Construction and properties of an “artificial” spleen focus-forming virus

VIROLOGY

183, 343-350

(1991)

Construction

and Properties

of an “Artificial”

Spleen Focus-Forming

Virus

ROLAND FRIEDRICH,’ URSULA FRIEDRICH, AND GABI MAENNLE Division of Molecular

Oncology,

Institute of Medical

Virology, lustus Liebig University,

Received January 29, 199 1; accepted

D-6300 Giessen, Germany

March 28, 199 1

The replication-defective Friend spleen focus-forming virus (F-SFFV) induces acute erythroblastosis in adult mice. The envelope-related (env) gene and LTR are the only functional elements of the viral genome. The env-coded glycoprotein gp55 has been shown to be responsible for target cell specificity and for the short latency of the disease caused by SFFV. This molecule closely resembles the envcoded proteins gp7O+pl5E of mink cell focus inducing viruses (MCFV). The only substantial differences between these two env genes are a large deletion spanning 585 nucleotides in the middle of the F-SFFV gene and a frameshift mutation near the 3’ end leading to a modified and shortened membrane anchor in the mature protein. To determine if the large deletion and/or the frameshift mutation are capable of changing the properties of a nonpathogenic MCFV into those of an acutely pathogenic SFFV we introduced these changes into the env gene of an MCFV. The results show that the mutated MCFV is as acutely pathogenic as F-SFFV. We therefore conclude that the modified membrane anchor of gp55 and the change caused by the large deletion are the essential determinants of the high pathogenicity of SFFV. o 1991 Academic PWSS. IX.

INTRODUCTION

tracellular interaction of the env gene product with the cellular erythropoietin receptor is an important step in the onset of the disease. The only functional elements of the genome of FSFFV are envelope gene and LTR. As in the case of MCFV, SFFV is believed to be a recombinant of the ecotropic F-MuLV and endogenously inherited sequences in mice. These acquired sequences contain the env gene and resemble those of the polytropic MCFVs (Clark and Mak, 1983; Amanuma et a/., 1983; Koch et al., 1983). The gag and pal sequences, as far as they are present, are highly homologous to F-MuLV (Friedrich et a/., 1990). The main differences of the env gene in SFFV compared to MCFV include: A number of point mutations (resulting in about 40 amino acid changes); a large deletion spanning 585 nucleotides in the middle of the gene; a short duplication and a frameshift mutation near the 3’ end of the gene leading to the substitution of 5 amino acids; premature termination and concomitant loss of 33 to 36 amino acids (depending on the virus strains compared) in the mature protein. The primary product of the env gene of the ecotropic MuLVs and the MCFVs is a glycoprotein, gp85, which is cleaved into gp70 (surface protein, SU) and p15E (transmembrane protein, TM) (Fig. 1). The pl5E molecule is further processed to pl2E and represents the membrane anchor of gp70 which forms a knob-like structure on the surface of the viruses. The large deletion in the SFFV env gene includes the normal gp70pl5E cleavage site resulting in a protein of an apparent molecular weight of 52,000 to 55,000 (gp52 or gp55). This protein could not be found in the viral membrane

Three different types of retroviruses are considered to be responsible for Friend erythroleukemias in mice: the ecotropic Friend murine leukemia virus (F-MuLV) causes erythroleukemia within l-2 months after injection into newborn susceptible mice. Some time after infection with F-MuLV, a new type of virus with polytropit host range appears in mice developing the disease. This virus is formed during the latency period by recombination of the F-MuLV genome and sequences endogenously inherited in mice. It is assumed that as in most other murine leukemias this newly generated second type of retrovirus, the mink cell focus forming virus (MCFV) is the ultimate inducer of murine leukemia. Finally, the replication-defective Friend spleen focus forming virus (F-SFFV) causes an acute erythroleukemia occurring as shortly as several weeks after infection of adult mice. F-MuLV acts as helper virus for replication of F-SFFV. The mechanism by which SFFV causes erythroleukemia remains unclear (for reviews on SFFV, see Famulari, 1983; Friend and Pogo, 1985; Ostertag et al., 1987; Kabat, 1989). Insertional activation of the putative oncogene spi-1 and inactivation of the p53 gene are frequently observed events at the cellular level. The env gene of SFFV has been shown to be responsible for the target cell specificity of the virus and the short latency of the disease. Recent studies by Li et al. (1990) and Yoshimura et a/. (1990) suggest that an in’ To whom correspondenc:e dressed.

