(2) NOTES VOL. 32, 1979 I Y .5 x E . gp701 30 40 50 60 70 80 Fraction number FIG. 1. Co-electrophoresis of uncloned WN1802B virus (0) with a clone from this stock (WN1802B CL Dl, 0). Electrophoresis was in 15% cylindrical acrylamide gels (13). A stacking gel of 4.5% acrylamide was used. Gels were fractionated into 2-mm portions in an Auto Gel Divider (Gilson) before scintillation counting. MuLV's were labeled in tissue culture with 3H- and "4C-labeled amino acid precursors and purified as previously described (23, 24). case, the B-tropic p30 appeared to migrate more slowly than the N-tropic p30, being comparable in this respect with the p30 phenotype of our uncloned WN1802B stock. The use of doublelabel counting with a WN1802N virus marker in cylindrical gels permitted routine quantitation of mobility differences, not only for p30, but also for other gag proteins and for gp7O (Table 1). In 11 clones prepared from the WN1802B stock, three distinct p30 mobility phenotypes were observed. The dominant p30 phenotype was similar to that reported by Schindler et al. (31). No differences were found in 16 clones prepared from the WN1802N stock. The p15 and gp7O proteins of all the B-tropic viral clones were distinguishable from their N-tropic counterparts as previously reported by Schindler et al. (31), the B-tropic p15 being faster and its gp7O being slower. The fact that p12 or plO mobility changes were not found within the set of Btropic clones appears to rule out an explanation of size changes in p30 based on a shift in the cleavage site between p30 and either p12 or plO in the processing of the gag translational product (4). The variations found in clones prepared from WN1802B, which has a long passage history in culture, prompted us to investigate other newly derived isolates of N- and B-tropic MuLV's from BALB/c mice. After isolation and initial characterization of the new clones in terms of tropism, protein patterns were compared by gel electrophoresis as before (Table 1). The dominant B-tropic phenotype differed markedly from that of clones from WN1802B, in terms of p30, p15, and p12 mobilities. Interestingly, however, one B-tropic clone displayed a p15, p12 phenotype identical to that of WN1802B, demonstrating that these features of p15 and p12 need not be a result of the long passage history of the latter virus. Two other B-tropic clones had additional alterations in p30 and p12 migrations. The new N-tropic isolates were all identical to each other and to WN1802N in their protein patterns, except that their gp7O proteins migrated more slowly than that of WN1802N. Hopkins and co-workers have found that, in several independent cases in which NB-tropic variants were selected from B-tropic BALB/c MuLV, a p30 mobility change occurred which was accompanied by an altered location of a single spot in the gag region of the RNA oligonucleotide maps of these viruses (10, 19). Since in our BALB/c isolates p30 mobility did not appear to be simply related to tropism, we asked whether some structural feature of p30 might Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest Fp15 x E C.) 351
(3) 352 NOTES J. VIROL. TABLE 1. Electrophoretic mobilities of BALB/c virus structural proteins Source WN1802B WN1802N Virus tropism gag phenotypeb env phenotypeb p15 p12 p30 plO gp7O p15(E) 8 2 B B B +1 +1 +1 0 0 0 +1 -1 -2 0 0 0 -1 -1 -1 0 0 0 16 N 0 0 0 0 0 0 7 1 1 1 5 B B B B N 0 +1 0 +1 0 -4 0 -4 +1 0 +2 +2 +1 -1 0 0 0 0 0 0 -1 (0)c -1 -1 -1 0 0 0 0 0 1 16-month-old BALB/c mouse Viruses were cloned by a modification of the microtiter technique of Stephenson et al. (36) as previously described (27). The prototype N- and B-tropic MuLV WN1802N and WN1802B were isolated from the spleen of a single 18-month-old BALB/c mouse by Hartley et al. (17). WN1802N and WN1802B viruses were obtained in 1972 from J. W. Hartley as pools 1898 and 1897, respectively. Clones were prepared directly from these pools. From the Poisson distribution of virus-positive wells in the replica microtiter XC