c myeloma mink cell focus-inducing (MCF) viruses

VIROLOGY

108, 474-483 (1981)

Envelope Proteins of the BALB/c Myeloma Mink Cell Focus-Inducing (MCF) Viruses DALE R . SPRIGGS' AND ROBERT G . KRUEGER2 Division of Tumor Biology, The Christ Hospital Institute of Medical Research, 2141 Auburn Avenue, Cincinnati, Ohio 45219, and the Department of Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Accepted August 19, 1980 A recent report from this laboratory demonstrated that the BALBIc myeloma retroviruses, MO-21 and FL-1, have a polytropic hostrange and exhibit "mink cell focus-inducing" (MCF) activity . Because previous MCF isolates have been shown to possess a recombinant envelope (env) gene, we undertook neutralization and peptide mapping studies to analyze the envelope proteins of MO-21 and FL-1 viruses and compare them to various prototype marine retroviruses . Like other MCF viruses, the infectivity of the myeloma viruses was inhibited by normal mouse serum . Two-dimensional peptide mapping data show that the viral envelope protein (gp70) of M0-21 and FL-1 has several distinct peptides which distinguish them from the gpi0 of any of the prototype viruses we analyzed . Unlike other MCF viruses, the gp70s of M0-21 and FL-1 are indistinguishable from one another . These results suggest that these MCF retroviruses may not be (env) gene recombinants . MO-21 and FL-1 viruses also contain another viral-coded envelope protein which has an apparent higher molecular weight than the gp7O . The possible significance of this gp70-like protein is discussed .

However, it is difficult to determine the identity of the exact xenotropic parent(s) of these recombinant viruses . Similar approaches have been taken to define the origins of the HIX (Fischinger et al ., 1975, 1978a, b) and MO-MCF (Vogt, 1979 ; Bosselman et al ., 1979) isolates, which originated from Moloney leukemia virus infections . Friend virus has also been used to generate MCF viruses which are (env) gene recombinants (Troxler et al ., 1978; Cloyd et al ., 1979) . In all the cases cited above, it would appear that the presence of ecotropic and xenotropic sequences in the (env) gene product enables the virus to infect cells of heterologous species, as well as murine cells . Thus, these viruses have been termed dual-tropic or polytropic (Hartley et al ., 1977 ; Fischinger et at ., 1978b) . MCF viruses also have interference and serum neutralization characteristics which are shared with ecotropic and xenotropic viruses . Determination of the genetic origin of MCF viruses has great importance since the evidence accumulated to date suggests that

INTRODUCTION

Several recent publications have dealt with the characterization of an unusual group of murine retroviruses which induce cytopathic effects in mink lung fibroblasts . These isolates are referred to as "mink cell focus-inducting" (MCF) viruses . These viruses were originally described by Hartley et al . (1977) who found these agents associated with thymic lymphomas in mice . Subsequent analysis of the gp70 tryptic peptides demonstrated that these viruses arose through recombination between ecotropic and xenotropic viruses within the envelope (env) gene (Elder et al ., 1977a, 1978b) . These studies have been confirmed by oligonucleotide mapping (Rommelaere et al ., 1978 ; Shih et al ., 1978) and by heteroduplex analysis (Chien et al ., 1978) . ' Present address : Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts. 2 To whom requests for reprints should be addressed at The Christ Hospital Institute of Medical Research . 0042-6822/81,'020474- 14J$02 .0014) Copyright n 1981 by Academic Press, lnc . All rights of ,,m xlnetinn in any f -

