A novel locus regulates both retroviral glycoprotein 70 and anti-glycoprotein 70 antibody production in New Zealand mice when crossed with BALB/c

Lupus-prone New Zealand Black and New Zealand White mice produce high serum levels of the endogenous retroviral envelope protein gp70 and develop an Ab response to this autoantigen as part of their autoimmune disease. Linkage analysis of two crosses involving New Zealand and BALB/c mice mapped these traits to a group of overlapping loci, including a novel locus on proximal chromosome 12. This locus was linked with serum gp70 and the autoimmune response against it. The linkage of serum gp70 levels to a previously described locus on distal chromosome 4 was also confirmed. Sequence analysis of a candidate gene on distal chromosome 4, Fv1, provided support that this gene may be associated with the control of serum gp70 levels in both New Zealand Black and New Zealand White mice. Linkage data and statistical analysis confirmed a close correlation between gp70 Ag and anti-gp70 Ab levels, and together gave support to the concept that a threshold level of gp70 is required for the production of anti-gp70 Abs. Serum levels of anti-gp70 Abs were closely correlated with the presence of renal disease, more so than anti-dsDNA Abs. Understanding the genetic basis of this complex autoantigen-autoantibody system will provide insight into the pathogenesis of lupus in mice, which may have implications for human disease.     

Glycoproteins of friend murine leukemia virus: separation and NH2-terminal amino acid sequences of gp69 and gp71.

The NH2-terminal amino acid sequences (initial 23 residues) of Friend murine leukemia virus gp71 and gp69 were determined and found to be different but highly related. Friend murine leukemia virus gp71 differed from Rauscher murine leukemia virus gp70 in only one position. Friend murine leukemia virus gp69 showed approximately 41% homology to these glycoproteins but lacked the glycosylation site (sequon) occurring at position 12 in Rauscher murine leukemia virus gp70.     

Identification of a mouse gene required for binding of Rauscher MuLV envelope gp70.

Mouse chromosome segregating somatic cell hybrids were established between a mouse thymic leukemai cell line (GRSL) and Chinese hamster E36 cells. The GRSL cells specifically bound purified Rauscher leukemia virus gp70 while the E36 cells exhibited no binding. The hybrids selectively bound Ruascher gp70 depending on the presence of a mouse cellular gene for the ecotropic murine luekemia gp70 receptor. A syntenic relationship was observed between the DIP-3 chromosome marker (on chromosome 5) and the gp70 receptor in primary clones and subclones of these hybrids; this was confirmed by chromosome analysis. The involvement of H-2 in the binding of Rauscher MuLV gp70 could be ruled out, because discordancies of the receptor presence and H-2 absence as well as of the receptor absence and H-2 presence type could be observed. Our results indicate that the Rec-1 (replication ecotropic MuLV) gene of Gazdar et al. (4) may well be the receptor gene for the ecotropic murine leukemia virus.     

Genetic control of glycoprotein 70 autoantigen production and its influence on immune complex levels and nephritis in murine lupus

The F1 hybrids of New Zealand Black (NZB) and New Zealand White (NZW) mice spontaneously develop an autoimmune disease that serves as a model for human systemic lupus erythematosus. Autoimmunity in (NZB x NZW)F1 mice includes the production of autoantibodies to the endogenous retroviral envelope glycoprotein, gp70, and gp70-anti-gp70 immune complexes (gp70 IC) have been implicated in the development of lupus nephritis in these animals. We used backcross and intercross combinations of C57BL/6 (B6; low gp70 levels) and NZB mice (high gp70 levels) to examine the contribution of serum gp70 Ag levels to the development of gp70 IC and nephritis. Analysis of (B6.H2z x NZB)F1 x NZB backcross mice and (NZB x B6)F2 mice showed a much stronger association of gp70 IC with kidney disease compared with IgG anti-chromatin autoantibodies in both populations of mice. Serum levels of gp70 correlated with production of gp70 IC in mice producing autoantibodies, although the overall effect on nephritis appeared to be small. Genetic mapping revealed three NZB-derived regions on chromosomes 2, 4, and 13 that were strongly linked with increased gp70 levels, and together, accounted for over 80% of the variance for this trait. However, additional linkage analyses of these crosses showed that loci controlling autoantibody production rather than gp70 levels were most important in the development of nephritogenic immune complexes. Together, these studies characterize a set of lupus-susceptibility loci distinct from those that control autoantibody production and provide new insight into the components involved in the strong association of gp70 IC with murine lupus nephritis.     