plaque assays, the probability that virus clones were derived from single infectious centers was more than 99.5% (26). New clones of N- and Btropic MuLV were isolated from the spleen of a single 16-month-old BALB/c mouse obtained from our own mouse colony. N-tropic MuLV's were cloned directly from a 10% (wt/vol) spleen extract on SC-1 cells. Ntropism of virus clones was determined by differential growth on NIH Swiss mouse embryo (ME) cells and BALB/c ME. Enrichment of B-tropic MuLV present in the spleen extract was achieved as follows. Infectious MuLV (840 PFU/ml) was amplified by a single passage in SC-1 cells, followed by two serial cell-free passages in BALB/c-ME cells. Evidence for enrichment of B-ecotropic MuLV was the change in relative XC plaque titers of SC-1 C) (C = cell-free passage number), BALB/c-ME Q, and BALB/c-ME ( virus stocks on NIHME and BALB/c-ME cells (N/B ratios: 43, 3.5, 0.02, respectively). Virus was then cloned from the BALB/cME () virus stock. Fv-1 tropism analysis of 30 clones indicated that 26 were typically B-tropic (N/B < 0.005), 2 were N-tropic (N/B = 10 to 30), and 2 appeared NB-tropic (N/B 5). The probability that N- and B-tropic virus clones were derived from single infectious centers was greater than 99.9% from replica plating statistics. No xenotropic MuLV was initially present in the spleen extract as determined by infection of mink CCL64 cells and assay 19 days after infection (three cell passages) by indirect immunofluorescence of fixed cells, using rabbit anti-p30 (Rauscher) serum which recognizes xenotropic MuLV p30 or by sedimentable RNA-directed DNA polymerase activity in culture fluids. b Numerical values refer to migrations relative to WN1802N viral proteins (in millimeters) on cylindrical gels in sodium dodecyl silifate-polyacrylamide gel electrophoresis (see Fig. 1). c Determination tentative. a - correlate with tropism. In view of the fact that p30 proteins of endogenous MuLV's are known to be very similar in protein sequence, as determined by peptide mapping (3, 15), a highly discriminating assay for p30 differences was needed. A clue to the development of such an assay was the finding that p30 carries one of the antigenic specificities of the type-specific Gross cell surface antigen system (34). We have found that Gross cell surface antigen typing serum, B6 anti-K36, employed in a competition radioimmunoassay (RIA) with p30 from the endogenous N-tropic virus of AKR mice as a labeled probe, provides the requisite type specificity in assaying p30. Results of a typical RIA competition test are shown in Fig. 2A. A WN1802N clone competed completely, whereas a WN1802B clone showed no appreciable absorption of antibody. Class a xenotropic virus from BALB/c also did not com- pete in the assay. The same lack of ability to compete was demonstrated with the class ,B xenotropic viruses AT124 and NZB (not shown). Figure 2B shows the results of competition RIAs testing B-tropic BALB/c isolates with four different p30 mobility phenotypes (+2, +1, -1, and -2). None of these viruses competed. Additional tests of a number of other BALB/c virus clones demonstrated a complete concordance between positivity and negativity in the RIA competition test and N- and B-tropism, respectively. Figure 2C shows results of tests with pairs of N- and Btropic viruses from two other mouse strains, C57BL/6 and AKR/Fv-lb/b. Again, complete competition was observed for the N-tropic viruses, and negligible competition was observed for their B-tropic counterparts. It is unlikely that the polymorphism which we found for BALB/c B-tropic MuLV is a consequence of the cloning procedure in SC-1 cells for Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest 16-month-old BALB/c mouse No. of clones isolateda