veers ed

474

MYELOMA VIRUS ENVELOPE PROTEINS

these recombinant viruses may be the agents which actually induce neoplasia in various murine systems . The AKR-MCF viruses described by Hartley et al . (1977) emerge from lymphoid tissue during the preleukemic or leukemic stage of lymphoma induction . Preliminary data cited by the authors suggested that the MCF-247 prototype isolate was highly lymphomagenic when inoculated into AKR mice . A recent publication has verified these findings and further showed that MCF lymphomagenicity correlated with the thymic origin of the virus as well as the ability of the MCF virus to replicate in the thymus (Cloyd et al ., 1980) . Similar studies with the HIX-MCF (Fischinger, 1977) and the MOMCF (Vogt, 1979) viruses, which were isolated after Moloney MuLV infection, have further shown that MCF viruses can be implicated in lymphomagenesis . We recently demonstrated that cloned preparations of two viruses originally produced by two different BALB/c myelomas, designated as MO-21 and FL-1, have a polytropic in vitro host range and they exhibit MCF activity (Spriggs et al ., 1980) . In light of the biochemical and biological evidence dealing with other MCF viruses, we undertook a series of studies to determine if these BALB/c retroviruses had any other features in common with previously described MCF agents . Normal BALB/c serum effectively neutralized the infectivity of MO-21 and FL-1 . This is in keeping with the polytropic host range of these viruses (Hartley et al ., 1977; Fischinger et al., 1978b), Two-dimensional peptide analysis of the viral gp70 envelope protein of these viruses demonstrated that it has several peptides in common with the gp70 of ecotropic and xcnotropic viruses, but the data suggest that the structure of the gp70 of MO-21 and FL-1 is not totally due to (env) gene recombination (i .e ., these MCF gp 70 proteins contained unique peptides not found in any of the other viruses we studied) . In addition, both myeloma retroviruses express a gp70-like protein which has an apparent higher molecular weight than the viral gp70 and presumably is an envelope protein . The expression of this gp70-like protein is regulated by the type of cell in which the virus is propagated .

475

MATERIALS AND METHODS

Viruses and Cell Cultures BALB:virus 1 (BV-1) and BALB :virus 2 (BV-2) were obtained from Dr . Stuart Aaronson . Dr. Janet Hartley provided the MCF-247 and 1504-A viruses . BALB-clone11 (B-C1-11) virus was provided by Dr . Naomi Rosenberg. FL-1 and MO-21 BALB/c myeloma viruses were isolated and cloned as reported previously (Kontor and Krueger, 1979 ; Spriggs and Krueger, 1979) . Briefly, the viruses were subjected to three cycles of limiting dilution passage in SC-1 cells and then passaged through various murine and nonmurine cell lines at limiting dilution . In addition, MO-21 was further cloned by isolating single cells 24 hr after infection with a limiting dilution of virus and then growing the cell clones to mass culture . BV-2 and 1504-A virus were grown in mink lung cells (Mvl, CCL64) and all other viruses were grown in wild mouse embryo SC-I cells . (NIH x BALB)F, and Pekin duck embryo cells (PDE) were infected and grown as described previously (Spriggs et al ., 1980) . Virus Purification Virus containing supernatant culture media was harvested from actively growing cells and centrifuged at 500 g for 20 min . The clarified media was then layered over a 30% sucrose cushion containing 5 µg/ml phenylrnethylsulfonylfluoride (PMSF, Sigma) and centrifuged at 100,000 g for 1.5 hr at 4°. The resultant pellets were resuspended and used as the source of virus for further analysis . We did not gradient purify the viruses since this procedure has been shown to greatly decrease the yield of 70 from various retroviruses (McGrath et al ., 1978) . The protein concentration of the preparation was determined by the method of Lowry et al . (1951) using bovine serum albumin as the standard . Aliquots of the resuspended viruses were stored at -80° . Iodination and Immunoprecipitation of Envelope Proteins Intact virus (10 µg in 10 µl) was added to 20 zl of 0 .5 M potassium phosphate

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buffer (pH 7.5) . Ten microliters of "5 1 (500 gCi) was then added, followed by 10 µl of chloramine T (10 µg) and the reaction was allowed to proceed for 1 min . Sodium metabisulfite (50 µl, 2 mg) was added to stop the oxidation and after 1 min of mixing, the virus was disrupted by adding 100 1d of 2x precipitation buffer [0 .2% SDS, 1 .0% NP-40, 0 .15, M KCI, 0 .01 M KI, 1 mg/ml ovalbumin, 5 µg/ml PMSF, 0 .02 M potassium phosphate (pH 7.4)] . After incubation at 4° for 1 hr with frequent mixing, 10 gl of heat-inactivated goat antiserum to Rauscher MuLV gp70 (supplied by Dr . Stuart Aaronson, Lot #4048) was added and antigen-antibody complexes were allowed to form overnight at 4° . The sample was centrifuged in a Brinkman microcentrifuge for 5 min and the supernate was added to a prewashed pellet of Protein ASepharose CL-4B (Pharmacia Chemicals, Piscataway, N . J .) containing enough Protein A to adsorb all the IgG in the serum . The sample was kept at 4° for 1 hr with frequent mixing and the immune complexes were isolated by centrifugation for 2 min in the microcentrifuge followed by four washes with 1 x precipitation buffer . The resultant pellet was either used immediately or stored at 4° for no longer than 2 days .

