Conversion of Friend mink cell focus-forming virus to Friend spleen focus-forming virus by modification of the 3'' half of the env gene.

The 3' half of the env gene of the dualtropic Friend mink cell focus-forming virus was modified by replacing the restriction enzyme fragment of the genome DNA with the corresponding fragment of the acutely leukemogenic, polycythemia-inducing strain of Friend spleen focus-forming virus (F-SFFVP) genome DNA. Replacement with the fragment of F-SFFVP env containing the 585-bp deletion, the 6-bp duplication, and the single-base insertion converted the resulting chimeric genome so that the mutant had a pathogenic activity like that of F-SFFVP. Replacement with the fragment containing only the 585-bp deletion did not result in a pathogenic virus. However, when this virus pseudotyped by Friend murine leukemia virus was passaged in newborn DBA/2 mice, we could recover weakly pathogenic viruses with a high frequency. Molecular analysis of the genome of the recovered virus revealed the presence of a single-base insertion in the same T5 stretch where the wild-type F-SFFV env has the single-base insertion. These results provided evidence that the unique genomic structures present in the 3' half of F-SFFV env are the sole determinants that distinguish the pathogenicity of F-SFFV from that of Friend mink cell focus-forming virus. The importance of the dualtropic env-specific sequence present in the 5' half of F-SFFV env for the pathogenic activity was evaluated by constructing a mutant F-SFFV genome in which this sequence was replaced by the ecotropic env sequence of Friend murine leukemia virus and by examining its pathogenicity. The results indicated that the dualtropic env-specific sequence was essential to pathogenic activity.     

Mutations in the env gene of friend spleen focus-forming virus overcome Fv-2r-mediated resistance to Friend virus-induced erythroleukemia.

Although Fv-2r homozygous mice are resistant to leukemias induced either by an erythropoietin-encoding virus or by wild-type Friend virus (FV) (M. E. Hoatlin, S. L. Kozak, F. Lilly, A. Chakraborti, C. A. Kozak, and D. Kabat, Proc. Natl. Acad. Sci. USA 87:9985-9989, 1990), they are susceptible to some variants of FV (R. A. Steeves, E. A. Mirand, A. Bulba, and P. J. Trudel, Int. J. Cancer 5:349-356, 1970; R. W. Geib, M. B. Seaward, M. L. Stevens, C.-L. Cho, and M. Majumdar, Virus Res. 14:161-174, 1989). To localize the virus gene involved in influencing the host range, we cloned and sequenced the env gene of the BB6 variant of FV (Steeves et al., Int. J. Cancer 5:349-356, 1970). In comparison with the wild-type env gene, the BB6 variant contains a 159-bp deletion that eliminates the membrane-proximal portion of the extracellular domain and 58 point mutations resulting in 13 amino acid changes. Substitution of the variant env gene for the wild-type env gene resulted in a recombinant virus that produced a Friend virus-like disease in Fv-2r homozygotes. Our results identify the spleen focus-forming virus env gene as the viral gene involved in this virus-host interaction. Additionally, they suggest that the product of the Fv-2r gene modifies the interaction between the spleen focus-forming virus envelope protein and the erythropoietin receptor.     

Both the changes of six amino acids and the C-terminal truncation caused by a one-base insertion in the defective env gene of Friend spleen focus-forming virus significantly affect the pathogenic activity of the encoded leukemogenic membrane glycoprotein (gp55).

Friend spleen focus-forming virus (F-SFFV) causes acute erythroleukemia in mice and encodes in its defective env gene an Env-like membrane glycoprotein (gp55). The F-SFFV env gene has three characteristic structures compared with that of ecotropic murine leukemia viruses (MuLVs): substitution by the polytropic MuLV env sequence, a 585-bp deletion, and a 1-bp insertion. All of these characteristic structures are essential for the leukemogenic potential of gp55 of polycythemia-inducing isolates of F-SFFV (F-SFFVp). The 1-bp insertion causes changes of six amino acids and truncation by 34 amino acids at the C terminus. In this study, we constructed 12 mutant F-SFFV genomes starting from the wild-type F-SFFVp and examined the effect of the C-terminal truncation and the six altered amino acids on the pathogenic activity of gp55. The results indicated that at least 18 to 24 amino acids must be deleted from the C terminus for the env product to be pathogenically active. We also found that the six altered amino acids contributed significantly to the pathogenic activity of gp55. Analyses of the cellular processing of these mutant gp55s supported a correlation between the pathogenic activity of gp55 and its efficiency in overall cellular processing.     