and reprint requests

should be ad-

343

0042.6822/91

$3.00

Copyright 0 1991 by Academic Press. Inc. All rights of reproducton I” any form reserved.

FRIEDRICH,

344

FRIEDRICH, AND MAENNLE

I ~dlfferentlal

F-MCFV

Env

i

s” region

TM +---I constant

(DFt)4?b

YY

YY

SPI

region

WY

QP7Q

I t--+,

4 196

aa

YY

W

p’*p15

deleted

1 bp +modlfled

Env

./

j PR I b

F-SFFV

(CR)

Ineertlon C -

terminus

I

j PR j

SPI I t-----+

b DR

QP55 -

+-----CR

4

FIG. 1. Schematic drawing of MCFV and SFFV env gene products. The two major differences introduced into the env gene of MCFV are represented on the protein level (fusion of SU and TM leading to the loss of 195 amino acids; carboy-terminal modification). Position of glycosylation sites and the protein domains (Koch et al., 1984) are also depicted. CR, constant region, common to xeno-, poly-, and ecotropic viruses; DR, differential region, characteristic for xeno- and polytropic viruses; PR, proline rich region; SP, signal peptide; SU, surface protein gp70; TM, transmembrane protein pl Z/p1 5E. Y, N-glycosylation site; 1, potential N-glycosylation site not substituted with carbohydrates in p12/pl5E and gp55.

and only very little is present in form of the more heavily glycosylated gp65 on the surface of cells of the strain SFFVp, a strain which causes polycythemia in mice. The gp55 of the strain SFFV, which causes severe anemia cannot be found in the cellular membrane. However, in cells infected with either virus, some gp65 is shed into the medium (for details, see the review of Kabat, 1989). The aim of the present study was to determine whether the env gene products of MCFVs carry all structural elements important for the acute disease induced by SFFV and, if so, whether the numerous point mutations or the large deletion and the carboxy-terminal variation in gp55 of SFFV or a combination of these are the essential factors responsible for the unique properties of this virus. To this end, we introduced into the env gene of an MCFV (which was nonpathogenic in the mouse strain used) the same two major changes as those present in the env gene of SFFV and compared the pathological properties of these new viral constructs to their progenitors. MATERIALS Cells, vectors,

AND METHODS

and viruses

Rat-l cells (Freeman eta/., 1973) were used for propagation of viruses and transfections; rat cells were chosen rather than mouse cells in order to avoid recombination of viral DNAs with closely related endogenous mouse viruses. Helper viruses were titrated on Rat-l cells by endpoint dilution and reverse transcrip-

tase assay. Titers of an ID,, of 106.5 were routinely obtained. The plasmid pAX327 was used for all cloning experiments used in this study. It is based on pBR327 (Soberon eta/., 1980)from which theAat andXmaIII sites were removed to facilitate cloning of viral sequences. Plasmid pAXMuLV57 consisting of the molecular FMuLV clone 57 (Oliff et a/., 1980; Koch er a/., 1983; Friedrich et a/., 1990) cloned in the Sphl site of pAX327 was the starting vector for the viral constructs. Cell line 8a + N (derived from uninfected Rat-l cells) producing SFFVp (Lilly-Steeve strain; Linemeyer et al., 1980) and the biologically cloned F-MuLV 22N were gifts from W. Ostertag, Hamburg. F-MuLV 22N (Ostertag et a/., 1980) is a highly effective helper virus for the SFFVs used in this study. pFP502, a molecular clone of the Axelrad strain of SFFV, was obtained from S. Clark (Clark and Mak, 1983). Since our experience showed that virus derived from pFP502 was either only weakly or not at all pathogenic, we replaced the Xmalll-Aatll fragment (env gene and LTR) by the corresponding sequence of a molecular clone of the Lilly-Steve strain (Wolff et a/., 1983) which was obtained as plasmid pMF1 (neoR SFFV) from W. Ostertag. This construct, termed pSAX in this study, led to a highly pathogenic SFFVp (SAX) V(malll to end of LTR, Wolff et a/., 1983; end of LTR to Aatll, sequenced by us, unpublished; all other nucleotides, Clark and Mak, 1983). The molecular clone p247w of MCF247 was obtained from C. Holland (Holland et al., 1989). MCF247 is the prototype of thymotropic MCFVs and was isolated from an AKR mouse during the preleukemic period. A small Xbal-