and Enzymatic Digestion Envelope Proteins

Isolation

of

the

The immune complex pellets were resuspended in 40 µl of disruption buffer [0 .5% SDS, 10% (3-mercaptoethanol, 6 M urea, 0.125 M Tris-HC1 (pH 6.7)] and incubated at 37° for 15 min . The samples were then placed in a boiling water bath for 3 min and after cooling to room temperature, they were loaded into a 12% SDSpolyacrylamide slab gel (Laemmli, 1970) and electrophoresed as described previously (Spriggs and Krueger, 1979) . Standard proteins were run in adjacent slots in the gel . The gel was stained overnight with Coomassie blue (25% isopropanol :10% acetic acid :0 .05% Coomassie blue R-250) and destained with 10% acetic acid . Kodak X-Omat R film was exposed to the wet gel at room temperature and the resultant autoradiograph was used as a template to identify the proteins with a molecular weight near

70,000 . Gel slices were cut from the gel and soaked overnight in 10% methanol . The gel slices were then dried under a stream of dry nitrogen with the aid of a heat lamp and they were then rehydrated with 0 .5 ml of 0 .05 M ammonium bicarbonate containing 25 µg of either TPCK-trypsin or TLCK-a-chymotrypsin . After incubation at 37° for 6 hr, another 0 .1 ml of fresh enzyme solution was added and the digestions were continued overnight . The resultant peptides were collected and dried down under a stream of nitrogen as indicated above and were stored at -20° until used . Peptide

Mapping

Two-dimensional peptide maps of the envelope proteins were generated by electrophoresis and chromatography on cellulose thin layer plates as described previously (Spriggs and Krueger, 1979 ; Spriggs et at ., 1980) . Neutralization Assays

Pseudotypes of the viruses tested were generated as indicated previously (Spriggs et al ., 1980) . Approximately 100 focusforming units of the pseudotypes were exposed to varying dilutions of normal BALB/c mouse serum for 30 min at 37° and assayed for residual focus-forming activity on monolayers of NRK cells . RESULTS

Neutralization Serum

Virus

by Normal

Mouse

Most murine sera contain a factor which preferentially inactivates xenotropic viruses (Levy et al ., 1975 ; Fischinger et al., 1976) as well as polytropic MCF viruses (Hartley et al., 1977; Fischinger et al., 1978b). The latter viruses are inactivated because the envelope glycoproteins contain xenotropic sequences (Fischingeret al ., 1977) . It has recently been shown that the inhibitory activity is contained in the very low-density lipoprotein fraction of normal mouse sera (Montelaro et al ., 1979 ; Kane et al ., 1980) . We generated MSV pseudotypes of MO-21 and FL-1 as well as other



MYELOMA VIRUS ENVELOPE PROTEINS TABLE 1 INACTIVATION OF MO-21 AND FL-1 VIRUSES BY NORMAL BALE/c SERUM

BALB/c serum dilution

Virus surviving fraction V„/V," BV-2

FL-1

MO-21

1504-A

B-C1-1P

1 :10 1 :20 1 :40 1 :100

0 .00 0 .00 0.00 0 .06

0 .00 0 .00 0.007 0 .018

0 .003 0 .003 0 .02 0.06

0 .38 0 .45 0 .64 0 .60

0 .67 1 .0 0.89 1 .0

° Neutralization was carried out by incubating MSV-pseudotypes of the listed viruses (approximately 100 focus-forming units) with normal BALB/c serum to give the final serum dilutions shown . The viruses were inoculated onto NRK cells and treated as indicated in Materials and Methods . B-C1-11 is a subclone of WN 1802-B .

viruses and determined the ability of normal BALB/c serum to neutralize their infectivity . As shown in Table 1, the infectivity of the myeloma viruses and the xenotropic virus, BV-2, was greatly reduced by the dilutions of serum tested . As expected, the ecotropic virus, B-Cl-11, was unaffected by this treatment (Levy et al ., 1975 ; Fischinger et al ., 1976) . The 1504-A amphotropic virus was neutralized at the serum dilution we tested, but the level of the inhibition was clearly less than that observed for MO-21, FL-1, or BV-2 . We are unaware of any data concerning the inhibition of the amphotropic class of viruses by normal murine serum, although Gardner (1978) stated that these viruses were unaffected by this treatment .