Radiommunoassays for the 70,000-molecular-weight glycoproteins of endogenous mouse type C viruses: viral antigen expression in normal mouse tissues and sera.

Genetic information coding for type C RNA viruses is transmitted within the DNA of mouse cells. At least three endogenous viruses have so far been immunologically distinguished by radioimmunoassays for their 12,000-molecular-weight polypeptides (p12). In the present study, the 70,000-molecular-weight glycoproteins (gp70) of three prototype viruses were purified, and competition radioimmunoassays were developed for each. By use of these immunoassays, the antigenic determinants of gp70's of different classes of endogenous virus, isolated from the same and from a variety of other mouse strains, were readily discriminated. In contrast, viruses of the same class were indistinguishable. These findings further document the existence of three distinct endogenous viruses of mouse cell. The levels of type C viral gp70 were quantitated in tissues and sera of several inbred strains. The pattern of immunological reactivity of the gp70 detected in serum was indistinguishable from that of the viral gp70 partially purified from tissues of the same strain. Moreover, in each case it was indistinguishable from that of a specific class of endogenous virus. In virus-negative tissues of BALB/c and NIH Swiss mice, the viral gp70 detected was shown to be representative of a class III endogenous virus whose p12 polypeptide was also expressed by the same cells.     

Establishment of monoclonal anti-retroviral gp70 autoantibodies from MRL/lpr lupus mice and induction of glomerular gp70 deposition and pathology by transfer into non-autoimmune mice

Several strains of mice, including MRL/MpJ mice homozygous for the Fas mutant lpr gene (MRL/lpr mice), F(1) hybrids of New Zealand Black and New Zealand White mice, and BXSB/MpJ mice carrying a Y-linked autoimmune acceleration gene, spontaneously develop immune complex-mediated glomerulonephritis. The involvement of the envelope glycoprotein gp70 of an endogenous xenotropic virus in the formation of circulating immune complexes and their deposition in the glomerular lesions have been demonstrated, as has the pathogenicity of various antinuclear, antiphospholipid, and rheumatoid factor autoantibodies. In recent genetic linkage studies as well as in a study of cytokine-induced protection against nephritis development, the strongest association of serum levels of gp70-anti-gp70 immune complexes, rather than the levels of antinuclear autoantibodies, with the development and severity of glomerulonephritis has been demonstrated, suggesting a major pathogenic role of anti-gp70 autoantibodies in the lupus-prone mice. However, the pathogenicity of anti-gp70 autoantibodies has not yet been directly tested. To examine if anti-gp70 autoantibodies induce glomerular pathology, we established from unmanipulated MRL/lpr mice hybridoma clones that secrete monoclonal antibodies reactive with endogenous xenotropic viral env gene products. Upon transplantation, a high proportion of these anti-gp70 antibody-producing hybridoma clones induced in syngeneic non-autoimmune and severe combined immunodeficiency mice proliferative or wire loop-like glomerular lesions. Furthermore, deposition of gp70 in glomeruli and pathological changes were observed after intravenous injection of representative clones of purified anti-gp70 immunoglobulin G, demonstrating pathogenicity of at least some anti-gp70 autoantibodies.     

Plasma membrane glycoproteins encoded by cloned Rauscher and Friend spleen focus-forming viruses.