CONSTRUCTION

OF AN ARTIFICIAL

Xmalll fragment of the end of the pal gene of FriendMCFV 54B (Koch et a/., 1984) was inserted as a linker to facilitate insertion of foreign env gene sequences into F-MuLV57.

Gel electrophoresis, oligonucleotides

DNA hybridization, and

Horizontal agarose gels (0.8 to 2.0%) were run in 40 mM Tris, 20 mM Na acetate, 1 mM EDTA (pH 7.8) to separate restriction enzyme fragments. DNA fragments visualized by ethidium bromide intercalation and uv transillumination were purified from agarose by the glass powder procedure (Vogelstein and Gillespie, 1979) or through electrophoresis in the Biotrap (Schleicher and Schuell, Dassel, Germany). The two oligonucleotides spanning the Kpnl-Bsml site in SFFV were a gift from Pharmacia (Freiburg, Germany). One contained 40 nucleotides of the noncoding DNA strand, and the other contained 38 nucleotides of the coding strand. Both oligonucleotides were purified on a 10% PAA urea gel and the sequences were verified by the method of Maxam and Gilbert (1980). The oligonucleotides were then hybridized, phosphorylated at their 5’ ends with polynucleotide kinase and used for ligation.

Transfection of viral DNA, virus rescue, and infection of mice Viral DNA was ligated to concatemers and was then transfected into Rat-l cells by the calcium phosphate method (Graham et a/., 1973) in the presence of a plasmid containing the neomycin resistance gene. Neomycin resistant colonies were selected and after 2-3 weeks, colonies were tested by hybridization and immunofluorescence for the presence of viral RNA and the env gene product. Positive cell lines were superinfected with F-MuLV 22N. Synthesis of intracellular viral RNA was monitored by blotting whole cells on nitrocellulose filters and by hybridization of denatured total RNA on these blots with specific probes according to the method developed by Paeratakul et a/. (1988). After superinfection of cells with F-MuLV 22N, the amount of virus present in the supernatant was estimated by measuring the activity of reverse transcriptase in the supernatant. Before using these preparations for the infection of mice, the numbers of viral particles present in the medium were estimated by hybridization of SFFV or F-MuLV specific probes to RNA extracted from the supernatant with Proteinase K and phenol/chloroform (Sambrook et al., 1989).

345

SFFV

Generally, 0.5 ml of supernatants from freshly confluent tissue cultures were injected into the tail veins of 8- to 16-week-old DBA/2J mice obtained from the Zentralinstitut ftir Versuchstierzucht at Hannover (Germany). At least 12 animals were infected with each viral construct. At the times indicated, 6 or more mice were killed by CO, suffocation and their spleen weights and hematocrits determined. Spleens were fixed in Bouin’s solution and then examined for foci.