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rupted, and the gp70 proteins were immune precipitated and isolated on SDS-polyacrylamide slab gels . An autoradiograph of a typical preparation is shown in Fig . 1 . It can be seen that the major proteins present have a molecular weight of approximately 70,000 although the absolute migration of the various proteins varies in different preparations . This latter phenomenon may be due to differing efficiencies of iodination . However, it should be pointed out that the relative migration of the various viral gp70 proteins was constant . For example, the BV-2 polypeptide always migrated faster than the BV-1 gp70 (Fig . 1) . As described by Schindler et at . (1977), the WN 1802-N gp70 migrated faster than the WN 1802-B gp70 (data not shown) . Lanes C and D contain the immune precipitated proteins from MO-21 and FL-1, respectively. It was obvious that these viruses contain two major high-molecularweight polypeptides which were precipitated by the antisera . We have previously demonstrated that these viruses were cloned (Spriggs and Krueger, 1979; Spriggs et al ., 1980), so it appears as though these viruses code for both the "gp70" proteins .

Envelope Protein Analysis

The normal serum neutralization data as well as our previous work demonstrating the polytropic host range and MCF activity of MO-21 and FL-1 (Spriggs et al ., 1980) suggested that these viruses may be (env) gene recombinants (Hartley et al ., 1977; Elder et al ., 1977a, 1978a) . We therefore carried out peptide mapping studies on the envelope glycoprotein (gp70) of the MO-21 and FL-1 viruses to determine their relationship to the gp70 of various prototype murine retroviruses . The viruses were purified, surface labeled with I?. I, dis-

FiC. 1 . Autoradiograph of ""T-labeled immuneprecipitated proteins from BV-1 (A), BV-2 (B), MO-21 (C), and FL-1 (D). Viruses were surface iodinated, disruped, and immune precipitated with goat antiRauscher MuLV gp7O as described in Materials and Methods . The samples were clectrophoresed on 12% SDS-polyacrylamide slab gels and subjected to autoradiography . Bovine serum albumin (68,000), ovalbumin (43,000), and cytochrome c (12,000) were used as molecular weight standards . The "a" and "b" designate the slower and faster migrating gp7O-like proteins of MO-21 and FL-1 .

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We have designated the slower migrating protein "a" and the faster migrating protein "b" . We have demonstrated that both proteins are glycosylated (data not shown) and therefore these two proteins probably correspond to the gp75 and gp9O we previously described (Spriggs and Krueger, 1979) . The two low-molecular-weight proteins observed in the autoradiographs were probably the viral proteins, p15E and p12E . These proteins form a noncovalent bond with the gp70 molecule on the surface of the vision (Jamjoom and Arlinghaus, 1978) and therefore may be precipitated with the anti-gp70 antisera . Figure 2 shows the chymotryptic peptide maps we obtained when we analyzed the gp70 proteins of three prototype murine retroviruses . BALB :virus I (BV-1) is an ecotropic virus, BALB :virus 2 (BV-2) is a xenotropic virus, and MCF 247 is the prototype AKR mouse MCF virus . These peptide maps show that viruses with different biological activities (e .g ., host range)

can be distinguished from one another by this technique . We have analyzed several other murine retroviruses and confirmed the observation that viruses in different host range classes have readily distinguishable gp7O proteins (data not shown) . These results are in accord with previous reports (Elder et al ., 1977b, 1978a ; Bryant et al ., 1978) . The chymotryptic peptide maps of the MO-21 and FL-1 "a" and "b" proteins are shown in Fig . 3. The data suggested that MO-21 and FL-1 contained "a" and "b" gp70 proteins which were virtually identical . The gp70-a proteins appeared to be more ecotropic-like and the gp7O-b proteins resembled the gp7O of the MCF 247 virus (Fig . 2) . The isolation of two gp70-like proteins from both of our my eloma virus populations suggested that these viruses may not have been truly cloned . To examine this possibility, we passed both MO-21 and FL-1 (grown in SC-1 cells) through (BALB

FIG. 2 . Autoradiographs of "'1-labeled gp70 chymotryptic peptides from BV-1, BV-2, and MCF 247 viruses. The gp7O proteins were isolated by immune precipitation and electrophoresis as described in Fig. 1 . Chymotryptic peptides were isolated and analyzed as described in Materials and Methods . The origin of each two-dimensional peptide map is in the bottom left corner . Electrophoresis was from left (anode) to right (cathode) . Chromatography was from the bottom to the top .