Fusion of the erythropoietin receptor and the Friend spleen focus-forming virus gp55 glycoprotein transforms a factor-dependent hematopoietic cell line.

The Friend spleen focus-forming virus (SFFV) gp55 glycoprotein binds to the erythropoietin receptor (EPO-R), causing constitutive receptor signaling and the first stage of Friend erythroleukemia. We have used three independent strategies to further define this transforming molecular interaction. First, using a retroviral selection strategy, we have isolated the cDNAs encoding three fusion polypeptides containing regions of both EPO-R and gp55. These fusion proteins, like full-length gp55, transformed the Ba/F3 factor-dependent hematopoietic cell line and localized the transforming activity of gp55 to its transmembrane domain. Second, we have isolated a mutant of gp55 (F-gp55-M1) which binds, but fails to activate, EPO-R. We have compared the transforming activity of this gp55 mutant with the EPO-R-gp55 fusion proteins and with other variants of gp55, including wild-type polycythemia Friend gp55 and Rauscher gp55. All of the fusion polypeptides and mutant gp55 polypeptides were expressed at comparable levels, and all coimmunoprecipitated with wild-type EPO-R, but only the Friend gp55 and the EPO-R-gp55 fusion proteins constitutively activated wild-type EPO-R. Third, we have examined the specificity of the EPO-R-gp55 interaction by comparing the differential activation of murine and human EPO-R by gp55. Wild-type gp55 had a highly specific interaction with murine EPO-R; gp55 bound, but did not activate, human EPO-R.     

Glycoprotein encoded by the Friend spleen focus-forming virus.

The Friend spleen focus-forming virus (F-SFFV) released from cultured erythroleukemia cells (cell line F4-6/K) was cloned free of its helper lymphatic leukemia virus (F-MuLV). After allowing adsorption to Sc-1 fibroblasts at a low multiplicity of infection, the cells were seeded individually into wells of a microtitier test plate and the resulting colonies were grown into large cultures. Among 14 of these cell cultures that have been analyzed thoroughly, 6 contained F-SFFV alone, 1 contained F-MuLV plus F-SFFV, and 7 were uninfected. Each of the Sc-1 cell lines which had been infected with cloned F-SFFV contained a glycoprotein with an apparent molecular weight of 55,000 (gp55) that was absent from the cell lines that lacked F-SFFV. gp55 was also present in Friend erythroleukemia cells and in fibroblasts infected with an F-SFFV that had been doubly cloned in another laboratory. These results indicate that gp55 is encoded by the F-SFFV genome. gp55 has the following additional properties. It can be immunoprecipitated with antiserum made to the F-MuLV virion envelope glycoprotein (gp75). Its unglycosylated polypeptide, formed in cells treated with 2-deoxy-D-glucose, has a molecular weight of approximately 45,000. Its tryptic peptide map contains peptides in common with F-MuLV gp75 but it also contains unique peptides. It appears to be absent or present in only low concentrations in erythroleukemia cell plasma membranes as determined by lactoperoxidase-catalyzed iodination, and it accumulates intracellularly in large amounts. In addition, it is absent from released virions. The majority of the cellular gp55 has an isoelectric point of 8.5 to 9.0. These results are consistent with the idea that an env gene recombination event was involved in the origin of F-SFFV.     

Requirement of the single base insertion at the 3'' end of the env-related gene of Friend spleen focus-forming virus for pathogenic activity and its effect on localization of the glycoprotein product (gp55).