(4) NOTES VOL. 32, 1979 N1802B0 CL;Dl 80 ~~~~~~~WN1802B CL 1 Q E 60 - E_ 20 - 100 60 - 10 40 _-< AKRFv-1 ( 20 ), B6-7(N) I 10 10 I I 102 103 Competing antigen (ng) X I~ ~ ~ I 104 FIG. 2. Competition RIAs with '251-labeled AKR p30 and Gross cell surface antigen typing serum, C57BL/6 anti-AKR spontaneous leukemia K36. p30 was purified from AKR N-ecotropic virus by either gel filtration in 6 M guanidine hydrochloride (14) or phosphocellulose chromatography (37). (We are indebted to A. Pinter of this laboratory for the latter preparation) In our hands, p30 purified by phosphocellulose chromatography was found to yield more reproducible results in RIAs with B6 anti-K36 serum. Purified AKR p30 was labeled with '25I by the chloramine T method as described by Hunter (20). Competition RIAs were carried out essentially as described by Strand and August (37), with addition of anti-mouse immunoglobulin (Pocono Rabbit Farms) to precipitate immune complexes. Competing virions lysed in 0.4% Nonidet P40 were added as indicated. (A) 0, WN1802B CL Di; E, BALB virus-2, class a xenotropic virus; 4 WN1802N CL B5. (B) , BALB, B.22, a new B-tropic clone; C, WN1802B CL D1; A, WN1802B CL D7; 0, WN1802B CL 1. p 30 sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility phenotypes were +2, +1, -1, and -2, respectively. (C) E, AKR Fv-1b'/b(N); , AKR Fv-lb/b(B); 0, B6-7(N); , B6-7(B). N- and B-tropic viruses from the AKR Fv-lh/b congenic strain, originally isolated by J. Hartley and W. Rowe, were obtained from Nancy Hopkins (Massachusetts Institute of Technology). The N- and B-tropic isolates from C57BL/6 (B6) mice, also from Hartley and Rowe, have been described previously (27). Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest co 353
(5) 354 NOTES alter separately or together.) The p12 of C57BL/6 B-tropic virus has been reported to share an antigen with the a subclass of xenotropic virus (5). However, in collaborative studies with M. Barbacid and S. Aaronson, no evidence was found for xenotropic p12 antigen in representative clones of our BALB/c B-tropic isolates. A role for MuLV structural protein in determining N- and B-tropism was first indicated by the findings of Rein et al. on the altered tropism of phenotypically mixed ecotropic viruses (29). The results of Hopkins and co-workers (19, 31) have shown that the p30 mobility phenotype is closely associated with tropism in N x B crosses and B -- NB tropic variant formation; these data have been reinforced by the detection of oligonucleotide spots in the gag region of viral RNA maps which cotype with tropism (10, 11). In this regard, the p30 RIA data for the N- and B-tropic pair of viruses from AKR/Fv-lb/b mice (Fig. 2) are especially striking, since the oligonucleotide maps of these isolates are virtually identical but differ in the typing spot for Nversus B-tropism (12; N. Hopkins, personal communication). At the present time, all N- and Btropic MuLV's which we have analyzed can be typed by the B6 anti-K36 p30 RIA test. Nowinski and collaborators (submitted for publication) have extended this correlation, using the same RIA test, to other viral isolates. Furthernore, peptide analysis of p30 proteins of N- and Btropic MuLV's from both BALB/c and C57BL/6 mice has revealed a specific peptide shift to be characteristic of B-tropic virus (7, 15; T. Albino, personal communication). Thus, whether brought about by recombination or by mutation, a specific change in p30 appears to be closely linked with the phenotype of B-tropism. We are indebted to Mariano Barbacid and Stuart Aaronson for performing type-specific p12 radioinununoassays on our isolates. We gratefully acknowledge the excellent technical assistance of H. Neufeld and T. Scholla and the help of A. Chege in preparing the manuscript. This research was supported by Public Health Service grants CA-08748 and CA-16599 from the National Cancer Institute. LITERATURE CITED 1. Aaronson, S. A., and C. Y. Dunn. 1974. High frequency C-type virus induction by inhibitors of protein synthesis. Science 183:422-424. 2. Aaronson, S. A., and J. R. Stephenson. 1973. Independent segregation of loci for activation of biologically distinguishable RNA C-type viruses in mouse cells. Proc. Natl. Acad. Sci. U.S.A. 70:2055-2058. 