MYELOMA VIRUS ENVELOPE PROTEINS

479

FIG . 3 . Autoradiographs of '251-labeled gp70 chymotryptic peptides from MO-21 and FL-1 . The "a" and "b" refer to the two species of gp7O isolated on SDS-polyacrylamide gels (see Fig. 1) . Twodimensional peptide analysis was performed as described in Fig . 2 .

x NIH)F,, Pekin duck embryo (PDE), and mink lung (Mvl) cells to determine if this procedure would eliminate expression of one of the gp7O proteins from the virus population . Virus isolated from Mel cells lacked the "a" protein, but viruses grown in F, or PDE cells still contained both gp70 proteins (data not shown) . The chymotryptic peptide maps of the gp7O proteins isolated from these viruses are shown in Fig. 4. First of all, these data showed that the gp70 proteins of MO-21 were viruscoded since their peptide maps were not detectably different when the virus was grown in different cell lines . FL-1 virus exhibited the same properties as MO-21 (data not shown) . These peptide maps also suggested that our original virus preparations may not have been cloned because virus isolated from Mv1 cells contained only one gp70 protein . We next passaged MO-21 virus from Mvl cells back into Mvl, SC-1 ., and PDE cells in an attempt to show that the virus was indeed cloned and it only contained one

gp7O protein. The results obtained from MO-21 virus isolated from MA and SC-1 cells are shown in Fig. 5 . Virus isolated from PDE cells was identical to MO-21 grown in SC-1 cells (data not shown) . It was clear that the virus isolated from SC-1 cells expressed two gp7O proteins while the virus propagated in Mvl cells contained one gp70 . Furthermore, the peptide maps showed that these gp70 species were the same as the original gp70 "a" and "b" proteins (Figs . 3 and 4) . Thus, these results are consistent with the assertion that our virus preparations were cloned when these experiments were initiated. . These findings suggested that the appearance of the gp70-a protein in mature virions may be determined by the cell in which the virus is propagated . Our previous studies, however, suggest that the "a" protein is probably not involved in the biological activity of the myeloma virus . For example . MO-21 grown. i n Mvl cells (gp70-b protein only) has a polytropic host range (Spriggs et al ., 1980) and is in-

SPRIGGS AND KRUEGER

4 80

FIG . 4 . Autoradiographs of the `"l-labeled ehymotryptic peptides generated from the MO-a and MO-b proteins isolated from MO-21 virus grown in various cells. F, cells = (NIH x BALB)F, cells, PDE cells = Pekin duck embryo cells, and Mvl = mink lung fihrohlasts . All experimental conditions were the same as described in Materials and Methods and Fig . 2.

activated by normal mouse serum (Table 1) . These properties are consistent with the gp 70 peptide mapping data described in this paper which showed that the gp70-b protein had an MCF-like chymotryptic peptide map profile . The possible significance of the gp70-a protein is discussed below . DISCUSSION

The data presented in this paper demonstrate that the BALB/c myeloma viruses, MO-21 and FL-1, possess envelope glycoproteins (gp7O) which resemble the gp70s isolated from the "mink cell focusinducing" (MCF) group of murine retrovirus . Our previous studies showed that MO-21 and FL-1 have MCF activity and a polytropic host range (Spriggs et al., 1980) . One unusual finding of the present study, however, was the observation that the gp70 proteins of these two myeloma MCF

viruses were virtually indistinguishable from one another (Fig. 3) . Previous peptide mapping studies of the gp70s of various MCF isolates have suggested that these agents are independent (env) gene recombinants which consequently express unique gp7O proteins (Elder et al ., 1977a, 1978b) . Recent studies in our laboratory have shown that another BALB/c MCF myeloma virus, CB 208 (Cloyd et al ., 1979), has a gp7O which is also apparently identical to the MO-21 and FL-1 gp7O-b protein (unpublished data) . We are presently studying the possible significance of these findings, but these preliminary studies suggest the possibility that the myeloma MCF virus (env) genes were not generated by independent reconibinational events . Another aspect of these studies was the demonstration that MO-21 or FL-1 viruses produced by various cell lines contained an additional gp70-like protein (gp70-a) .

