in the CNS of C58 and AKXD-16 mice have not been identified as to cell type. However, the localization of the positive cells in white-matter tracts suggests that cell types exclusively populating the white matter, that is, glial cells (5, 13, 18), express ecotropic MuLV RNA.
We previously determined that expression of ecotropic MuLV in spinal cord glial cells of C58 and AKR mice was due to active infection with ecotropic MuLV, probably during em-bryogenesis (2). Thus, it seemed likely that, with the exception of expression in the SVZ, the expression we observed in the brains of C58 mice was also due to active infection limited to the period of embryonic or early postnatal development. In support of this hypothesis, in utero infection of CE/J mice with ecotropic MuLV resulted in expression of the MuLV in the CNS. CE/J mice were used in this study because they are devoid of any ecotropic MuLV proviruses (6). In a previous study, we found that two fetuses from two litters became in-fected when injected on embryonic day 8.5 (E8.5) with the
infectious ecotropic MuLV clone AKR-623 (2). At 7 months of age, the mice of both litters were sacrificed and their brains were analyzed by Northern or in situ hybridization for eco-tropic MuLV RNA expression. High-level expression was ob-served in the brains of the two ecotropic MuLV-infected mice but not in the brains of the uninfected littermates. Figure 5 shows representative brain sections of one infected CE/J mouse. As in C58 mice, most ecotropic MuLV RNA-contain-ing cells in the brain (and the spinal cord [2]) of this mouse were located in white-matter tracts. An especially high density of positive cells in the brain was present in the corpus callosum (Fig. 5A). Other white-matter tracts with many positive cells were the stria terminalis (data not shown) and the fimbria hippocampi (Fig. 5B). No positive cells were detected in the thalamic grey matter adjacent to the stria terminalis or in the deep grey matter of the neocortex (data not shown).
[image:4.612.62.554.73.308.2]Overall, the distribution of ecotropic MuLV RNA-positive cells in the in utero-infected CE/J brain was similar to that observed in C58 and AKXD-16 mice of the same age. How-ever, the density of positive cells in the CNS was considerably higher than that in the C58 mice in which the ecotropic MuLV in the CNS was derived from spontaneously activated germ line proviruses. In addition, ecotropic MuLV RNA-expressing cells were found in the hippocampal dentate gyrus (data not shown) and the internal granule cell layer of the cerebellum of the in utero-infected CE/J mouse (Fig. 5C), but not in the cerebella of C58 mice. This finding is of interest, as the neurons of the hippocampal dentate gyrus and the cerebellar internal granule cells are the last neurons to proliferate and differen-tiate within the CNS (13). They finish this process by postnatal day 20, whereas the generation of all other brain neurons is completed by E17 (13). Figure 5C shows that Purkinje neurons (see arrows), which are generated by E17, did not contain ecotropic MuLV RNA in the in utero-infected mice. Thus, while it appears that some CNS neurons are infected in in utero-infected CE/J mice, the infection seems to be limited to the late-differentiating neurons.
FIG. 3. Distribution of ecotropic MuLV RNA-containing cells (arrows) in grey matter of the brain of an 8-month-old C58 mouse. In situ hybridization was conducted as described in the legend to Fig. 1. (A) st, stria terminalis. (B) fi, fimbria hippocampi; E, ependymal cells; LV, lateral ventricle; cp, choroid plexus. Magnification for both panels,3400 (magnification pertains to the microscope; see text).
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The higher density of ecotropic MuLV-expressing cells in the CNS of in utero-infected CE/J mice than in the CNS of C58 mice could reflect a difference in the virus concentration at the time during embryogenesis when maximum infection of CNS cells occurs. The study by Rowe and Pincus (26) indicated that the concentration of infectious MuLV in AKR fetuses before
E17 was below detectable levels (,2 focus-forming units/0.1 g
of tissue), whereas we injected the fetuses of CE/J mice with about 400 focus-forming units at E8.5 (2). Thus, infection of the fetuses at E8.5 may have resulted in the earlier infection and of a greater number of cells with ecotropic MuLV than in C58 and AKR mice in which the ecotropic MuLV is generated by spontaneous activation of a germ line provirus. That acti-vation of germ line proviruses in C58 mouse fetuses generally occurs late during embryogenesis is suggested by the finding that neurons of the hippocampal dentate gyrus and cerebellar granule cells expressed ecotropic MuLV RNA in in utero-infected CE/J mice but not in C58 mice. However, infection of CNS cells with endogenously derived ecotropic MuLV seems to be rather restricted to certain times during development. In a previous study, no ecotropic MuLV RNA-containing cells
were detected in CE/J mice that were infected as newborns with AKR-623 MuLV (2).
[image:5.612.60.561.72.456.2]In conclusion, our results demonstrate that ecotropic MuLV RNA-containing cells in the CNS are located primarily in white-matter tracts both in the brains and spinal cords of C58 and AKXD-16 mice as well as of CE/J mice that have become infected as midgestation fetuses. The number of ecotropic MuLV RNA-containing cells increases progressively with the age of the mice, apparently because of activation of proviruses acquired during embryogenesis (2). The positive cells are found in distinctive patterns throughout the CNS and often in clusters. Since the ecotropic MuLV RNA-positive cells are found almost exclusively in white-matter tracts, they are glial cells, but further work is required to identify the type(s) of glial cells involved and to determine to what extent these cells produce ecotropic MuLV proteins and infectious virions. Re-gardless of the nature of these cells, it would be surprising if the massive expression of ecotropic MuLV in the CNS of old mice were not associated with some, perhaps subtle, effects on CNS functions. This possibility has not been investigated as yet. It has been reported that at least one type of endogenous
FIG. 4. Ecotropic MuLV RNA-positive cells in the SVZ of 8-month-old C58 (A and B) and 12-month-old DBA/2J (C and D) mice. In situ hybridization of the sagittally cut sections was conducted as described in the legend to Fig. 1. cc, corpus callosum; N, neocortex; CPu, caudate putamen. Panels B and D are dark-field exposures of panels A and C, respectively. Magnification for all panels,3200 (magnification pertains to the microscope; see text).