In order to obtain evidence for the essential role of the single base insertion occurring at the 3' end of the env-related gene of Friend spleen focus-forming virus (SFFV) encoding the leukemogenic glycoprotein (gp55) a mutant SFFV genome was constructed in which the segment of the gp55 gene of the polycythemia-inducing strain of SFFV containing the single base insertion and the 6-base-pair duplication was replaced by the corresponding sequence of the Friend murine leukemia virus env gene. The mutant SFFV-Friend murine leukemia virus complex did not induce symptoms of the erythroproliferative disease in adult DBA/2 mice. During passage through newborn DBA/2 mice, the mutant virus complex invariably gave rise to weakly pathogenic variant SFFVs. All of the variant SFFVs induced in adult DBA/2 mice a transient mild splenomegaly associated with normal or slightly low hematocrit value, and they produced gp55 with a molecular weight similar to that of gp55 of the wild-type SFFV. For the two isolates of variant SFFV, the 3' portion of the viral DNA intermediate containing the 3' portion of the gp55 gene was molecularly cloned. Nucleotide sequences of these biologically active cloned DNAs were determined and showed that the variant SFFV genomes arose from the mutant SFFV genome by regaining the single base insertion, indicating that the single base insertion is essential for the biological activity of gp55. Evidence is presented indicating that the single base insertion which causes a loss of the cytoplasmic domain of the env-related protein is not related to the localization of the further-glycosylated form of gp55 in the plasma membrane but is involved with the release of gp55 from cells.     

Glycosylation of glycoprotein 55 encoded by the anaemia-inducing strain of Friend spleen focus-forming virus

Normal rat kidney cells, non-productively infected with the anaemia-inducing variant of Friend spleen focus-forming virus (F-SFFV_A), were metabolically labelled with [2-³H]mannose. The primary translation product of the viral envelope gene (env), representing a glycoprotein with an apparent molecularM _r of 55 000 (gp55), was isolated from cell lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Radiolabelled oligosaccharides, released from tryptic glycopeptides by treatment with endo--N-acetylglucosaminidase H, were characterized chromatographically, by enzymic digestion and by acetolysis. The results revealed that F-SFFV_A gp55 obtained from this source carried predominantly oligomannose type sugar chains with five to nine mannoses. As a characteristic feature, glycans with seven to nine mannoses contained, in part, an additional glucose residue. Although the amount of glucosylated species found was higher in F-SFFV_A gp55 (about 25% of total endo-H-sensitive oligosaccharides) than in gp55 of the corresponding polycythaemia-inducing variant (F-SFFV_P, 16.3%), the overall glycosylation pattern of the F-SFFV_A env product closely resembled that of F-SFFV_P gp55 [Strubeet al. (1988)J Biol Chem 263:3762–71]. Hence, our results demonstrate that the different intracellular processing and transport of the primary F-SFFV_A env product cannot be attributed to aberrant trimming of its oligomannose type glycans.Abbreviations endo H endo--N-acetylglucosaminidase H fromStreptomyces griseus - env envelope gene - Env protein translation product ofenv - F-SFFV Friend spleen focus-forming virus - F-SFFV_A anaemia-inducing variant of F-SFFV - F-SFFV_P polycythaemia-inducing variant of F-SFFV - Hex hexose - NRK normal rat kidney - PNGase F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase F fromFlavobacterium meningosepticum     

Integration of spleen focus-forming virus proviruses in Friend tumor cells.

Using the Southern blot procedure, we studied the presumed spleen focus-forming virus (SFFV) provirus integration sites in the genome of the premalignant and the malignant cells isolated during the course of Friend erythroleukemia. Two restriction endonucleases, PstI and BamHI, discriminated the presumed integrated SFFV proviruses from the endogenous xenotropic-mink cell focus-forming viral sequences. No SFFV integration sites were detectable in the premalignant cells, suggesting a random integration of SFFV proviruses in the genome of these cells. In contrast, SFFV proviruses were detected at a single or very few sites in the genome of all malignant cells we analyzed. These results indicate that the event leading to the malignant transformation in acute Friend leukemia is clonal. In two of the six animals examined, tumors cells isolated from the spleens and the livers of individual mice showed identical SFFV integration patterns. This last result suggests that in some cases different tumors in a same leukemic animal could be derived from a unique clonal event.     

The hydrophobic membrane-spanning sequences of the gp52 glycoprotein are required for the pathogenicity of Friend spleen focus-forming virus.

