in Japanese Wild N 8-Tropic Friend

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1 Genetic Resistance molossinus) to an in Japanese Wild N 8-Tropic Friend Mice (Mus musculus Murine Leukemia Virus 1. Takeshl Odaka,3 Hldetoshl Ikeda,3 Kazuo Morlwakl,4 Aklo Matsuzawa,3 Mlchlru Mizuno, 5 and Kyo)1 Kondo 6. 7 ABSTRACT-Wild mice (Sk, Hz-VI, Hz-IV, Om, Mol.A, Fu, Te, and Sn) trapped In various areas of Japan were crossed with mice of Inbred strains (C57BL/6, C57L, BALB/c, and C57BL/10), and their progeny were Infected with NB-troplc Friend murine leukemia virus. Ten days after Infection, the spleens were weighed, examined for macroscopic focal lesions, and assayed for Infectious virus by the XC test. Genetic analysis Indicated that 4 of 8 mice tested had a dominant gene that suppresses the virus replication; the gene resembles the Fv-4' allele. No mice with the Fv-' allele were found.-j Natl Cancer Inst 61: , Several MuLV have been isolated from wild mice and characterized (1-7). In wild mice in some areas of California, amphotropic and N-tropic MuLV are maternally transmitted and the leukemia of nonthymic origin occurs. This finding contrasts with that of the laboratory strain AKR mice, which have a high incidence of leukemia and in which N-tropic MuLV is genetically transmitted and the leukemia is of the thymic type (4, 5, 8). Therefore, the mechanism of leukemogenesis may differ between laboratory mice and wild mice. In laboratory mice, several host genes affect leukemogenesis remarkably (9-14), but we know little about such genes in wild mice (15). F-MuLV replicates well and rapidly induces spleen foci and splenomegaly in adult mice (16). With this virus, three host genes were detected. The Fv-l locus (10, 17) and Fv-4 locus (14, 18) affect propagation of not only F-MuLV but also various other strains of MuLV. At both loci, the resistance alleles are dominant. The most conspicuous difference between these two loci is that the replication of NB-tropic F-MuLV is suppressed by the Fv-4 locus but not by the Fv-l locus. However, the Fv- locus scarcely affects the replication of MuLV. The locus controls the susceptibility of mice to spleen focus formation and splenomegaly induction by F MuL V. The susceptibility allele Fv- s is dominant (9, 19). However, the Fv- s phenotype is partially or completely masked by the resistance allele (Fv-4r) at the Fv-4 locus (0). We report here a dominant gene that suppresses replication of NB-tropic F-MuLV in japanese wild mice (Mus musculus molossinus). This gene is similar to the Fv-4 r allele that was found in G mice by Suzuki (14). The Fv-4 r allele of G mice suppresses the expression of endogenous AKR virus (Odaka T. Ikeda H:. Unpublished data). Therefore. the gene described here may have an important role in the natural occurrence and propagation of MuLV in the wild mice. MATERIALS AND METHODS Mice.-Most inbred strains and their hybrids were described in (0). The C57BL/IO strain is from the colony of K. Kondo. Genotypes and responses to NBtropic F-MuLV of these mice (0) are shown in table I (see also text-fig. I). G and DDD mice were included here to show the effects of Fv-4 r and Fv- s alleles. Wild mice were from various areas of japan: 8 3 (Sk, Hz-VI. Hz-IV) from Kyushu. 3 (Om, MoI.A, Fu) from Honshu, and (Te, Sn) from Hokkaido. Among them, 3 were trapped in the fields and 4 were born in the laboratory of K. Moriwaki from mothers that had been pregnant when captured. Wild mice Mol.A were in the 16th-d generation of brother X sister mating conducted by K. Kondo. Virus.-NB-tropic F-MuLV that had been serially passaged 4 times in BALB/c mice was used. The methods of preparation and storage were described in (19). Mice of both sexes, mostly at 4 weeks of age, were inoculated ip with 0. ml of 10% spleen extract (.5XI05 PFU as assayed by the XC test on C-18 cells and median infective dose as assayed by splenomegaly induction in susceptible ddy mice) and killed 10 days later. The spleens were weighed and examined for spleen foci. The virus in the individual spleens was extracted and assayed by the XC test on C-18 cells as described in (19, 1). Titers were expressed as PFUI spleen. RESULTS Wild mice were crossed with inbred mice. The progeny were challenged with NB-tropic F-MuLV and killed on day 10 of infection for the assay of virus in the spleens. According to the responses of inbred mice (table I), we divided the infected mice into 3 groups: ABBREVf,\T10NS lise[): F-MuLV = Friend murine leukemia virus; MuLV=murine leukemia virus(es); PFU=plaque forming unit(s). I Received February ; accepted june Supported by a Grant-in-Aid for Cancer Research from the Ministry of Education. Science. and Culture. japan. 