lmmunodetection

of env gene products

For immunoblots, cellular extracts were prepared from freshly confluent virus-infected cells or from Rat-l cells as negative controls. Cells were grown on 5-cm cell culture dishes. They were washed three times with PBS and then incubated on ice for 15 min with 0.5 ml extraction buffer [lo mM Na phosphate, pH 7.6, 100 mM NaCI, 1 mM EDTA, 19/oTriton X-l 00, 0.59/o deoxycholate, 0.1% SDS, 0.5% Trasylol (Bayer, Leverkusen, Germany), 1 mM dithiothreitol and 10 mM Leupeptin (Sigma)]. The extracted supernatants were clarified by centrifugation at 12,000 g at 0” for 10 min. Extracts were then mixed with the same amount of 2X sample buffer (125 mR/I Tris, pH 6.8, 4% SDS, 2% 2-mercaptoethanol, 10% glycerol with bromphenol blue) and boiled for 5 min. SDS-polyacrylamide gels (10%) were run and blotted onto lmmobilon P (Millipore) for 90 min at 0.8 mA/cm2 in a semidry blotting apparatus. The membranes were blocked for at least 2 hr or overnight in PBS/O.5% Tween 20. Incubation with a 1:200 dilution of rabbit anti gp70 serum in blocking buffer was for 1 hr at 37”. Blots were washed in blocking buffer three times for 5 min and were then incubated with a 1:200 dilution of a gold conjugate of an anti-rabbit antibody (AuroProbe BL, Amersham) in blocking buffer for 2 hr at room temperature. After two washes with blocking buffer and two washes with distilled water, visibility of the bands was enhanced by silver staining (IntensSE BL, Amersham). For immunofluorescence, cells were seeded in Flexiperm chambers (Heraeus, Hanau, Germany) onto sterile glass slides and allowed to grow for 24 hr. The slides were then thoroughly washed with PBS, fixed for 20 min at -20” in a 3:l mixture of methanol-acetone and air-dried. They were then incubated for 45 min at 37” with polyclonal anti-gp70 goat or rabbit serum diluted 1:200 in PBS. After three washes with PBS, they were incubated for 45 min at 37” with FITC-labeled second antibody (anti-rabbit or goat IgG, Sigma) and visualized using a Zeiss fluorescence microscope. Anti-gp70 goat and anti-gp70 rabbit sera were gifts from Heinz Bauer, Giessen, Gerhard Hunsmann, Gbttingen, and Rudolf Geyer, Giessen.

346

FRIEDRICH,

FRIEDRICH.

RESULTS Outline of cloning strategies To construct a virus with sequences similar to FSFFV, gag and pol sequences from F-MuLV and env sequences from MCF247 were combined. MCF247 was preferred over Friend-MCFV since it is not pathogenic in the mouse strain used here. It accelerates leukemia in AKR mice but is not pathogenic in Balb/c, NIH Swiss or DEW2 mice (Famulari, 1983; Holland et a/., 1983, 1989). LTRs were used from either MCF247 or SFFV, (pMF1). Most of the cloning steps were done with subclones which contained only env and LTR of the respective viruses. For the sake of brevity, only the important cloning steps are described here (Fig. 2). pAXMuLV57 (F-MuLV57 in the Sphl site of pAX327) was cut with Xbal and Aatll and env and LTR replaced with the corresponding sequences taken from MCF247. The resulting plasmid pMuMM2 was then digested partially with Kpnl and &ml and the 623 bp long envfragment removed by electrophoresis. A double-stranded oligonucleotide containing 40/38 nucleotides of the corresponding sequence of SFFV, was ligated into the resulting gap giving rise to plasmid pMu1 MM. Plasmid pMu2MS, an F-MuLV/MCFV construct with LTR and 3’ end of env from SFFV,, was derived from pMuMM by replacing the EcoRV-Aatll fragment (containing 3’ end of env gene, LTR and part of U,) with the corresponding fragment of pSAX (SFFV,). Similarly, the plasmid pMu1 .2MM, carrying both SFFV-like deletions in its env gene, was derived by replacing the EcoRV-fspl fragment of pMu1 MM (containing 3’ end of env) with the corresponding fragment of pSAX. Finally, in pMu1 .2MS, the MCFV-LTR of pMu1 -2MM was exchanged with the LTR of SFFV. As a control, the plasmid pMuSS was constructed which contained the full env gene and LTR from pSAX (SFFV,) in a gag and pol environment of F-MuLV.