Rauscher spleen focus-forming virus (SFFV) was cloned free of its helper virus into normal rat kidney and mouse fibroblasts, and the resulting nonproducer fibroblast clones were analyzed. Our results suggested that Rauscher SFFV encodes a glycoprotein with an apparent Mr of 54,000 (gp54) that reacts with antisera made to the envelope glycoprotein (gp70) of ecotropic murine leukemia viruses, as well as with a rat antiserum that reacts with the gp70's of dual-tropic mink cell focus-inducing and HIX viruses but not with the gp70's of ecotropic viruses. In these respects and in its tryptic peptide map, Rauscher SFFV-encoded gp54 is nearly identical to the gp55 glycoprotein which we previously reported to be encoded by Friend SFFV (Dresler et al., J. Virol. 30:564--575, 1979). However, gp54 is slightly smaller, and it lacks one methionine-containing tryptic peptide that occurs in gp55. Studies with cytotoxic antiserum in the presence of complement and with a rosetting technique which employed sheep erythrocytes coupled to protein A suggested that the gp54 and gp55 glycoproteins are weakly expressed on the surface membranes of SFFV-infected cells. In addition, the Rauscher SFFV genome also encodes gag polyproteins which appear to be identical to the gag polyproteins encoded by helper Rauscher murine leukemia virus, but differ from the antigenically related polyproteins encoded by some but not all clones of Friend SFFV. Furthermore, the glycosylated gag polyproteins encoded by Rauscher SFFV and by some Friend SFFVs also appear to be expressed on the surface membranes of infected cells. These results suggest that similar env gene recombination and partial deletion events were involved in the independent origins of two different strains of acute erythroleukemia virus.     

Interspecies determinants of Friend leukemia virus antigens involved in cytolysis of virus-pfoducing cells.

The cytolytic reactivity of a complex goat anti-feline leukemia virus (FeLV) antiserum for mouse cells (Eveline) releasing large quantities of Friend leukemia virus (FLV) was analyzed by the sensitive [14C]nicotinamide release microcytotoxicity assay. Whereas this interspecies killing reactivity could be blocked by absorption of the goat anti-FeLV serum with a preparation of disrupted FLV, absorption with purified FLV gp71, the major envelope glycoprotein, had no effect. Subsequent serum absorptions with the individual FLV structural polypeptides revealed that the lysis of the Eveline cells by the goat anti-FeLV serum is mediated by antibodies recognizing the interspecies determinant of p30, the major internal capsid protein. The expression of this internal viral component at the surface of virus-producing cells is discussed further. The results also demonstrated that removal of approximately 70% of the carbohydrate portion of gp71 with a preparation of glycosidases did not affect the integrity of its interspecies determinant; these results are in agreement with an earlier study (Bolgnesi et al., 1975) that examined primarily the group- and type-specific sites.     

Isolation and characterization of a mouse cell line containing a defective Moloney murine leukemia virus genome.

A culture of mouse cells containing a 1,000-nucleotide deletion mutant of Moloney murine leukemia virus has been isolated. The deletion did not affect the size or function of the 21S mRNA that encodes the env gene products. Both the deleted RNA and the 21S mRNA were recovered in polyribosomes. Cells containing the deleted virus made no detectable Pr180gag-pol. Pr65gag synthesis with also absent, but a 45,000-molecular-weight gag gene product was found that might be encoded by the deleted genome. Biosynthesis of Pr80env proceeded normally in these cells; the intracellular precursor was cleaved and migrated to the cell surface as gp70. The cells could not be superinfected by homologous Moloney murine leukemia virus presumably because of surface restriction due to the gp70. Although the cells express the Moloney murine leukemia virus gp70 on their surface, they will not make pseudotypes after infection with vesicular stomatitis virus implying that Pr65gag may play a critical role in pseudotype formation. Induction of endogenous virus expression in the cells carrying the deletion mutant generated an N-tropic murine leukemia virus that can fuse XC cells. This may represent a recombinant between the deletion mutant and an endogenous virus.     

Origin of the minor glycoproteins of murine leukemia viruses.