3 Institute of Medical Science. University of Tokyo. P.O. Takanawa. Tokyo \08. japan. National Institute of Genetics. Mishima 41 I. japan. 5 Shizuoka Agricultural Cooperative Association for Laboratory Animals. Hamamatsu 435. japan. 6 Laboratory of Animal Genetics. Faculty of Agriculture. Nagoya University. Nagoya 464. japan. 7 We thank Miss Michiko Yamashita for excellent technical assistance. 8 The nomenclature for the wild mice was established by Dr. Moriwaki. VOL. 61. NO.5. NOVEMBER j NATL CANCER INST

2 130 Odaka, Ikeda, Moriwakl, et al TABLE 1.-Virus titer and splenomegaly in mice with various genotypes 10 days after infection of NB-tropic F-MuLV Mice" Genotypeb Virus titer' Splenomegalyd G (1) Fv-4" Fv-" Low No C57BL/6 () Fv-4" Fv-" High No C57BL/IO Fv-4" Fv-" High No C57L (3) Fv-4 " Fv-" High No BALB/c (4) Fv-4" Fv-" High Yes DDD (5) Fv-4" Fv-" High Yes (G X C57BL/6)F. (6) Fv-4"Fv-" Low No (G X C57L)F. (7) Fv-4"Fv-" Low No (G X DDD)F. (8) Fv-4" Fv-" Low No " Numbers in parentheses correspond to those in text-fig. 1. b Fv-4' and Fv-' are dominant genes (9, 14). The expression of Fv-' is masked by Fv-4' (0)., Low: <5,000 PFU/spleen; high: ':5,000 PFU/spleen. d No: <0.5 g (Fv-4" Fv-'- mice may develop focal lesions on spleen surface); yes: ':0.5 g. producers of high levels of virus (;:::5,000 PFU/spleen) with splenomegaly (;:::0.5 g), producers of high levels of virus (;:::5,000 PFU/spleen) without splenomegaly «0.5 g), or producers of low levels of virus «5,000 PFU/ spleen) without splenomegaly «0.5 g). From the responses of the infected progeny, the genotypes of the wild mice were deduced. Inasmuch as the crosses were done for other purposes, the inbred partner strains varied. Therefore, the wild mice will be described individually. The Te Mouse All 9 (C57BL/6 X Te)F\ mice developed splenomegaly and had high titers of virus in their spleens (text-fig. A). These responses corresponded to those of Fv-4 ss Fv-s- mice (text-fig. I). Inasmuch as C57BL/6 mice have the Fv-4 ss Fv-" genotype, the Te mouse was considered to be of the Fv-4 ss Fv- ss genotype. If the Te mouse had the Fv-4' allele, low virus producers without splenomegaly should have appeared in the F\ generation. Furthermore, if the Te mouse had the Fv-4 ss Fv-rs genotype, half the F \ generation would have been high virus producers without splenomegaly. No such mice were detected. The Om Mouse Similar results were obtained with 1 (C57BL/6 X Om)F\ mice (data not shown). Therefore, the Om mouse was considered to have the Fv-4 ss Fv- ss genotype. The Sk Mouse Eleven (C57BL/6 X Sk)F\ mice showed responses intermediate between those of (C57BL/6 X Te)F\ and (C57L X Hz-VI)F\ mice (text-fig. B). Virus titers were not as high as those in (C57BL/6 X Te)F\ mice, and the spleen weights- varied from 0.13 to 0.77 g. Focal lesions were clearly observed on the surfaces of all spleens. The lower virus titers and the spleen foci 7 6 "0 g. 5 c a: is. III ::; u. 4 a Spleen weight (g) TEXT-Fl(aIRE I.-Weights and virus contents of the spleens of mice infected with NB-tropic F-MuLV 10 days previously. Numbers represent resuits of individual mice of laboratory strains or their F. hybrids described in table I. I. G;. C57BL/6; 3. C57L; 4, BALB/c; 5, DOD; 6, (G X C57BLl6)F.; 7. (G X C57L)F.; 8. (G X DDD)F G.6,6 7.7,1,7 suggested the involvement of Fv-4' and Fv- s alleles, respectively, of SK origin. To confirm the genotype of the Sk mouse, we backcrossed 10 uninfected (C57BL/6 X Sk)F\ mice with C57BL/6 mice and tested the progeny for their susceptibility to F-MuLV (text-fig. D). Three types of mice were observed: low virus producers without splenomegaly, high virus producers without