Properties

of the new env gene products

Our experiments were performed with an MCFV not pathogenic in DBA/2 mice. The only differences in env gene and LTR between the original MCF247 and the

’ Nomenclature of the viral constructs: Mu, gag and pal sequences originating from F-MuLV; M, MCFV sequences (env or LTR), S, SFFV sequences (env or LTR); 1, large internal deletion in the envgene; 2, frameshift mutation in env, leading to the loss of 36 and the substitution of 5 carboxy-terminal amino acids. The order of the designations determines the order of the sequences (gag and pal; env; LTR).

AND MAENNLE

“artificial” SFFV (Mu 1 -2MM) are the large deletion of 585 nucleotides, a single basepair insertion near the 3’ end of the env gene leading to the premature termination of the gene product and three amino acid differences (positions 355, 359, and 362 in SFFV) near the EcoRV site used to construct these mutants. These three amino acid changes are conservative exchanges (T + A, S --, P, L + I); they vary in many SFFV and MCFV strains and are thus not characteristic of SFFV or MCFV. The env genes of the newly constructed viruses result in different electrophoretic mobilities of the env gene products on SDS polyacrylamide gels. Figure 3 shows an immunoblot of extracts of cells infected with wild-type SFFVs or mutated viruses pseudotyped with F-MuLV (22N). Cells infected with the MCF247/FMuLV hybrid (MuMM) yielded gp70 and the precursor protein gp85. Exchange of the carboxy terminus of the MCFV env gene product with that of gp55 led to a shorter gp85 which could not be discriminated from gp85 by immunoblotting (a small size difference could sometimes be observed on SDS polyacrylamide gels; data not shown). Mutant MulMM carrying the large deletion of 195 amino acids produced a protein of an apparent molecular weight of about 60,000. Both double mutants (Mu1 -2MS and Mu1 -2MM) produced a protein that migrated at a slightly slower rate than gp55 of wild type SFFVs. This difference in migration is probably due to an additional amino acid at position 134 in the mature gp55 in addition to the sequence difference of 38 amino acids distributed throughout the gene product. Pathogenicity

of newly constructed

viruses

All viral constructs were tested in DBAI2J mice for their pathogenicity. Spleens were examined for the appearance of foci 14 days after infection; spleen weights and hematocrits were determined 21 days after infection (Fig. 4). Mice infected with the viral construct missing the internal 585 nucleotides in the env gene (Mu1 MM) or carrying the frameshift mutation near the 3’terminus of the gene leading to the carboxy-terminal deletion of the env gene product (Mu2MS) were indistinguishable from those that had received the helper virus only. Mice injected with the construct encompassing both the large deletion and the changed carboxyterminus specific for the SFFV env protein (Mu1 -2MM) resembled animals infected with SFFV, (SAX) or MUSS (SFFV, env and LTR in F-MuLV environment): their spleens displayed foci and were grossly enlarged after three weeks. The number and appearance of foci were indistinguishable from those induced by wild-type

CONSTRUCTION

OF AN ARTIFICIAL

SFFV

347

pMuMM

pMulMM

pMu2M-S

pMui.PMM

pMul.PMS

pMuSS 4

4J

QPQQ

SFFV, FIG. 2. Restriction enzyme maps of constructed viruses. The two upper drawings both represent plasmid pMuMM, consisting of gag andpolof F-MuLV57 and env and LTR of MCF247. Viral sequences were cloned into the Sphl site of pAX327. At the bottom is a map of the SFFV, construct pMuSS which contains the envgene and LTR of pSAX (SFFV,, Lilly-Steeve strain), and gag and polof F-MuLV. F-MuLV sequences are depicted as a thick line, SFFV sequences as dashed boxes or lines and MCFV sequences as open boxes or lines. SP, signal peptide. All other abbreviations are explained in text and footnote.