Friend spleen focus-forming virus (SFFV) codes for a transport-defective envelope glycoprotein designated gp52, which is responsible for the leukemogenic properties of the virus. gp52 is a monotopic integral membrane protein anchored in the membrane by a stretch of hydrophobic amino acid residues located near the carboxy terminus of the molecule. We have constructed a mutant SFFV envelope gene in which the sequences that code for the hydrophobic membrane-spanning domain have been deleted, and we expressed this gene by using recombinant vaccinia virus vectors or retroviral vectors. The mutant SFFV envelope gene was found to encode a truncated glycoprotein (gp52t) which was also transport defective; a majority of gp52t remained cell associated, while a small proportion of the molecules underwent oligosaccharide processing. The processed form of gp52t was secreted from the cells. Retroviral vectors carrying the mutant SFFV envelope gene were found to be nonpathogenic in adult mice. These results indicate that the hydrophobic membrane-spanning region of gp52 is required for pathogenicity of SFFV and suggest that these sequences may play a role in signal transduction. The results also indicate that the transport defect of SFFV gp52 is due to structural features of the ectodomain of the molecule.     

Analysis of spleen focus-forming virus-specific RNA sequences coding for spleen focus-forming virus-specific glycoprotein with a molecular weight of 55,000 (gp55).

The 32S RNA of the Friend strain of spleen focus-forming virus (SFFV) contains two sets of sequences: about half is specific to SFFV, and the other half is in common with the sequence of the helper lymphatic leukemia virus. Fingerprinting analysis of RNase T1 oligonucleotides showed that the SFFV-specific sequences were located in two distinct regions: in the 3' half and near the 5' terminus of the genome. Translation of SFFV RNA in a cell-free system yielded three SFFV-specific polypeptides: two main products with molecular weights of about 47,000 (P47) and 16,000 (P16) and a variable amount of a product with a molecular weight of 40,000 (P40). P47 was translated from polyadenylic acid-containing fragments of 1,500 to 3,000 nucleotides with SFFV-specific sequences from the 3' half of the genome, whereas P16, which contained peptides in common with those of P47, was synthesized by smaller RNA. P47 formed in vitro was found to be structurally related to the protein portion of a glycoprotein, gp55, specifically found in SFFV-infected cells in vitro. It is concluded from the results that a defective env gene containing SFFV-specific sequences in the 3' half of the genome codes for SFFV-specific gp55.     

Molecular cloning of biologically active Rauscher spleen focus-forming virus and the sequences of its env gene and long terminal repeat.

Rauscher and Friend spleen focus-forming viruses (R- and F-SFFVs) cause similar progressive erythroleukemias dependent upon a virus-encoded membrane glycoprotein. Moreover, these SFFV glycoproteins are immunologically related to each other and to the recombinant-type glycoproteins encoded by the env genes of dual tropic murine leukemia viruses. To better understand these diseases and the viral origins, we isolated a pathogenically active molecular clone of R-SFFV proviral DNA, sequenced its 3'-terminal 2,163-base-pair (bp) region, and compared these sequences with previously determined sequences of F-SFFV. The 516-bp R-SFFV long terminal repeat is highly homologous to those of F-SFFV and Friend murine leukemia virus, although only the latter contains a 65-bp direct repeat in its U3 region. The env gene of R-SFFV encodes a glycoprotein with 408 amino acids that is identical in its basic domain organization to the glycoprotein of F-SFFV. Thus, the junctions between the dual tropic-related and ecotropic sequences occur at the same nucleotide, and both SFFV env genes contain identical 585-bp deletions in their ecotropic domains and single-bp insertions which cause premature terminations at the same amino acid in their ecotropic p15E domains. Consistent with their independent origins, however, the env sequences of R- and F-SFFV are distinctive in both their 5' dual tropic-related and 3' ecotropic-related domains. Furthermore, there are several consistent amino acid differences between the polycythemic F-SFFV sequences and the anemia-inducing R-SFFV sequence. The striking similarities of the independently formed F- and R-SFFV env genes imply that all of the glycoprotein domains arranged in a precise organization may be required for its leukemogenic activity     

Specific neutralization of defective spleen focus-forming virus in Friend virus complex by rat antiserum.

The spleen focus-forming virus (SFFV), a rapidly transforming, replication-defective virus in Friend virus (FV) complex that is readily neutralized by antisera directed against its helper virus, was examined for the presence of SFFV-specific antigens. Antisera prepared in Fisher rats against an SFFV-infected Fisher rat embryo fibroblast line (SFFV-FRE) neutralized SFFV effectively, but not Friend-associated murine leukemia virus (F-MuLV) whether the latter was tested alone or was mixed with SFFV in the FV complex. In contrast, serum from mice immunized with SFFV-infected nonproducer mouse cells had little or no neutralizing activity against SFFV. Both absorption and immunoprecipitation studies indicate that the SFFV-specific antigen is immunologically related to xenotropic murine leukemia virus antigens. The role of both SFFV- and F-MuLV-specific antigens in the neutralization of SFFV suggests that this defective virus could be an antigenic mosaic and that viruses in the FV complex may participate in a undirectional form of phenotypic mixing.     