splenomegaly, and high virus producers with splenomegaly. The distinction among these three types was clear. Table shows the segregation in individual families. Except for #IA, all (C57BL/6 X Sk)F\ mice yielded both low virus producers and high virus producers, and a portion of high virus producers developed splenomegaly. In families of #A, B, and 6, no mice developed splenomegaly, but clear focal lesions were observed on the surfaces of some spleens. Therefore, all the (C57BL/6 X Sk)F\ mice, except for the #IA mouse, were considered to have the Fv-4rs Fv-rs genotype. Theoretically, the cross of mice of genotype Fv-4 rs Fv- rs with the C57BL/6 strain should yield equal numbers of mice of four genotypes: Fv-4 rs Fv-", Fv-4 rs Fv- rs, Fv-4 ss Fv-", and Fv-4ssFv-rs. Because the Fv-4' allele masks the Fv-s phenotype, three phenotypes of mice are expected in the ratio : I: l. These phenotypes are: low 4 VOL. 61. NO.5, NOVEMBER 1978

3 ~." ~ it4.!!.. II) :; U. Il. A B o o 00 c (C57BL/6 X To)F, ICS7BLl6 X Sk)f, o _.... _. ~... E F ICS7BL/6 x SkI X C57BL/6 IC578LJ10 X MoI.A) XCS7BL/l0 (C57L X Hz-VI)F, IF" X BALB/c)F Spleen weight Ig) TEXT FlGllRE.-Weights and virus contents of the spleens of hybrid mice infected 10 days previously. virus producers without splenomegaly (Fv-4 rs Fv-r-), high virus producers without splenomegaly (Fv-4 SS Fv-"), and high virus producers with splenomegaly (Fv-4 ss Fv- rs ). These three phenotypes were observed in a ratio of 37: 17: 15 (except for the #IA family) as predicted (0.7<P<0.8). Inasmuch as we could not explain the appearance of the #IA mouse, we assigned the Fv-4r-Fv-s- genotype to the Sk mouse. The Hz-VI Mouse In 7 (C57L X Hz-VI)FI mice, virus production was strongly suppressed (text-fig. C). This suppression suggested an involvement of the Fv-4' allele ~f Hz-VI origin. Four uninfected (C57L X Hz-VI)Fl mice were backcrossed with C57L mice, and the progeny were challenged with the virus. Three types of mice appeared: 30 low virus producers without splenomegaly, 3 high virus producers without splenomegaly, and 16 high virus producers with splenomegaly. The last type of mice must have acquired the Fv- s allele from the Hz-VI mouse. Although the segregation ratio deviated from a : I: I ratio (P<O.OI), the appearance of the three types suggested the genetic similarity between Sk and Hz-VI mice. To determine the genotype segregation in the first Genetic Resistance in Japanese Wild Mice to F-MuLV backcross generation, we again crossed 9 uninfected mice in this generation with C57L or C57BL/6 mice, and the susceptibility of the resulting generation was determined (table ). The data can be explained by the involvement of both Fv-4' and Fv- s alleles of Hz-VI origin. If the Hz-VI mouse had the Fv-4" genotype, mice with Fv-4 rs and Fv-4 ss genotypes were expected to appear in the first backcross generation with equal frequency. Six mice from the first backcross generation (#7A, 8B, 8C, 9, 11 and 1) yielded both low virus producers and high virus producers with ratios compatible with the I: I expectation. Therefore, their genotype would be Fv-4rs. The remaining 3 mice from the first backcross generation (#7B, 8A, and 10) yielded high virus producers only. These 3 mice might be of genotype Fv-4ss. Together with the findings for the infected (C57L X Hz-VI)Fl mice, the Hz-VI mouse was considered homozygous for the Fv-4' allele. Not enough data were available to confirm the Fv- s homozygosity. The Hz-IV Mouse The virus production and splenomegaly induction in 8 (C57BL/6 X Hz-IV)Fl mice were more strongly suppressed than in (C57BL/6 X Sk)Fl mice, but foci were observed on the spleen surfaces in 4 of 8 mice. These results indicated the Fv-4"Fv- s - genotype for the Hz-IV mouse. Inasmuch as the Hz-IV mouse was from the same area as the Hz-VI mouse, the Hz-IV mouse was not studied further. The Sn Mouse Of the (C57L X Sn)Fl mice, I mouse was a low virus producer without splenomegaly and 4 were high virus producers with splenomegaly. Six uninfected (C57L X Sn)Fl mice were backcrossed with C57L mice. Of these I produced 3 high virus producers without splenomegaly and 4 high virus producers with splenomegaly. The