MYELOMA VIRUS ENVELOPE PROTEINS

481

FIG . 5 . Autoradiographs of 126 I-labeled immune precipitates and the corresponding chymotryptic peptide maps obtained from the designated proteins . MO-21 virus replicating in Mvl cells was used to infect SC-1 and MV-1 cells . The virus from these cells was then isolated and the gp70 proteins were analyzed by immunoprecipitation and chymotryptiepeptide analysis as described in Figs . '1 and 2 . Lane A shows the immune precipitate of MO-21 virus isolated from Mvl cells and Lane B shows the immune precipitate of MO-21 isolated from SC-1 cells . The corresponding chymotryptic maps are indicated in the figure.

This protein could be distinguished from the functional viral gp70 (gp7O-b) by its apparent higher molecular weight as well as the two-dimensional maps of the peptides generated by a-chymotrypsin (Figs . 3-5) . Viruses grown in SC-1, F„ and PDE cells contained the gp70-a and b proteins, but viruses isolated from Mvl cells contained only the gp70-b species (Fig . 4). Surprisingly, passage of the MA grown virus back through SC-1 or PDE cells resulted in the reappearance of the gp70-a protein on the virus (Fig . 5) . This latter observation seems to rule out the possibility that the expression of the two gp70 proteins was due to the presence of a mixture of viruses in our virus preparations . We are pres

ently attempting to confirm the genomic homogeneity of the MO-21. and FL-1A virus stocks since it is remotely possible that viruses which have been shown to be biochemically or biologically homogeneous could still contain low levels of contaminating genomes . The possibility that the expression of the gp70-a protein is due to the rescue of an endogenous virus by the myeloma virus seems remote since we obtained the same protein from three different cell lines (SC-1, F,, and PDE) . However, if rescue were involved, it would suggest that the MO-21 and FL-1 are very unusual in this ability since we have never observed the gp70-a protein on any other retrovirus we have



482

SPRIGGS AND KRUEGER

studied that has been grown in these cells . Another possibility we are investigating is that the gp70-a protein is an incompletely modified or processed precursor to the gp70-b protein . It has been well established that the viral gp70 is generated by a series of posttranslational processing steps (Jamjoom and Arlinghaus, 1978 ; Naso et al ., 1976), therefore it is not unreasonable to propose that the gp70-a and gp7O-b are different forms of the (env) gene precursor protein . We would therefore suggest that most of the cells we tested were unable to efficiently convert one gp70 species to another, but Mvl cells were the exception . Comparative pulse-chase type experiments (Naso et al ., 1976; Jamjoom and Arlinghaus, 1978) as well as further biochemical analyses are currently underway to investigate these proposals. It is also possible that the expression of the gp70-a protein is related to the MCF properties of the virus (Cloyd et al ., 1979; Stockert et al ., 1979) . We are also studying the relationship of the gp70-a protein to various well-defined differentiation antigens (Elder et al ., 19776, 1978a, Lerner et al ., 1976; Tung et al ., 1978) . Regardless of the outcome, the studies we have presented have clearly shown that the BALB/c myeloma MCF viruses have unusual properties which warrant further study .

ACKNOWLEDGMENTS We wish to thank Dr . Stuart Aaronson for providing the goat anti-Rauscher gp70 antiserum and Dr . Janet Hartley for providing the various prototype viruses used in this study . This research was supported in part by Public Health Service grant CA-18936 from the National Cancer Institute and by the Elizabeth Gamble Deaconess Home Association . REFERENCES BOSSELMAN, R. A ., VAN GRIENSVEN, L . J . L . D ., VOGT, M., and VERMA, I . M . (1979) . Genome organization of retroviruses. VI . Heteroduplex analysis of ecotropic and xenotropic sequences of Moloney mink cell focus-inducing viral RNA from either a cloned isolate or a thymoma cell line. J . Virol . 32, 968-978 . BRYANT, M . L ., PAL, B . K ., GARDNER, M . B ., ELDER, J . H., JENSEN, F . C., and LERNER, R. A . (1978) . Structural analysis of the major envelope gly-