SFFV (“+” in Fig. 4). Hematocrits were also similar to the positive controls. No differences were observed whether the LTRs originated from SFFV or from MCF247 (Mu1 -2MS or Mu1 -2MM). Similar results were obtained when mice were sacrificed 2 weeks after infection (data not shown); spleen weights and hematocrits, however, were increased less than after 3

weeks. Mice that were infected with MuMM, Mu1 MM, or Mu2MS and observed for up to 6 months did not develop any sign of disease. Thus, the two major mutations (deletion and 3’-terminal alteration) discriminating the env gene of SFFV from that of the MCF viruses are necessary and are also sufficient for the highly and acutely pathogenic effects induced by SFFV.

348

FRIEDRICH,

FRIEDRICH,

AND MAENNLE

DISCUSSION

FIG. 3. Biosynthesis of Env proteins in virus-infected cells (immunoblot). All cells were superinfected with F-MuLV and therefore produced F-MuLV gp70 and its precursor gp85. Env proteins produced by the MCF247/F-MuLV hybrid (MuMM) were indistinguishable from F-MuLV Env proteins. The slightly shorter gp85 in Mu2MS (carrying the frameshift mutation at the carboxy terminus of gpl5) cannot be distinguished from gp85 of the helper virus in this immunoblot. The gp70/p15E fusion protein of MulMM (carrying the large deletion only) can be seen as a gp60. Proteins migrating at a slightly slower rate than SFFV gp55 were found in cell lines producing Env-related proteins carrying both mutations (Mu1 a2M.S and Mu1 -2MM). Two different SFFV, producing cell lines were used as reference for gp55.

2

The two major characteristics distinguishing the SFFV env gene from the MCFV env gene were introduced into an F-MuLV construct containing env gene and LTR of MCF247 (a virus not pathogenic in DBA/2 mice). Deletion of 195 amino acids spanning the carboxy-terminal region of the SU protein (gp70) and the amino-terminal region of TM (pl5E) did not by itself result in a pathogenicity that was characteristic of SFFV. However, when the frameshift mutation found at the 3’ end of the SFFV env gene was concomitantly introduced, the new viral construct showed pathological properties characteristic for SFFV (grossly enlarged spleens with foci and high hematocrit values 2 to 3 weeks after infection). We therefore conclude that the two major differences in the env gene of SFFV are sufficient to change the env gene of a nonpathogenic MCFV into a highly pathogenic envgene and are therefore responsible for the characteristic pathology of this virus. The only other possible candidate as a genetic determinant of the acute erythroleukemia, the LTR, did not

191

[%I 80

60

F-MuLV Weight St.

IZB f

dev.

Hematocrit Foci

m

SFFV

1 MUSS

MUMM

MulMM

Mu2MS

Mul.PMS

Mul.PMM

0.26

1.11

1.79

0.13

0.14

0.14

1.31

1.46

0.16

0.40

0.26

0.03

0.06

0.03

0.30

0.66

48

nd

48

49

48

63 +

74 +

+

61 +

FIG. 4. Pathology of virus-infected mice. Mice were examined 2 weeks (foci) and 3 weeks (spleen weight, hematocrits) Hematocrits are given in percentages, weights in grams + standard deviation. I’+” denotes spleen foci undistinguishable appearance from those induced by wild-type SFFV. For explanation of the virus designations, see text and footnote.