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.     

Sequence comparisons of the anemia- and polycythemia-inducing strains of Friend spleen focus-forming virus.

A nucleotide sequence analysis carried out on the envelope gene of the anemia-inducing strain of the Friend spleen focus-forming virus (F-SFFVA) reveals that its product has some unique features in common with previously described polycythemia-inducing strains of F-SFFV (F-SFFVP). (i) It contains an amino terminus that is highly related to the gp70 of mink cell focus-inducing viruses, (ii) it is a fusion protein containing the amino terminus of gp70 and the carboxy terminus of p15E, and (iii) it lacks the R-peptide normally found at the carboxy end of the p15E region. Although the envelope genes of F-SFFVA and F-SFFVP are quite similar overall, they do show sequence variation, particularly at the 3' end in the p15E-related region. These variations may contribute to previously observed differences in the response of F-SFFVP- and F-SFFVA-infected erythroid cells to regulatory hormone or to differences in the way the envelope glycoproteins are processed. The long terminal repeat regions of F-SFFVA and the Lilly-Steeves strain of F-SFFVP were also sequenced and compared with each other and with a previously published sequence of another F-SFFVP long terminal repeat. The sequences were found to be reasonably similar to each other but different from their ecotropic parent, Friend murine leukemia virus, as a result of a deletion of one copy of the direct tandem repeat in the enhancer regions. The observation that all SFFVS have this common change in the long terminal repeat enhancer region raises the possibility that it is required for pathogenicity.     

Sequence Flexibility in the Polytropic env gp70-Derived Region of the Membrane Glycoprotein (gp55) of Friend Spleen Focus-Forming Virus Affects Its Biological Activity