coprotein (gp7O) of the amphotropic and ecotropic type C viruses of wild mice . Virology 84, 348-358 . CHIEN, Y ., VERMA, I . M ., SETH, T . Y ., SCOLNICR, E . M ., and DAVIDSON, N . (1978) . Heteroduplex analysis of the sequence relations between the RNAs of "mink cell focus-inducing" (MCF) and murine leukemia viruses. J . Viral . 28, 352-360 . CLOYD, M. W., HARTLEY, J . W., and ROWE, W . P. (1979) . Cell surface antigens associated with recombinant mink cell focus-inducing murine leukemia viruses . J. Exp . Med. 149, 702-712 . CLOYD, M. W., HARTLEY, J . W ., and ROWE, W . P. (1980) . Lymphomagenicity of recombinant mink cell focus-inducing murine leukemia viruses . J . Exp . Med. 151, 542-552. ELDER, J . H ., GAUTSCH, J . W ., JENSEN, F . C ., LERNER, R . A., HARTLEY, J . W ., and ROWE, W . P. (1977a). Biochemical evidence that MCF murine leukemia viruses are envelope (env) gene recombinants . Proc . Nat . Acad . Sci . USA 74, 4676-4680 . ELDER, J . H ., GAUTSCH, J . W., JENSEN, F . C ., and LERNER, R . A . (1978a) . Multigene family of endogenous retrovirus : Recombinant origin of diversity . J . Nat . Cancer Inst . 61, 625-633 . ELDER, J . H ., G .AUTSCII, J . W., JENSEN, F . C ., and LERNER, R . A . (1978b) . Generation of diversity among murine C-type viruses via envelope gene recombination . In "Differentiation of Normal and Neoplastic Hematopoietic Cells" (B . Clarkson et al ., ed .), pp . 553-560 . Cold Spring Harbor Laboratory, New York . ELDER, J . H ., JENSEN, F . C ., BRYANT, M . L ., and LERNER, R . A . (1977b) . Polymorphism of the major envelope glycoprotein (gp70) of murine C-type viruses: Virion associated and differentiation antigens encoded by a multi-gene family. Nature (London) 267, 23-28 . FISCHINGER, P. J ., BLEVINS, C . S ., and DUNLOP, N . M. (1978a). Genomic masking of nondefective recombinant murine leukemia virus in Moloney virus stocks . Science 201, 457-4 .59. FISCHINGER, P. J ., FRANEEL, A . E ., ELDER, J . H ., LERNER, R . A ., IHLE, J . N., and BOLOGNESI, D . P . (1978b) . Biological, immunological, and biochemical evidence that HIX virus is a recombinant between Moloney leukemia virus and murine xenotropic C type virus . Virology 90, 241-254. FISCHINGER, P . J ., IHLE, J . N ., BOLOGNESI, D . P ., and SHAFER, W. (1976) . Inactivation of murine xenotropic oncornavirus by normal mouse serum is not immunoglobulin-mediated . Virology 71, 346-351 . FISCHINGER, P. J ., IHLE, J . N ., DE NORONHA, F ., and BOLOGNESI, D . P . (1977) . Oncogenic and immunogenic potential of cloned HIX viruses in mice and cats . Med . Microbiol . Immunal . 164, 119-129. FISCHINGER, P . J ., NOMURA, S ., and BOLOGNESI, D . P . (1975) . A novel murine oncornavirus with