Polyacrylamide gel electrophoretic analysis and immunoprecipitation were used to study glycoproteins from purified Rauscher murine leukemia virus (R-MuLV) and from AKR thymic lymphoblastoid cell membranes. In addition to gp70, a minor glycoprotein of approximately 52,000 daltons (gp52) was demonstrated in purified R-MuLV preparations, which was antigenically related to gp70. Analysis of R-MuLV glycopeptides obtained after exhaustive Pronase digestion showed that gp70 has at least two different glycopeptide size classes with molecular weights of 5,100 and 2,900, respectively. gp52, however, contained only a single glycopeptide size class of approximately 5,100 daltons, indicating that the two glycoproteins contain distinct carbohydrate components. Trypsin treatment of R-MuLV converted gp70 into a product with a molecular mass of approximately 52,000 daltons as well as a 45,000-dalton minor product, with little effect on virus infectivity. Similarly, trypsin treatment of 125I-labeled glycoproteins derived from AKR mouse lymphoblastoid cell membranes generated fragments antigenically related to gp70 and similar in size to those obtained by trypsin treatment of R-MuLV. In both cases, the appearance of cleavage products was accompanied by a decrease in gp70 during trypsin treatment. The occurrence of glycosylated components antigenically related to gp70 in AKR membrane glycoprotein preparations and in purified R-MuLV preparations which were similar to those generated by trypsin treatment supports the concept that these minor components arise from proteolytic cleavage of gp70.     

Characterization of murine-specific leukemia virus receptor from L cells.

The host cell receptor for Moloney murine leukemia virus was solubilized from murine L-cell membranes and characterized. In initial studies designed to identify a receptor-rich cell line, different mouse cells were screened for binding to Moloney gp70, the viral envelope glycoprotein which determines host cell-binding specificity. gp70 binding to murine L cells was specific and saturable, with an apparent affinity constant (Ka) of 4 X 10(8) M-1, and the number of receptors per cell (6 X 10(5)) was similar to that of other mouse fibroblast cell lines. Characterization of the gp70 receptor with regard to extraction by detergents, protease sensitivity, and heat denaturation suggests that the receptor is an intrinsic membrane protein. Upon extraction of L-cell membranes with 0.2% deoxycholic acid and precipitation with acetone, specific and saturable binding of gp70 could be detected. The solubilized gp70-binding component was eluted upon gel filtration on Sephacryl S-300 into a species with an approximate molecular weight of 110,000.     

Purification and serological characterization of the major envelope glycoprotein from AKR murine leukemia virus and its reactivity with autogenous immune sera from mice.

The major envelope glycoprotein (gp71) from AKR murine leukemia virus (MuLV) was purified and its serological reactivity with heterologous and autogenous immune mouse sera was examined. Homologous and interspecies competition radioimmunoassays using antisera to Rauscher-MulV gp69/71 or Friend-MuLV gp71 or antisera to feline leukemia virus to precipitate 125I-labeled gp71 from various MuLV showed that distinct differences exist between Rauscher- or Friend-MuLV and AKR-MuLV glycoproteins. Characteristically the AKR-MuLV gp71, in contrast to FLV or RLV gp71, does not compete fully in homologous or interspecies radioimmunoassays with iodinated Friend of Rauscher glycoproteins. Purified 125I-labeled AKR-MuLV gp71, in contrast to the Rauscher- or Friend-MuLV glycoproteins, reacts with normal (autogenous immune) mouse sera in direct radioimmune precipitation assays. Competition experiments further demonstrate that this is a predominant immunological reactivity of normal mouse sera which had previously been detected by radioimmune precipitation assay against intact virions.     

Posttranslational modifications distinguish the envelope glycoprotein of the immunodeficiency disease-inducing feline leukemia virus retrovirus.

The envelope glycoprotein (gp70) of a molecularly cloned, replication-defective feline leukemia virus (FeLV-FAIDS clone 61C) carries determinants for induction of fatal immunodeficiency disease, whereas the gp70 of its companion replication-competent, probably parent virus (clone 61E) does not. Immunoprecipitation analysis of the extracellular glycoproteins of 61E and EECC, a replication-competent viral construct composed of the 61C env and 3' long terminal repeat fused to the 61E gag-pol genes, demonstrated that the gp70 of EECC could be distinguished from that of 61E by both feline immune serum and a murine monoclonal antibody. Molecular weights of both the envelope precursor polyprotein (gp80) and the mature extracellular glycoprotein (gp70) of 61E were smaller than the corresponding proteins from the pathogenic EECC. Both the molecular weight disparity and monoclonal antibody discrimination of the two gp80s were abolished by inhibition of envelope protein glycosylation with tunicamycin, whereas the apparent gp70 size differences were resolved by enzymatic removal of N-linked oligosaccharides. Pulse-chase studies in EECC-infected cells demonstrated that processing of gp80 to gp70 was delayed and that this retardation of envelope glycoprotein processing could be simulated in 61E-infected cells by treatment with the glucosidase inhibitor N-methyldeoxynojirimycin, a compound that causes retention of oligosaccharides in the high-mannose form. The resultant 61E gp70 then could be recognized by sera from EECC-immunized cats. The presence of a higher content of sialic acid on the apathogenic 61E gp70 indicated that oligosaccharides of 61E and EECC gp70 were processed differently. These data suggested that the unique biochemical properties which distinguish the envelope glycoproteins of the FeLV-FAIDS variant from its companion apathogenic parent virus were responsible for T-cell cytopathicity and induction of immunodeficiency disease. Further biochemical characterization of these glycoproteins should be useful in understanding the pathogenic mechanisms of immunodeficiency disease induced by retroviruses.     