We previously reported (N. Watanabe, M. Nishi, Y. Ikawa, and H. Amanuma, J. Virol. 65:132–137, 1991) that the mutant Friend spleen focus-forming virus (F-SFFV_MS), which encodes a mutant gp55 membrane glycoprotein with an ecotropic env gp70 sequence, was nonpathogenic. Here we injected the F-SFFV_MS–Friend murine leukemia virus (F-MuLV) clone 57 complex into newborn DBA/2 mice. We obtained four groups of pathogenic variant F-SFFV complexes, each showing a different degree of pathogenicity in adult mice and a different gp55 profile. Of these, group 1 variant F-SFFV was particularly interesting, because it was the most frequently obtained and because it produced doublet bands of gp55 (59 and 57 kDa), neither of which reacted with the nonecotropic gp70-specific monoclonal antibody, and because its DNA intermediate did not hybridize with the nonecotropic env-specific probe. Cloning and DNA sequence analysis of the env region of one isolate of the group 1 variant F-SFFV revealed that this virus consisted of two distinct F-SFFV genomes; one (clone 117) differed from the other (clone 118) due to the presence of a 39-bp in-frame deletion. Reconstitution to full-length F-SFFV genomes and a pathogenicity assay showed that each reconstituted F-SFFV was pathogenic, with clone 117 showing a higher degree of pathogenicity than clone 118. Both reconstituted F-SFFVs caused activation of the mouse erythropoietin receptor in the factor-independent cell proliferation assay, although much less efficiently than the wild-type polycythemia-inducing isolate F-SFFVp. Clone 118 produced a gp55 of 59 kDa, while clone 117 produced one of 57 kDa. Clone 118 had a substitution by the F-MuLV clone 57 gp70 sequence, indicating that it was derived from the F-SFFV_MS env gene by a homologous recombination with the F-MuLV clone 57 env gene. The site of the 39-bp deletion in clone 117 corresponded to the portion of the clone 118 sequence which was unique to the ecotropic env genes. These results indicated the importance for the biological activity of gp55 of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.Friend spleen focus-forming virus (F-SFFV), a replication-defective mouse type C retrovirus contained in the Friend virus complex, causes an acute erythroleukemia in adult mice of susceptible strains in the presence of a helper virus, such as the replication-competent Friend murine leukemia virus (F-MuLV) (reviewed in references 8 and 23). F-SFFV does not encode a viral oncogene, but its defective env gene product, gp55, plays a critical role in inducing the disease. In vitro biochemical evidence demonstrated that gp55, a membrane glycoprotein encoded by the polycythemia-inducing isolate of F-SFFV (F-SFFVp), specifically binds to a mouse erythropoietin receptor (EPO-R) and activates it, causing mitogenic signal transduction in the absence of the natural ligand erythropoietin (EPO) (12). Since the EPO-R is expressed in the erythroid progenitor cells, and the interaction between EPO and EPO-R regulates the level of erythropoiesis (15), it is assumed that the continuous expression of F-SFFV gp55 results in an abnormal proliferation of these cells, leading to leukemic transformation after several additional cytogenetic changes (3).gp55, although closely related to the Env protein of MuLV, is not incorporated into the retrovirus particles, but stays inside the cells, mainly in the rough endoplasmic reticulum membrane, with a small fraction of the molecules (3 to 5%) processed through the Golgi apparatus to the cytoplasmic membrane (6, 21, 24). Cell surface-localized gp55 is then shed from the cells, probably after proteolytic cleavage from the transmembrane domain (6, 20, 21).There are three major differences between the primary structures of gp55 of F-SFFVp and the Env protein of ecotropic MuLV (1, 4, 31). These are, from the N terminus, a substitution by the polytropic (dualtropic) env gp70 sequence, a 585-bp deletion which eliminates the proteolytic cleavage site for gp70 and p15E, and a 6-bp duplication and a single base insertion which cause premature termination of translation and loss of a 34-amino-acid peptide at the C terminus. By constructing F-SFFV encoding a mutant gp55, analyzing its pathogenicity in vivo, and also obtaining spontaneous revertant F-SFFVs from the mutant F-SFFV, we demonstrated that each of these three structural differences is essential for the pathogenicity of gp55 (2, 27–29). We previously reported (28) that the constructed F-SFFV (F-SFFV_MS) encoding the mutant gp55, in which the polytropic gp70 sequence had been replaced by the ecotropic F-MuLV clone K-1 gp70 sequence, was nonpathogenic in adult mice. The polytropic gp70-derived sequence in gp55 appears to contain a binding site for EPO-R (32). The purpose of the present study was to obtain spontaneous revertants from mice neonatally injected with F-SFFV_MS and to analyze the structure of their gp55s, with the goal of pinpointing the sequence required for pathogenicity and activation of EPO-R. The results indicated the importance of the sequences in the gp70 differential region, which are contained in both polytropic and ecotropic env genes.     

Spleen focus-forming Friend virus: identification of genomic RNA and its relationship to helper virus RNA.

The genome of the defective, murine spleen focus-forming Friend virus (SFFV) was identified as a 50S RNA complex consisting of 32S RNA monomers. Electrophoretic mobility and the molecular weights of unique RNase T1-resistant oligonucleotides (T1-oligonucleotides) indicated that the 32S RNA had a complexity of about 7.4 kilobases. Hybridization with DNA complementary to Friend murine leukemia virus (Fr-MLV) has distinguished two sets of nucleotide sequences in 32S SFFV RNA, 74% which were Fr-MLV related and 26% which were SFFV specific. By the same method, SFFV RNA was 48% related to Moloney MLV. We have resolved 23 large T1-oligonucleotides of SFFV RNA and 43 of Fr-MLV RNA. On the basis of the relationship between SFFV and Fr-MLV RNAs, the 23 SFFV oligonucleotides fell into four classes: (i) seven which had homologous equivalents in Fr-MLV RNA; (ii) six more which could be isolated from SFFV RNA-Fr-MLV cDNA hybrids treated with RNases A and T1; (iii) eight more which were isolated from hybrids treated with RNases A and T1; and (iv) two which did not have Fr-MLV-related counterparts. Surprisingly, the two class iv oligonucleotides had homologous counterparts in the RNA of six amphotropic MLV's including mink cell focus-forming and HIX-MLVs analyzed previously. The map locations of the 23 SFFV T1-oligonucleotides relative to the 3' polyadenylic acid coordinate of SFFV RNA were deduced from the size of the smallest polyadenylic acid-tagged RNA fragment from which a given oligonucleotide was isolated. The resulting oligonucleotide map could be divided roughly into three segments: two terminal segments which are mosaics of oligonucleotides of classes i, ii, and iii and an internal segment between 2 and 2.5 kilobases from the 3' end containing the two oligonucleotides shared with amphotropic MLVs. Since SFFV RNA consists predominantly of sequence elements related to ecotropic and amphotropic helper-independent MLVs, it would appear that the transforming gene of SFFV is not a major specific sequence unrelated to genes of helper viruses, as is the case with Rous sarcoma and probably withe other defective sarcoma and acute leukemia viruses.     