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dual eco- and xenotropic properties . Proc . Nat . Aced . Sci. USA 72, 5150-5155 . GARDNER, M . B. (1978) . Type C viruses of wild mice : Characterization and natural history of amphotropic, ecotropic, and xenotropic MuLV . In "Current Topics in Microbiology and Immunology" (W. Arber et al ., ed .), Vol . 79, pp. 215-259 . Springer-Verlag, Berlin/New York . HARTLEY, J. W., WOLFORD, N . K ., OLD, L . J ., and RowE, W . P . (1977) . A new class of murine leukemia virus associated with development of spontaneous lymphomas . Proc . Nat. Acad . Set . USA 74, 789-792 . JAMJOOM, G . A ., and ARLINGHAUS, R . B . (1978). Synthesis and processing of the translational products of RNA tumor viruses . In "Methods in Cancer Research" (H . Busch, ed .), Vol. 15, pp . 3-69 . Academic Press, New York . KANE, .1 . P., HARDMAN, D . A ., DIMPEL, J . C ., and LEVY, J . A. (1979) . Apolipoprotein is responsible for neutralization of xenotropic type C virus by mouse serum . Proc . Nat . Acad . Sci . USA 76, 5957-5961 . KONTOR, E . J ., and KRUEGER, R . G. (1979) . Characterization of an amphotropic murine C-type virus that is NB-tropic . Virology 94, 451-459 . LAEMMLI, U . K . (1970) . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature (London) 227, 680-685 . LERNER, R . A., WILSON, C . B ., DEL VILLANO, B . C ., MC CONANEY, P. J ., and DIXON, F . J . (1976). Endogenous oncoviral gene expression in adult and fetal mice : Quantitative, histologic and physiologic studies of the major viral glycoprotein, gp70 . J . Exp . Med. 143, 151-166. LEVY, J. A ., IHLE, J . N ., OLESZKO, 0 ., and BARNES, R . D . (1975) . Virus-specifle neutralization by a soluble non-immunoglobulin factor found naturally in normal mouse sera. Proc . Nat. Acad . Sci. USA 72, 5071-5075 . LOWRY, 0 . H ., ROSEBROUGH, N . J ., FARR, A . L ., and RANDALL, R . J. (1951). Protein measurement with the Folin phenol reagent . J . Biel . Chem . 193, 265-275 . Mc GRATH, M ., WITTE, 0 ., PINCUS, T ., and WEISSMAN, 1 . L . (1978) . Retrovirus purification : Method that conserves envelope glycoprotein and maximizes infectivity. J . Virol . 25, 923-927. MONTELARO, R . C ., FISCHINGER, P . J ., LARRICK, S . B ., DUNLAP, N . W., IHLE, J . N ., FRANK, H ., SHAFER, W ., and BOLOGNESi, D . P . (1979) . Fur-

483

ther characterization of the oncornavirus inactivating factor in normal serum . Virology 98, 20-34. NASO, R . B ., ARCEMENT, L . J ., KARSHIN, W . L ., JAMJOOM, G . P., and ARLINGHAUS, R . B . (1976) . A fueose-deficient glycoprotein precursor to Rauscher leukemia virus gp69/71 . Proc . Nat . Acad . Sci . USA 73, 2326-2320 . ROMMELAERE, J ., FALLER, D . V ., and HOPKINS, N . (1978) . Characterization and mapping of RNase TIresistant oligonucleotides derived from the genomes of AKV and MCF murine leukemia viruses . Proc . Nat . Acad . Sci . USA 75, 495-499 . SCHINDLER, J ., HYNES, R ., and HOPKINS, N . (1977) . Evidence for recombination between N- and Btropic murine leukemia viruses : Analysis of three virion proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . J . Viral . 23, 700-707 . SHIN, T . Y ., WEEKS, M . 0 ., TROXLER, D . H ., COFFIN, J . M ., and SCOLNICK, F . M . (1978) . Mapping host range-specific oligonucleotides within genomes of the ecotropic and mink cell focus-inducing strains of Moloney murine leukemia virus . J . Viral . 26, 71-83 . SPRIGGS, D . R., DIEBOLD, M . A., and KRUEGER, R . G. (1980) . BALB/c myeloma retroviruses have "mink cell focus-inducing (MCF) activity. J. Viral ., in press . SPRIGGS, D . R ., and KRUEGER, R . G . (1979). Identification of the major structural proteins of two BALB/c myeloma C-type viruses . Virology 98, 35-44. STOCKERT, E ., DE LEO, A . B ., O'DONNELL, P . V ., OBATA, Y ., and OLD, L . J . (1979) . G, aks ,,, : A new cell surface antigen of the mouse related to the dual tropic mink cell focus-inducing class of murine leukemia virus detected by naturally occurring antibody . J. Exp. Med. 1149, 200-215 . TROXLER, D . H ., YUAN, F ., LINEMEYER, D ., RUSCETTI, S., and ScoLNICK, F . M . (1978) . Helperindependent mink cell focus-inducing strains of Friend type-C virus : Potential relationship to the origin of replication-defective spleen focus-forming virus. J . Exp. Med . 148, 639-653. TUNG, J . S ., O'DONNELL, P . V ., FLEISSNER, F ., and BoYSE, E . A . (1978). Relationships of gp70 of MuLV envelopes toegp70 components of mouse lymphocytes plasma membranes . J . Exp . Med . 147, 1280-1284 . VOGT, M . (1979) . Properties of a "mink cell focusinducing' (MCF) virus from spontaneous lymphoma lines of BALB/c mice carrying Moloney leukemia virus as an endogenous virus . Virology 93, 226-236 .