after infection. in number and

CONSTRUCTION

OF AN ARTIFICIAL

play any significant role in the disease: the LTR of MCF247 as well as the SFFV LTR were equally effective in induction of acute erythroleukemia in connection with the “artificial” SFFV env gene (Mu1 .2MM and Mu1 .2MS, respectively). A similar result was obtained by Wolff and Ruscetti (1986) and Wolff et al. (1988) who showed that the LTR of Moloney MuLV could replace the LTR of SFFV without changing the pathogenicity of the virus. MuLVs that induce chronic disease behave differently because in this case it is the LTR that determines the type of leukemia (Chatis eta/., 1984; lshimoto el al., 1987). The rather unspecific role of the LTR in SFFV induced disease is surprising since nine nucleotides in the U, domain of the LTRs are consistently different when MuLVs causing erythroleukemia are compared to thymotropic MuLVs (Koch et a/., 1984; Bestwick et a/., 1984). The data presented here and those of Wolff and Ruscetti (1986) and Wolff et al. (1988) clearly show that these differences are not functionally significant. Our data thus unequivocally demonstrate that no other differences between MCFV and SFFV apart from the two alterations in the env gene contribute to the specificity of the disease. MCFVs contain all of the genetic information necessary to induce acute ery-throleukemia; however, their TM and SU proteins do not fulfill all essential requirements. Only after structural changes can the env gene product cause acute leukemia. One might envision different mechanisms for the changed action of the newly constructed gp55 versus the original MCFV Env proteins: the conformation of gp55 and its changed subcellular location due to the removal of the protein processing site and the partial deletion of its membrane anchor might lead either to the binding to a different cellular receptor or to an altered affinity to the MCFV receptor. Some evidence exists that SFFV gp55 and MCFV gp70 both can bind to the same receptor (Chesebro and Wehrly, 1985; Li et al., 1987). Whether this receptor is identical with the erythropoietin receptor which has recently been reported to bind strongly to gp55 (Li et a/., 1990; Yoshimura et al., 1990) is not yet clear. Whether the erythropoietin receptor is involved in disease induction by SFFV and whether it has different binding properties for MCFV gp70 remains to be determined. One might also speculate that the impaired incorporation into the cellular membrane shown for gp55 (for references, see Kabat, 1989; Ostertag eT al., 1987) and the concomitant high concentration of this protein within the cell can cause intracellular effects not invoked by gp70 of MCFV, therefore resulting in the exceptional pathological properties of this virus. We have recently found a sequence in gp55 and MCFV gp70 which is involved in pathogenicity of SFFV and is very similar to the nuclear

349

SFFV

location signal of the large T antigen of polyoma virus (manuscript in preparation). Whether a change of the Env protein or its subcellular location caused by the major mutations in SFFV env leads to a functional alteration of this signal sequence is presently being investigated.

ACKNOWLEDGMENTS We thank Heinz Bauer, Steven Clark, Christie Holland, Gerhard Hunsmann, Nick Hunt, Wolfram Ostertag, and Josef Schneider for supplying materials: Wolfgang Zimmermann and members of our laboratory for helpful discussions; Bruce Boschek for help with the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft. Note added in proof. After preparation of this manuscript, comparable results regarding the unique elements of gp55 were published by Watanabe et al. (1991).

REFERENCES AMANUMA, H., KATORI, A., OBATA, M., SAGATA, N., and IKAWA, Y. (1983). Complete nucleotide sequence of the gene for the specific glycoprotein (gp55) of Friend spleen focus-forming virus. Proc. Nat/. Acad. Sci. USA 80, 3913-3917. BESTWICK,R. K., BOSWELL, B. A., and KABAT, D. (1984). Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat. /. Viral. 51, 695-705. BONNER,W. M.. and LASKEY,R. A. (1974). A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46, 83-88. CHATIS, P. A., HOLLAND, C. A.. SILVER, J. E.. FREDERICKSON,T. N., HOPKINS, N., and HARTLEY,J. W. (1984). A 3’end fragment encompassing the transcriptional enhancers of nondefective Friend virus confers erythroleukemogenicity on Moloney leukemia virus. J. Viral. 52, 248-254. CHESEBRO. B., and WEHRLY. K. (1985). Different murine cell lines manifest unique patterns of interference to superinfection by murine leukemia viruses. virology 141, 119-l 29. CLARK, S. P., and MAK, T. W. (1983). Complete nucleotide sequence of an infectious clone of Friend spleen focus-forming provirus: gp 55 is an envelope fusion glycoprotein. Proc. Narl. Acad. Sci. USA 80, 5037-5041. FAMULARI, N. G. (1983). Murine leukemia viruses with recombinant env genes: Discussion of their role in leukemogenesis. Curr. Top. Microbial. Immunol. 103, 75-108. FREEMAN, A. E., GILDEN, R. V., VERNON, M. L., WOLFORD, R. G., HuGUNIN, P. E., and HUEBNER, R. J. (1973). 5-Bromo-2’.deoxyuridine potentiation of transformation of rat-embryo cells induced in vitro by 3-methylcholanthrene: Induction of rat leukemia virus gs antigen in transformed cells. Proc. Nat/. Acad. Sci. USA 70, 24152419. FRIEDRICH,R. W., KOCH, W., VON MAYDELL-LIVONIUS.U., SCHREWE,H., and ZIMMERMANN, W. (1990). Complete sequence of the Friend murine leukemia virus genome. GenBank and EMBL Nucleofide Sequence Libraries, Accession No. X02794. FRIEND, C., and POGO, B. G. T. (1985). The molecular pathology of Friend erythroleukemla virus strains. An overview. Biochim. Biophys. Acta 780, 181-l 95. GRAHAM, F. L., and VAN DER Ea. P. I. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52,456471.