3. Albino, A., L Korngold, and R. C. Meliors. 1979. Tryptic peptide analysis of gag gene proteins of endogenous mouse type C viruses. J. Virol. 29:102-113. 4. Barbacid, M., J. R. Stephenson, and S. A. Aaronson. 1976. gag gene of mammalian type-C RNA tumour Downloaded from http://jvi.asm.org/ on November 10, 2019 by guest several reasons. (i) The effect would have to be specific for B-tropic, and not N-tropic, MuLV. (ii) B-tropic clones isolated from two sources, from the WN1802B stock and fresh from BALB/c mice, displayed quite different polymorphic patterns. (iii) B-tropic MuLV's from individual BALB/c radiation leukemias, cloned in SC-1, reveal a still wider range of polymorphisms, and in repeated clonings of virus from the same leukemia extracts particular viral phenotypes are reproduced (R. W. Ellis, N. Hopkins, and E. Fleissner, J. Virol., submitted for publication). It is quite possible, however, that some genetic changes may occur as a result of prolonged passage of virus in BALB/c fibroblasts (e.g., in the case of WN1802B [Table 1]) or due to extensive replication of B-tropic virus in BALB/c mice before virus isolation. Whatever the source of the polymorphism which we observed it may be caused by a mechanism which is distinct from that responsible for other viral variants which exhibit defects in replication (18, 33). We infer this because positivity in the XC plaque test was the basis on which we selected clones and because none of our clones displayed any gross lesions in replication or in expression of the three main classes of viral proteins (gag, pol, and env products). If one takes into account the limited ecotropic viral information present in mouse genomes (9), the extent of variation which we found among B-tropic isolates from normal BALB/c mice, as well as from BALB/c radiation leukemias (Ellis et al., submitted for publication), suggests that B-tropic MuLV's may result from genetic alterations of an endogenous N-ecotropic viral genome. Recombination between inducible Ntropic virus and other endogenous viral genes is one mechanism by which B-tropic viruses might arise; mutational processes could also account for N -- B conversion. Xenotropic virus in BALB/c cells can be induced under a variety of conditions (1, 2, 32). Cells producing this virus would not be resistant to superinfection with Necotropic virus and could offer opportunities for recombinational events. The RIA data in Fig. 2A can be regarded as compatible with substitution of xenotropic sequences in the p30 proteins of these viruses; moreover, the mobility phenotypes +1, -4, and +2 for p15, p12, and p30 (Table 1), respectively, are characteristic of inducible BALB/c xenotropic virus (Ellis et al., submitted for publication). A limited substitution of xenotropic information coding for a particular protein might suffice to alter tropism. (Depending on the crossover points involved in a particular recombinational event, the antigenicity and mobility marker of a protein could J. VIROL.
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Hopkins. 1977. Evidence for recombination between N- and B-tropic murine leukemia viruses: analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Virol. 23:700-707. 32. Sherr, C. J., M. M. Lieber, and G. J. Todaro. 1974. Mixed splenocyte cultures and graft versus host reactions selectively induce an "S-tropic" murine type C virus. Cell 1:55-58. 33. Shields, A., 0. N. Witte, E. Rothenberg, and D. Baltimore. 1978. High frequency of aberrant expression of Moloney murine leukemia virus in clonal infections. Cell 14:601-609. 34. Snyder, H. W., Jr., E. Stockert, and E. Fleissner. 1977. Characterization of molecular species carrying Gross cell surface antigen. J. Virol. 23:302-314. 35. Stephenson, J. R., and S. A. Aaronson. 1976. Induction of an endogenous B-tropic type C RNA virus from SWR/J mouse embryo cells in tissue culture. Virology 70:352-359. 36. Stephenson, J. R., R. K. Reynolds, and S. A. Aaronson. 1972. 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