The spleen focus-forming virus (SFFV)-specific neoantigen shares cross-reactive determinants with mink cell focus-inducing (MCF) virus gp70.

We recently reported that the spleen focus-forming virus [(SFFV), a replication-defective leukemogenic virus] coded for a cell surface neoantigen which could be detected by lymphocyte-mediated cytolysis. Because the SFFV genome has recently been characterized as a recombinant between both ecotropic and xenotropic viral information, we examined whether the SFFV-specific neoantigen, as defined by in vitro cytolysis, shared determinants with antigens coded for by mink cell focus (MCF)-inducing viruses (also xenotropic/ecotropic recombinants). As detected by direct cytolysis of SFFV- and MCF-infected target cells, the SFFV-specific neoantigen was found to be partially cross-reactive with determinants on the surface of MCF virus-infected cells. The cross-reactive determinants appeared to be coded for by the envelope region of both AKR and Moloney MCF viruses, in that antisera specific for MCF gp 70 were capable of blocking SFFV-specific neoantigen-directed cytolysis. These results provide further evidence in support of the recombinant nature of the SFFV genome and its close relationship to MCF viral genetic information. However, in that the SFFV-specific neoantigen was not totally cross-reactive with MCF gp70, it remains possible that additional antigenic determinants associated with SFFV-encoded transformation may be present.     

Structural analysis of the spleen focus-forming virus envelope gene product

The structure of the envelope gene product (gp52) of the spleen focus-forming virus was analyzed and compared to that of envelope proteins (gp70 and p15E) of another pathogenic Friend virus recombinant, Friend mink cell focus-inducing virus (F-MCF). This has enabled us to confirm and extend previous nucleotide sequence data regarding the make up of specific domains of the SFFV glycoprotein. Amino-terminal 23,000-Da V8 protease fragments from gp52 of the Lilly-Steeves strain of SFFV and from gp70 of a pathogenic F-MCF isolate produced tryptic peptide fingerprints in which the mobilities of the trypsin-generated peptides were identical. The carboxyl-terminal, 21,000 Da, V8 protease fragment of gp52, however, has a unique fingerprint that contained a single highly charged trypsin-generated peptide. This peptide migrated to the same position as a peptide in F-MCF p15E, thus indicating that the p15E-related nucleotide sequences, that follow the large envelope deletion, are translated in the same reading frame as those in the standard p15E. Although R peptide determinants can be detected in F-MCF Pr15E, they could not be detected in gp52. The amino-terminal 23K domain of gp52, like that of MCF gp70, contains two oligosaccharide attachment sites. The other two attachment sites are located within the 21K carboxyl-terminal domain.     

Monoclonal antibodies against murine leukemia viruses: identification of six antigenic determinants on the p 15(E) and gp70 envelope proteins.

Hybrid cells that produced monoclonal antibodies against the envelope proteins of murine leukemia virus (MuLV) were prepared by the polyethylene glycol-mediated fusion of a mouse myeloma cell line with lymphocytes from mice immunized with allogeneic MuLV-producing leukemia cells. Twenty-three independent cell lines were cloned and inoculated into syngeneic mice for the production of ascites fluids that contained high-titered (20–75 mg/ml) monoclonal antibodies. Six serologically distinct specificities were detected when these ascites fluids were tested on a broad panel of MuLV and non-murine retra iruses. Prototype cell lines producing monoclonal antibodies that were representative of each pattern of reaction were selected for further study. In immune precipitation assays each of the prototype antibodies reacted with viral envelope proteins; three of these identified antigenic determinants on p15(E), while three others identified antigenic determinants on gp70. The p15(E) antigenic determinants were shared by a diverse panel of MuLV. One of these p15(E) antigenic determinants was also found in feline leukemia virus. The gp70 antigenic determinants, on the other hand, had a more restricted distribution and were found in only selected isolates of MuLV.     