Monoclonal antibody to spleen focus-forming virus-encoded gp52 provides a probe for the amino-terminal region of retroviral envelope proteins that confers dual tropism and xenotropism.

Monoclonal antibodies which recognize a region common to Friend spleen focus-forming virus encoded gp52 and Friend mink cell focus-inducing viral gp70 were isolated. One such antibody from hybridoma 7C10 was tested extensively in immune precipitation and was found to react with a determinant on envelope gp70s of all mink cell focus-inducing, xenotropic, and amphotropic mouse retroviruses tested, but not with envelope gp70s of ecotropic viruses, including Friend, Moloney, and AKR murine leukemia viruses. Monoclonal antibody from hybridoma 7C10 precipitated a 23,000-molecular-weight fragment, derived by V8 protease digestion of Friend mink cell focus-inducing gp70. This 23,000-molecular-weight peptide was determined to derive from the amino terminus of the molecule. These results correlate well with other genetic data which indicate that endogenously acquired sequences of mink cell focus-inducing viruses are found at the 5' end of the envelope gene.     

Antigenic determinants of the 70,000 molecular weight glycoprotein of woolly monkey type C RNA virus.

The 70,000 molecular weight glycoprotein (gp70) of a type-C RNA virus originally isolated from a woolly monkey has been partially purified and immunologically characterized. Evidence that this viral protein is viral coded was derived from studies showing its antigenic properties to be unaltered by virus passage in cells of different species. A broadly reactive competition immunoassay was developed utilizing antiserum prepared against feline leukemia virus to precipitate 125I-labeled woolly monkey virus gp70. Gibbon and woolly viruses, as well as feline and several mouse type-C viruses, all reacted with equal efficiency in this assay. In contrast, an endogenous virus of the baboon failed to cross-react, suggesting that viruses of this latter group are less immunologically related to the others. In a homologous competition immunoassay for the woolly viral glycoprotein, the woolly virus was readily distingusihed from otherwise colsely related viruses of gibbon apes. These findings demonstrate the pronounced type-specific antigenic dterminants possessed by this viral protein. The antigenic determinants of gp70 responsible for neutralization have also been investigated.     

Characterization of structural and immunological properties of specific domains of Friend ecotropic and dual-tropic murine leukemia virus gp70s.

A detailed comparison of the gp70 proteins of cloned ecotropic Friend murine leukemia virus (FLV) and dual-tropic Friend mink focus-forming virus (FrMCF) was performed by analyzing the structural and immunological properties of amino- and carboxy-terminal domains of these molecules generated upon controlled trypsinization. The two gp70s gave characteristic fragmentation patterns; the amino-terminal fragments of FrMCF gp70 were smaller than the corresponding fragments of FLV and contained a trypsin site which resulted in a 19,000-dalton amino-terminal fragment not observed for FLV, whereas both molecules yielded an identically sized carboxy-terminal fragment. All amino-terminal fragments of both gp70 molecules contained an endo H-sensitive oligosaccharide chain; for FrMCF, a second endo H-sensitive carbohydrate was present as well at a carboxy-terminal site for approximately 50% of the molecules. Several aspects of the disulfide interactions of the two gp70s were conserved; in both cases the carboxy-terminal fragments were disulfide bonded to p15(E), there were no disulfide bonds between amino- and carboxy-terminal fragments, and the amino-terminal fragments exhibited a significant increase in mobility upon analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. Analysis of the immunoreactivity of the different domains of the proteins by immunoprecipitation of the fragments with antisera prepared against xenotropic murine leukemia virus and feline leukemia virus gp70s indicated major differences in antigenicity for the amino-terminal domains of FLV and FrMCF gp70, whereas the carboxy-terminal domains were immunologically conserved. Similar analyses with antibodies specific for p15(E) and Pr15(E) demonstrate that these components are conserved as well. These data provide direct evidence that p15(E) and the C-terminal gp70 domain of FrMCF gp70 are related to the corresponding regions of the ecotropic FLV parent and indicate that the acquisition of MCF-specific properties is due to the replacement of the ecotropic amino-terminal gp70 domain with sequences related to those of xenotropic gp70s.     