remaining 5 mice had the three types of progeny: 1 low virus producers without splenomegaly, 0 high virus producers without splenomegaly, and 30 high virus producers with splenomegaly. However, this ratio was not compatible (P<O.Ol) with the : 1: I segregation that was expected from the cross of mice of the genotype Fv-4 rs Fv- rs with C57L mice. Five (C57L X Sn) X C57L mice derived from I (C57L X Sn)Fl mouse that had produced the three types of progeny were again backcrossed with C57L mice. Thus far, 5 mice in the second backcross generation were tested. Except for I unhealthy mouse, all mice were high virus producers. Thus the resistance factor(s) seemed to have diluted out upon backcrossing. The Sn mouse was considered to have the Fv- ss genotype, but whether the mouse had the Fv-4' allele was not clear. The Fu Mouse The test of the Fu mouse was started with the (Fu X BALB/c)F generation. In the tests of the Te VOL. 61, NO.5, NOVEMBER 1978

4 1304 Odaka, Ikeda, Morlwakl, et al TABLE.-Progeny testing Generation of mice Mouse No. No. of progeny with:" Genes derived from wild mice Low virus production High virus production High virus production without splenomegaly without splenomegaly with splenomegaly Fv-4 Fv- b (C57BL/6 x Sk)F. la 0' 0 14 s IB 1 r s IC r s A r (s) B 7 0 r (s) C 3 r s r r s r r (s) (C57L x Hz-VI)F.xC57L 7A d 1 0 r 7B 0 3 s 8A 0 1 s 8B r 8C r r s r r s (Fu X BALB/c)F 1 3' 0 6 r 0 r r r 7' r " Low virus production: <5,000 PFU/spleen; high virus production: ~5,OOO PFU/spleen. Without splenomegaly: <0.5 g; with srlenomegaly: ~0.5 g. (s)=deduced from focus formation on the surfaces of spleens of some low virus producers without splenomegaly., Numbers of (C57BL/6 x Sk)F. x C57BL/6 mice. d Numbers of [(C57L x Hz-VI)F. x C57L] x C57L (or C57BL/6) mice., Numbers of (Fu X BALB/c)F X BALB/c mice., One offspring had a spleen that weighed l.58 g, but it contained 13 PFU. Whether this is due to an error of recording is not clear. The mouse was omitted from the tabulation. and Sn mice, inbred mice with the Fv-" genotype were crossed with the wild mice, whereas here BALB/c mice with the Fv-ss genotype were used. In the F generation, 18 mice were low virus producers without splenomegaly and 4 were high virus producers with splenomegaly (text-fig. F). One can explain the data by postulating that the Fu mouse has the genotype Fv-4"Fv- ss ; consequently, all the (Fu X BALB/c)F mice have the Fv-ss genotype, and the resistance to splenomegaly induction is determined by a single dominant gene, Fv-4'. The observed ratio was compatible with a 3: 1 expectation (0.30<P<0.50). Six uninfected (Fu X BALB/c)F mice were crossed with BALB/c mice, and the progeny were tested for the susceptibility to F-MuLV (table ). Of 6 (Fu X BALB/c)F mice, 5 (#1,, 4, 6, and 7) had the Fv-4 rs genotype and 1 (#5) had the Fv-4 ss genotype. If the Fu mouse has the Fv-4" genotype, then the foilowing genotypes are expected in this generation: 1 Fv-4", Fv-4 rs, and 1 Fv-4ss. Although no mice with the Fv-4" genotype were found, the observed results were still compatible with the expectation (0.0<P<0.30). From the above results, the Fu mouse was considered to have the Fv-4"Fv- ss genotype. DISCUSSION Wild mice were crossed with inbred mice and the progeny were infected with a large amount of NBtropic F-MuLV. Ten days later the spleens were weighed, examined for focal lesions on the surfaces, and assayed for infectious virus by the XC test. Progeny of wild mice may have had the endogenous MuL V (1,, 8) that can be detected by our assay system, but we did not test this possibility. However, if the endogenous virus does not interfere with the replication of exogenous F-MuLV to decrease its yield below the level of 5,000 PFU/spleen, the presence of the endogenous virus will not seriously affect our conclusion. A portion of the progeny mice strongly suppressed the replication of NB-tropic F-MuL V. The resistance is not due to the Fv-1 locus, because NB-tropic MuLV replicate well in cells with any Fv-1 genotype (17). The only known gene that can suppress NB-tropic MuL V is the Fv-4' allele (14, 18). The Fv- locus must be also taken into consideration when the resistance to F MuL V is assessed by spleen focus formation and splenomegaly induction (9, 19). Therefore, we at- VOL. 61, NO.5, NOVEMBER 1978