Different pseudotypes of Friend spleen focus-forming virus induce polycythemia and erythropoietin-independent colony formation in serum-free medium

The polycythemia-inducing strain of Friend leukemia virus (FV-P) is a complex, consisting of a replication-defective spleen focus-forming virus (SFFV_P) and a replication-competent Friend murine leukemia helper virus (F-MuLV_P). It has previously been shown that infection of susceptible adult mice with FV-P rapidly results in the appearance of spleen cells capable of forming small erythroid colonies in a semisolid medium in the absence of exogenous erythropoietin (Epo). To determine whether the SFFV_P or F-MuLV_P components of FV-P are important for the appearance of these cells, we examined erythroid colony-forming cells from spleen cell populations infected with different pseudotypes of SFFV_P. These SFFV_P pseudotypes were obtained by rescuing a SFFV_P nonproducer fibroblast cell clone with different murine leukemia helper viruses. Large numbers of Epo-independent erythroid progenitor cells were observed, independent of the helper virus used to rescue the SFFV_P genome; furthermore, the murine leukemia helper viruses alone had no effect on either the number or Epo dependence of erythroid colony-forming cells. In addition, these experiments were carried out in serum-free culture conditions to eliminate the small amounts of Epo present in fetal calf serum. Taken together, we conclude that the appearance of truly Epo-independent progenitor cells can be attributed to the defective SFFVP and not the replication-competent F-MuLV_P component of FV-P complex.     

Replication of spleen focus-forming friend virus in fibroblasts from C57BL mice that are genetically resistant to spleen focus formation

Induction of erythroleukemia and spleen focus formation by the defective spleen focus-forming component of Friend virus (SFFV) is governed by a genetic locus of the mouse termed Fv-2 (F. Lilly, J. Nat. Cancer Inst.45, 163–169, 1970). Subsequently, the Fv-2 gene was reported to act by directly controlling SFFV, but not helper virus replication in the animal (R. A. Steeves, R. J. Eckner, M. Bennett, E. A. Mirand, and P. J. Trudel, J. Nat. Cancer Inst.44, 1209–1217, 1971; R. A. Steeves, F. Lilly, G. Steinheider, and K. Blank, In “Differentiation of Normal and Neoplastic Hemopoietic Cells” (B. Clarkson, P. A. Marks, and J. Tills, eds.), pp. 591–600. Cold Spring Harbor Conference on Cell Proliferation, Cold Spring Harbor, N. Y., 1978) or in cultured cells (N. M. Teich and T. M. Dexter, In “Oncogenic Viruses and Host Cell Genes” (Y. Ikawa and T. Odaka, eds.), pp. 263–276. Academic Press, New York, 1979). To determine how Fv-2 controls SFFV-induced erythroleukemia, we have examined its effect on SFFV replication in fibroblasts of resistant C57B1 mice infected with an ecotropic SFFV-Friend leukemia virus pseudotype. It was found that virus produced by C57B1 cells had spleen focus-forming activity in susceptible mice. RNA isolated from virus produced by C57Bl cells was indistinguishable in electrophoretic mobility and RNase T,-resistant oligonucleotides from authentic SFFV RNA described previously. We conclude that the Fv-2 gene does not inhibit SFFV replication in fibroblasts of Fv-2-resistant C57BI mice. Based on previous work of others and on our results, we suggest that Fv-2 acts either by controlling in hematopoietic cells susceptibility to helper virus, or by controlling a target necessary for transformation. Loss of SFFV from SFFV-helper virus stocks passaged in resistant animals would be a consequence of selection against, rather than direct inhibition of, SFFV. In susceptible animals, rapidly proliferating, SFFV-transformed cells would maintain a high ratio of SFFV to helper virus. Lack of transformed cells in resistant animals, and the consequent lack of selection for SFFV, would favor helper virus over SFFV.     