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FIG. 5. Distribution of ecotropic MuLV RNA-containing cells (arrows) in the brain of a 7-month-old CE/J mouse that had been infected in utero with the ecotropic MuLV clone AKR-623. In situ hybridization of brain sections was conducted as described in the legend to Fig. 1. (A) Corpus callosum. Magnification,3200. (B) Fimbria hippocampi. Magnification,3400. (C) Cerebellum in sagittally cut section. Purkinje cells are indicated by the arrows. ML, molecular layer; IGL, internal granule layer; ml, medullary layer. Magnification,3200. (All magnifications pertain to the microscope; see text.)
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mice predisposes mice to paralytic infection by lactate dehydrogenase-ele-vating virus. J. Virol. 69:308–319.
3. Bartman, T., D. M. Murasko, T. G. Sieck, A. Turturro, R. Hart, and K. J.
Blank.1994. A murine leukemia virus expressed in aged DBA/2 mice is derived by recombination of the emv-3 locus and another endogenous gag sequence. Virology 203:1–7.
4. Blum, H. E., A. T. Haase, and G. N. Vyas. 1984. Molecular pathogenesis of hepatitis B infection: simultaneous detection of viral DNA and antigen in paraffin embedded liver sections. Lancet ii:771–775.
5. Branks, P. L., and M. C. Wilson. 1986. Patterns of gene expression in the murine brain revealed by in situ hybridization of brain-specific mRNAs. Brain Res. 387:1–16.
6. Coffin, J. M. 1985. Endogenous retroviruses, p. 357–404. In R. Weiss, N. Teich, H. Varmus, and J. Coffin (ed.), RNA tumor viruses, vol. 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
7. Contag, C. H., and P. G. W. Plagemann. 1989. Age-dependent poliomyelitis of mice: expression of an endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons. J. Virol. 63:4362–4369.
8. Copeland, N. G., H. G. Bedigian, C. Y. Thomas, and N. A. Jenkins. 1984. DNAs of two molecularly cloned endogenous ecotropic proviruses are poorly infectious in DNA transfection assays. J. Virol. 49:437–444. 9. Copeland, N. G., N. A. Jenkins, B. Nexo, A. M. Schultz, A. Rein, T.
Mikkel-son, and P. Jorgenson.1988. Poorly expressed endogenous ecotropic provi-rus of DBA/2 mice encodes a mutant Pr65gagprotein that is not myristylated.
J. Virol. 62:479–487.
10. Gilbert, D. J., P. E. Neumann, B. A. Taylor, N. A. Jenkins, and N. G.
Copeland.1993. Susceptibility of AKXD recombinant inbred mouse strains to lymphoma. J. Virol. 67:2083–2090.
11. Gravel, C., D. G. Kay, and P. Jolicoeur. 1993. Identification of the infected target cell type in spongiform myeloencephalopathy induced by the neuro-tropic Cas-Br-E murine leukemia virus. J. Virol. 67:6648–6658.
20. Lewis, P. F., and M. Emerman. 1994. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J. Virol.
68:510–516.
21. Lindeskog, M., P. Medstrand, and J. Blomberg. 1993. Sequence variation of human endogenous retrovirus ERV9-related elements in an env region cor-responding to an immunosuppressive peptide: transcription in normal and neoplastic cells. J. Virol. 67:1122–1126.
22. Lowy, D. R., E. Rands, S. K. Chattopadhyay, C. F. Garon, and G. L. Hager. 1980. Molecular cloning of infectious, integrated murine leukemia virus DNA from infected mouse cells. Proc. Natl. Acad. Sci. USA 77:614–618. 23. Murphy, W. H., J. J. Mazur, and S. A. Fulton. 1987. Animal model for motor
neuron disease, p. 135–155. In W. M. H. Behan, P. O. Behan, and J. A. Aarli (ed.), Clinical neuroimmunology. Blackwell Scientific Publications, Oxford. 24. Plagemann, P. G. W. 1996. Lactate dehydrogenase-elevating virus and re-lated viruses, p. 1105–1120. In B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Virology, 3rd ed. Raven Press, New York.
25. Portis, J. L. 1990. Wild mouse retrovirus: pathogenesis. Curr. Top. Micro-biol. Immunol. 160:11–27.
26. Rowe, W. P., and T. Pincus. 1972. Quantitative studies of naturally occurring murine leukemia virus infection of AKR mice. J. Exp. Med. 135:429–436. 27. Stoye, J. P., and J. M. Coffin. 1987. The four classes of endogenous murine
leukemia virus: structural relationships and potential for recombination. J. Virol. 61:2659–2669.
28. Stoye, J. P., C. Moron, and J. M. Coffin. 1991. Virological events leading to spontaneous AKR thymoma. J. Virol. 65:1273–1285.
29. Wilkinson, D. A., N. L. Goodchild, T. M. Saxton, S. Wood, and D. L. Mager. 1993. Evidence for a functional subclass of the RTVL-H family of human endogenous retrovirus-like sequences. J. Virol. 67:2981–2989.
30. Wong, P. K. Y., and P. H. Yuen. 1992. Molecular basis of neurological disorders induced by a mutant, ts1, of Moloney murine leukemia virus, p. 161–197. In R. P. Roos (ed.), Molecular neurovirology: pathogenesis of viral CNS infections. The Humana Press Inc., Totowa, N.J.