5 Genetic Resistance in Japanese Wild Mice to F-MuLV 1305 tempted to explain the resistance in terms of Fv-4 and Fv-. In this study, the crosses of G mice with inbred mice (0) served as a model, because the G strain is the sole laboratory strain with the Fv-4' allele. In mice carrying the Fv-4' allele, the expression of the Fv- s allele is masked, because spleen cells do not proliferate in the absence of virus replication. This epistatic effect of the Fv-4' allele allows assortment of laboratory mice into three types: low virus producers without splenomegaly, high virus producers without splenomegaly, and high virus producers with splenomegaly (table I, text-fig. I). The progeny of wild mice could be assorted in a similar way (text-fig. ). The nature of the resistance observed in the progeny of the Sn mouse-(c57l X Sn)F.. (C57L X Sn)F. X C57L, and [(C57L X Sn)F. X C57L] X C57L-is not clear. I) The number of low virus producers without splenomegaly in the first backcross generation is far smaller than expected from the Fv-4Fv- genotype model. ) The resistance factor(s) seemed to dilute out upon advanced backcrossing. 3) Many progeny of the Sn mouse were not healthy, and many died before the end of observation. 4) We often observed unusually small XC plaques. These findings suggest nongenetic interfering factor(s). Whether this factor was viral was not determined. Transmission of the nongenetic interfering factor by milk is unlikely because the Sn mouse was a male. Among (C57BL/6 X Sk)FI mice, a female (#IA) was exceptional (table ). All of her 14 offspring were high virus producers with splenomegaly. The #IA female may be a mutant or we may have observed a rare segregation in her progeny. The remaining (C57BL/6 X Sk)FI mice that were progeny tested gave data that indicated the Fv-4"Fv- ss genotype for the Sk mouse. If this genotype is true, the #IA mouse rr""st have the Fv-4 rs Fv- rs genotype and should yield progeny with a ratio of low virus producers without splenomegaly, I high virus producer without splenomegaly, and 1 high virus producer with splenomegaly. The obtained results deviated remarkably from those expected. Except for the above-mentioned instances, the resistance observed in the progeny of wild mice could be explained by the Fv-4Fv- model. By repeating backcrosses, we are now introducing the resistance gene of Hz-VI and Fu mice into the genetic background of inbred strains. In these backcrossings, we can clearly observe the effect of a single dominant gene. The resistance gene is similar to the Fv-4' allele of G mice in that the resistance is dominant and the replication of NB-tropic F-MuLV is suppressed. However, both loci are not yet mapped on chromosomes. The identity of the resistance gene of wild mice with the Fv-4' allele or other known genes therefore awaits further confirmation. The present study demonstrated the Fv-4' allele or a similar gene in 4 of 8 wild mice tested; 3 (Sk, Hz-VI, Hz-IV) of 3 from Kyushu and I (Fu) of 3 from Honshu. Thus this gene seems to be common in wild mice, at least in areas west of Tokyo. In this respect, observations on G mice are interesting. We searched for infectious ecotropic MuLV in normal G mice 1-14 months of age. No mice were positive. In (G X AKR)F. mice, the expression of the AKR virus was strongly suppressed and no leukemias developed during 1 year of observation (Odaka T, Ikeda H: Unpublished data). The gene described here must have an important role in the natural occurrence and propagation of MuLV in wild mice. This consideration may provide an explanation for the low expression of MuLV in some populations of wild mice (8, ). REFERENCES (I) BWIGIAN HG. 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6 1306 Odaka, Ikeda, Morlwakl, et al in mice: Masking of Fv- phenotype by an epistatic gene. Fv-4'. Jpn ] Exp Med 47: (1) ROWE WP. PUGH WE. HARTLEY ]W: Plaque assay techniques for murine leukemia viruses. Virology 4: II ; () AN[)ERVONT HB. DUNN TB: Occurrence of tumors in wild house mice. J Natl Cancer Inst 8: VOL. 61. NO.5. NOVEMBER 1978