Rapid emergence of mink cell focus-forming (MCF) virus in various mice infected with NB-tropic friend virus

Mink cell focus forming (MCF) virus was always detected in the enlarged spleens of NFS, BALB/c, SL, and C₃H mice infected with NB-tropic ecotropic Friend virus. The MCF virus emerged from the spleens as early as 7 days postinfection. The MCF viruses isolated from various mouse strains were NB-tropic dualtropic and highly oncogenic to newborn NFS and BALB/c mice. When cells infected with MCF virus were overlaid with XC cells without uv irradiation, the viruses were XC positive. Interference and host-range properties suggested that the MCF viruses were recombinants between exogenous NB-tropic ecotropic Friend virus and endogenous xenotropic virus.     

Analysis of the expression of spleen focus-forming virus (SFFV)-related RNA and gp55, a friend and rauscher virus-specific protein

A 55,000-dalton glycoprotein, gp55, is the major intracellular species precipitable with anti-envelope glycoprotein (gp70) sera in murine erythroleukemia cells transformed by either the anemia- or the polycythemia-inducing strains of Friend virus. Similarly, all Friend erythroleukemia cell lines studied contained similar levels of spleen focus-forming virus (SFFV)-specific RNA in the cytoplasm as assessed by hybridization to a Friend SFFV-specific eDNA probe. Neither SFFV-specific RNA nor gp55 is detectable in helper virus-infected cells or in chemically induced rat erythroleukemia cell lines. SFFV non-producer cell lines were isolated and analyzed for viral gene expression by immunoprecipitation and molecular hybridization. SFFV nonproducer cells could be grouped into two classes: those which constitutively synthesize gp55 and those which synthesize gp55 after superinfection with type-C helper virus. No gag gene-related proteins were detected in any of these nonproducer cell lines. Characterization of Friend virus-specific RNAs in SFFV nonproducer cells indicates that they can express both a 32 S and a 21 S RNA species related to Friend virus. SFFV nonproducer cells expressing 21 S SFFV-specific RNA constitutively synthesize gp55.     

The isolation of hybrid cell lines producing monoclonal antibodies against the p15(E) protein of ecotropic murine leukemia viruses.

Hybrid cell lines were prepared by the fusion of mouse myeloma cells with the spleen cells of C57BL/6 mice that were immunized with the AKR leukemia K36. Approximately 10% of the hybrid cells produced immunoglobulins that reacted in antibody binding assays with AKR MuLV. By the combined use of low-density passage and cloning, seven independent cell lines were isolated. These cells produced antiviral antibodies at a level of 3–15 μg/ml of culture fluid. Inoculation of the hybrid cells into syngeneic mice resulted in the formation of tumors (hybridomas) that secreted extremely high levels of monoclonal antibodies (5–15 mg/ml) into the serum or ascites fluid. Five of the hybrid cell lines produced immunoglobulins of the IgM subclass, one produced IgG_2a, and one produced IgG_2b. In high-resolution two-dimensional polyacrylamide gels, these immunoglobulins showed the limited heterogeneity in heavy and light chains that would be expected for monoclonal products. Radioimmune precipitation assays demonstrated that the monoclonal antiviral antibodies reacted with the p15(E) protein of ecotropic MuLV; these antibodies did not react with the p15(E) protein of xenotropic MuLV. In contrast, rabbit antiserum prepared against purified p15(E) reacted equally well with ecotropic and xenotropic MuLV. The sera or ascites fluids from hybridoma-bearing mice had antibody titers 75- to 100-fold higher than the sera from conventionally immunized mice or rabbits. Serological analysis demonstrated that the monoclonal antibodies reacted with the cell surface of virus-producing leukemia cells, but not with normal thymocytes. Furthermore, monoclonal anti-pl5(E) antibodies of the IgG_2a subclass mediated lysis of the virion of ecotropic MuLV in the presence of complement.     