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FRIEDRICH,

FRIEDRICH, AND MAENNLE

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FRANZ, T., and HESS, N. (1987). Transforming genes and target cells of murine spleen focus-forming viruses. Adv. CancerRes. 48, 193-355. OSTERTAG,W., VEHMEYER,K., FAGG, B., PRAGNELL,I. B., PAETZ,W., LE BOUSSE, C., SMADJA-JOFFE,F., KLEIN, B., JASMIN, C., and EISEN, H. (1980). Myeloproliferative virus, a cloned murine sarcoma virus with spleen focus-forming properties in adult mice. /. Viral. 33, 573-582. PAERATAKUL,U., DE STASIO, P. R., and TAYLOR, M. W. (1988). A fast and sensitive method for detecting specific viral RNA in mammalian cells. 1. Viral. 62, 1 132-l 135. SAMBROOK, J., FRITSCH, E. F., and MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” 2nd ed. Cold Spring Harbor Laboratories, Cold Spring Harbor, NY. SOBERON,X., COVARRUBIAS,L., and BOLIVAR, F. (1980). Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene 9, 287-305. VOGELSTEIN,B., and GILLESPIE,D. (1979). Preparative and analytical purification of DNA from agarose. Proc. Nafl. Acad. Sci. USA 76, 615-619. WATANABE, N., NISHI, M., IKAWA, Y., and AMANUMA, H. (1991). Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3’ half of the envgene. /. Viral. 65, 132-137. WOLFF, L., CHUNG, S. W., and RuscE~-~, S. (1988). Molecular basis for the pathogenicity of the Friend spleen focus-forming virus. Mod. Trends Virol. 123-l 33. WOLFF, L., and RUSCEI-~, S. (1986). Tissue tropism of a leukemogenie murine retrovirus is determined by sequences outside of the long terminal repeats. Proc. Natl. Aced. Sci. USA 83, 3376-3380. WOLFF, L., SCOLNICK. E., and RUSCETTI,S. (1983). Envelope gene of the Friend spleen focus-forming virus: Deletion and insertions in 3’ gp7O/pl5E-encoding region have resulted in unique features in the primary structure of its protein product. Proc. Narl. Aced. Sci. USA 80,4718-4722. YAMAMOTO, Y., GAMBLE, C. L., CIARK. S. P., JOYNER,A.. SHIBUYA, T., MAC DONALD, M. E., MAGER, D., BERNSTEIN,A., and MAK, T. W. (1981). Clonal analysis of early and late stages of erythroleukemia induced by molecular clones of integrated spleen focus-forming virus. Proc. Natl. Acad. Sci. USA 78, 6893-6897. YOSHIMURA, A., D’ANDREA, A. D., and LODISH, H. F. (1990). Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. Proc. Natl. Acad. Sci. USA 87, 4139-4143.