Retrovirus transduction: segregation of the viral transforming function and the herpes simplex virus tk gene in infectious Friend spleen focus-forming virus thymidine kinase vectors.

A series of deletions and insertions utilizing the herpesvirus thymidine kinase gene (tk) were constructed in the murine retrovirus Friend spleen focus-forming virus (SFFV). In all cases, the coding region for the SFFV-specific glycoprotein (gp55), which is implicated in erythroleukemic transformation, was left intact. These SFFV-TK and SFFV deletion vectors were analyzed for expression of tk and gp55 after DNA-mediated gene transfer. In addition, virus rescued by cotransfection of these vectors with Moloney murine leukemia virus was analyzed for infectious TK-transducing virus, gp55 expression, and erythroleukemia-inducing ability. The experiments demonstrated that deletions or insertions within the intron for the gp55 env gene can interfere with expression of gp55 after both DNA-mediated gene transfer and virus infection. In contrast, the gene transfer efficiency of the tk gene was unaffected in the SFFV-TK vectors, and high-titer infectious TK virus could be recovered. Revertant viruses capable of inducing erythroleukemia and expressing gp55 were generated after cotransfection of the SFFV-TK vectors with murine leukemia virus. The revertant viruses lost both tk sequences and the ability to transduce TK- fibroblasts to a TK+ phenotype. These experiments demonstrate that segregation of the TK and erythroleukemia functions can occur in retrovirus vectors which initially carry both markers.     

The membrane glycoprotein of Friend spleen focus-forming virus: evidence that the cell surface component is required for pathogenesis and that it binds to a receptor.

The leukemogenic membrane glycoprotein of Friend spleen focus-forming virus (SFFV) has an apparent Mr of 55,000 (gp55), is encoded by a recombinant env gene, and occurs on cell surfaces and in intracellular organelles. There is evidence that the amino-terminal region of gp55 forms a dualtropic-specific domain that is connected to the remainder of the glycoprotein by a proline-rich linker (C. Machida, R. Bestwick, B. Boswell, and D. Kabat, Virology 144:158-172, 1985). Using the colinear form of a cloned polycythemic strain of SFFV proviral DNA, we constructed seven in-phase env mutants by insertion of linkers and by a deletion. The mutagenized SFFVs were transfected into fibroblasts and were rescued by superinfection with a helper murine leukemia virus. Four of the mutants cause erythroblastosis. These include one with a 6-base-pair (bp) insert in the ecotropic-related sequence near the 3' end of the gene, two with a 12- or 18-bp insert in the region that encodes the proline-rich linker, and one with a 6-bp insert in the dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific region. The other mutants (RI, Sm1, and Sm2) are nonpathogenic and contain lesions in dualtropic-specific sequences that are highly conserved among strains of SFFV. A pathogenic revertant (RI-rev) was isolated from one mouse that developed erythroblastosis 3 weeks after infection with RI. RI-rev contains a second-site env mutation that affects the same domain as the primary mutation does and that increases the size of the encoded glycoprotein. All pathogenic SFFVs encode glycoproteins that are expressed on cell surfaces, whereas the nonpathogenic glycoproteins are exclusively intracellular. The pathogenic SFFVs also specifically cause a weak interference to superinfection by dualtropic MuLVs. These results are compatible with the multidomain model for the structure of gp55 and suggest that processing of gp55 to plasma membranes is required for pathogenesis. The amino-terminal region of gp55 binds to dualtropic murine leukemia virus receptors, and this interaction is preserved in the SFFV mutants that cause erythroblastosis.