The avian sarcoma virus PRCII lacks 1020 nucleotides of the fps transforming gene

Fujinami sarcoma virus (FSV) and PRCII avian sarcoma virus both encode gag-fps transforming proteins associated with tyrosine-specific protein kinase activity; however, PRCII has a lower oncogenic potential than does FSV. In this study, the genomes of PRCII and FSV have been compared. By hybridization of PRCII [32P]RNA to FSV DNA on Southern blots, a large internal deletion in the 5' half of the fps gene in PRCII has been mapped. To determine the exact size and location of the deletion in PRCII, dideoxy sequencing of PRCII RNA with FSV DNA fragments as primers was used. The FSV sequence corresponding to the deletion in PRCII was flanked by 6-base direct repeats ( AGCTGG ) at 1614-1619 and 2634-2639 nucleotides. One copy of the direct repeat was retained in the PRCII genome. The length of the deleted region was 1020 nucleotides. The deletion in fps did not alter the kinase domain or ATP-binding site of the P105 transforming protein of PRCII. It was shown that the specific kinase activity of P105 was as high as that of FSV P130 . The sequence deleted from PRCII was found to encode part of a large hydrophilic domain. In the accompanying paper [J. Woolford and K. Beemon (1984) Virology 135, 168-180], evidence that the PRCII and FSV proteins have different subcellular locations and solubility properties, possibly due to the loss of this domain, is presented. These alterations in the structure and location of the PRCII protein may prevent it from phosphorylating certain substrates involved in oncogenic transformation.     

Structural analysis of Rauscher virus Gp70 using monoclonal antibodies: sites of antigenicity and P15(E) linkage

A linear map of 19 monoclonal antibody-binding domains on Rauscher retroviral gp70 was generated using a technique we have designated PEC-MAP (partial enzymatic cleavage-monoclonal antibody precipitation). We used eight proteolytic enzyme preparations in limited digests to produce 39 gp70 fragments. Immune precipitation of these fragments by monoclonal antibodies from 51 cell lines allowed us to define 19 binding sites by virtue of overlapping fragments and differential precipitation patterns. The sites most accessible to proteolytic attack and antibody binding were then mapped by analyzing the apparent molecular weights of the various gp70 fragments. This analysis revealed three hyperreactive regions in gp70 located approximately within the first 2000 daltons of the amino end of gp70 as well as 18,000 and 38,000 daltons from the amino end of the M_r 47,000 deglycosylated gp70 molecule. In addition, we used monoclonal antibodies directed against the disulfide-linked p15(E) molecule to localize its linkage site to Domain XVII, estimated to be between 34,000 and 38,000 daltons from the amino end of deglycosylated gp70. These data place certain constraints on the tertiary structure of gp70 and suggest a mechanism for the generation of leukemogenic MCF recombinants.     

Generation of AKR mink cell focus-forming virus: nucleotide sequence of the 3' end of a somatically acquired AKR-MCF

The 3' end of an AKR-MCF provirus (MCFr35) was cloned and found to be biologically active. Comparison of the nucleotide sequence of MCFr35 with the sequence of other MuLVs revealed that the MCFr35 was most likely derived from the same xenotropic and ecotropic parents, which were involved in the generation of AKR-MCF247. Ecotropic sequences are present around the XbaI site at position 7.9 on the genomic map, and in the long terminal repeat. Most of the T1 oligonucleotide sequences, characteristic for the leukemogenic "class I" MCFs, are also present in MCFr35, with the exception of T1 oligonucleotides 108 and 18. The MCFr35 LTR contains a duplicated enhancer sequence from a xenotropic-like provirus, which is present only once per haploid genome equivalent. The 3' end of MCFr35 consist predominantly of nonecotropic sequences, thereby delimiting the positions of recombination in various MCF viruses.     

Restricted expression of integrated primate type-C virus (gibbon ape leukemia virus-simian sarcoma virus) proviral DNA in nonproductively infected human B lymphoblasts

Using the Southern blotting technique, we have analyzed the presence and state of woodchuck hepatitis virus (WHV) DNA in liver and serum of woodchucks. Among chronic carriers essentially two forms of infection could be distinguished: in the first one WHV DNA was detected in the liver only, integrated in cellular DNA, with no detectable WHV DNA in the serum; in the second one, large amounts of unintegrated WHV DNA were present in the liver, often accompanied by WHV DNA in the serum. Very similar results have been reported with hepatitis B virus infection in man. This analogy further underlines the possibility of using WHV infection of woodchucks as a model for the understanding of hepatitis B virus-associated diseases, especially hepatocarcinoma.     