Pathogenicity of a Mutant Friend Spleen Focus-Forming Virus Encoding an Env-Like Membrane Glycoprotein (gp55) with Substitution by a Xenotropic Murine Leukemia Virus Env gp70 Sequence

Friend spleen focus-forming virus (F-SFFV) is a replication-defective acutely leukemogenic mouse retrovirus and encodes an envelope protein (Env)-like membrane glycoprotein (gp55) in its defective env gene, which is responsible for the early stage of the viral leukemogenesis. Gp55 is a modified Env protein and contains a polytropic mink cell focus-inducing (MCF) murine leukemia virus (MuLV) Env gp70-derived sequence in its amino-terminal region. To evaluate the possibility that the presumed binding of gp55 to an MCF MuLV receptor protein has some role in leukemogenesis, we examined the biological activities of a mutant gp55 (XE gp55), which has a xenotropic MuLV Env gp70 amino-terminal region. XE gp55 displayed almost the same biological activities as the wild-type gp55, excluding the above possibility.     

Biological, Chemical, and Immunological Studies of Rauscher Ecotropic and Mink Cell Focus-Forming Viruses from JLS-V9 Cells

Two murine leukemia viruses were isolated from JLS-V9 cells which had been infected with Rauscher plasma virus. One virus was XC positive and failed to grow on mink or cat cells and thus was an ecotropic virus. The other virus formed cytopathic foci on mink cells, was XC negative, and fell into the mink cell focus-forming (MCF) viral interference group and was thus an MCF virus. The glycoproteins of the two viruses could be distinguished immunologically, by peptide mapping, and by size in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The MCF virus produced gp69, and the ecotropic virus produced gp71, explaining the origin of the heterogeneous glycoprotein (gp69 and gp71) of Rauscher leukemia virus. Amino-terminal sequences of gp69 and gp71 were determined. The MCF sequence was distinct from the ecotropic sequence, but retained partial homology to it. The data show that the glycoproteins are encoded by related yet distinct genes. The protein structural data support the proposal that MCF virus gp70 molecules have nonecotropic sequences at the amino terminus, with ecotropic sequences occurring at the 3' end of the gene. The Rauscher MCF virus glycoprotein lacks a glycosylation site found at position 12 of the ecotropic sequence.     

Anomalous reactions of mouse alloantisera with cultured tumor cells. II. Cytotoxicity is caused by antibodies to leukemia viruses.

Certain alloantisera prepared in mice against H-2 region membrane antigens were found to be unexpectedly cytotoxic for murine sarcoma and leukemia cells in culture. This anomalous cytotoxicity was shown to be the result of antibody in these alloantisera directed against the p15 and gp70 envelope proteins of Mu LV which were present on the surface of the tumor target cells. Sera from aged unimmunized mice of strains used for the preparation of alloantisera also contained antibodies against MuLV protein p15 and gp70 that were cytotoxic for sarcoma and leukemia cells, which indicates that these antibodies occurred naturally in mice. These results independently confirm earlier findings of the widespread occurrence in mouse serum of antibodies reactive with MuLV. The presence of antibody against MuLV in mouse serum which can cause cytotoxic reactions with tumor cells points to the fact that particular caution should be used during the typing of murine sarcomas or leukemias for cell surface antigens, since mouse antisera may yield cytotoxicity (or other serologic reactions) based on anti-MuLV specificities, rather than on anticipated antigens.