The purification of rubella virus (RV) and determination of its polypeptide composition

Rubella virus (RV) has been propagated in murine fibroblasts (L cells) and purified using two Renografin gradients. The virus was grown in the presence of 2 μCi/ml [³H]uridine, pelleted from tissue culture media 6 days postinfection, and applied to a 25–45% discontinuous gradient. A single, sharp band was observed at the interface. This band was collected and applied to a 30–45% continuous gradient which separated intact labeled virions from ³H-labeled, light density material. Infectivity was measured using a modified hemadsorption assay. A recovery of 90% of the RV infectivity was achieved by these methods. Purified virions obtained in this way were dissociated, labeled with ¹²⁵I, immune precipitated with rubella-specific antiserum, and subjected to polyacrylamide slab gel electrophoresis and autoradiography. Four polypeptides were observed with molecular weights of 44, 41, 24, and 19 × 10³ designated as VP44, VP41, VP24, and VP19, respectively.     

Generation of a mouse mammary tumor virus (MMTV) pseudotype of Kirsten sarcoma virus and restriction of MMTV gag expression in heterologous infected cells.

C3H mouse mammary tumor cells producing mouse mammary tumor virus (MMTV) were cocultivated with nonproducer mouse cells (KNIH) transformed by Kirsten sarcoma virus (KiSV). These cocultivated cells were then treated with mitomycin C and overlayed onto human embryonic skin and muscle cells. The virus resulting from this cocultivation could be titrated in a focus-forming assay on Fischer rat embryo (FRE) cells exhibiting one-hit kinetics. Furthermore, focus formation on FRE cells was neutralized specifically by antiserum directed against MMTV and the major MMTV external glycoprotein gp52, but not against a broadly reactive antiserum directed murine leukemia virus (MuLV) gp70 and MMTV gp36, p27, p14, and p10. These results demonstrate the generation of a KiSV(MMTV) pseudotype and further demonstrate that gp52 is a target antigen for neutralization of MMTV. This pseudotype possessed a wide host range, transforming cells of human, rat, mouse, mink, and rabbit origin. MMTV but not MuLV antigen expression was demonstrated in the KiSV(MMTV) pseudotype-infected cells. Analysis of intracellular MMTV protein synthesis in these in vitro-infected cells has indicated that the low yield of extracellular MMTV produced by the transformed cells may be the result of the poor expression of the MMTV gag precursor polyprotein relative to the expression of the env gene polyprotein. These studies thus provide the basis for an in vitro infectivity assay for neutralization and host range studies of MMTV.     

Amplified expression of murine leukemia virus (MuLV)-coded antigens on thymocytes and leukemia cells of AKR mice after infection by dualtropic (MCF) MuLV

Recently, we have shown that several isolates of recombinant, dualtropic (MCF) murine leukemia virus (MuLV) can induce amplification of MuLV gag and env gene-coded antigens on thymocytes of young AKR mice and, subsequently, can accelerate leukemia in these same animals. With respect to env gene-coded antigens, it is unclear whether antigen amplification represents expression of env gene products of the input virus on the surface of infected thymocytes or whether infection by MCF viruses induces the expression of other endogenous env gene sequences. Infection by antigenically marked viruses has allowed us to decide between these alternatives. We have found that AKR MCF isolates 69L1,13, and 247 can be distinguished serologically by specific patterns of reactivity with mouse and rat monoclonal antibodies that recognize 10 distinct epitopes (single antigenic determinants) of AKR ecotropic MuLV gp70 and p15(E) proteins. Accordingly, 1-monthold AKR mice were injected intrathymically with either MCF 69L1, MCF 13, or MCF 247 viruses and thymocytes of virus-injected mice were then assayed for amplification of specific epitopes at 32—36 days postinjection. Quantitative expression of MuLV antigens was determined by flow microfluorometry using a fluorescence-activated cell sorter. The patterns of reactivity with monoclonal antibodies which were originally determined for each virus in vitro were found to breed true on thymocytes infected in vivo. Moreover, the phenotype of virus recovered in vitro from virus accelerated leukemias was identical to that of the injected virus. Thus, the preleukemic change of MuLV antigen amplification appears to represent expression on thymocytes of env gene products encoded by the infecting MCF viral genome which continues to be expressed in the resulting leukemias without apparent changes in the recombinant env gene.     

Characterization of a papilloma virus from the European elk (EEPV)

A novel papilloma virus (EEPV), morphologically resembling other members of the papovavirus group has been isolated from elk papillomas. The EEPV DNA is superhelical and has a molecular weight of 5.4 × 10⁶. Cleavage with the restriction endonucleases HaeIII, HindII, HindIII, and BglII suggests that the EEPV DNA is different from all bovine papilloma virus DNAs characterized so far. Hybridization between EEPV DNA and bovine papilloma virus type 1 or 2 DNA revealed no sequence homology under stringent hybridization conditions.     

Viral expression in two myelomonocytic cell lines obtained from long-term bone marrow culture infected with the friend polycythemia-inducing virus (FV-P)

Two subclones of monomyelocytic cell lines (317 and G-31), issued from mouse long-term bone marrow cultures infected with Friend polycythemia-inducing virus, were studied for virus release and viral protein expression. The 317 cell line permanently produced biologically active spleen focus-forming virus (SFFV) and expressed the gp52^SFFV which has been described as encoded by the SFFV genome. In contrast, the G-31 cell line was devoid of any SFFV expression although it released Friend helper leukemia virus (FMuLV). Both lines expressed a new gp70-related glycoprotein with an apparent molecular weight of 66,000. This gp66 species was metabolically unstable and was not packaged into viral particles nor released in culture medium. It was recognized by an anti-Rauscher gp70 antiserum but not by anti-MCF gp70 or anti-p15E antisera. gp66 was not found in erythroblastic Friend tumor cell lines, in fibroblasts infected with virus released by lines 317 and G-31, or in nonmyelomonocytic infected hematopoietic cells. However, it was constantly observed in cell lines with a myelomonocytic origin established in vivo and in vitro. Therefore, gp66 is a F-MuLV gp70-related product specifically linked to the myeloid lineage.     

Biochemical studies of the maturation of the small Sindbis virus glycoprotein E3

A small glycoprotein (E3) was purified from the culture fluid of Sindbis virus-infected primary chick embryo fibroblasts. Tryptic peptide mapping and pulse-chase studies verified that this protein was produced as a by-product of the cleavage of the precursor protein PE2 to produce the envelope glycoprotein E2. A 2600-fold purification was achieved via a procedure which used differential ethanol precipitation, gel filtration, ion-exchange chromatography, and affinity chromatography on a lentil lectin column. Amino acid composition analysis, N-terminal microsequencing, and labeling studies yielded information about the fine structure of E3 and its relationship to E2 and virion maturation. The N-terminal sequence of E3 is identical to that of PE2, including the result that 90% of the molecules appear to be blocked. The first 19 amino acids are uncharged and presumably serve as the signal sequence for the insertion of PE2 into the membrane of the endoplasmic reticulum, but this sequence is unusual in that it is not immediately cleaved from PE2 and is glycosylated at the asparagine at position 14. The two residues at the C-terminus of E3, Lys-Arg, are removed during or shortly after cleavage from PE2. Labeling studies imply that, although the PE2----E2 + E3 cleavage is necessary for virion budding, these two events are not closely coupled. E3 is cleaved and released into the culture fluid under conditions where virions do not bud, and the kinetics of the appearance of E3 in the culture fluid and E2 in virions are quite dissimilar. The maturation of E3 is discussed as it relates to the processing of cellular membrane and secretory glycoproteins.