Biomedical subjects

D Kabat

Publications and source records attributed to D Kabat.

At least 73 records · Page 4Linked to original sources

PubMed1992

Role of the PU.1 transcription factor in controlling differentiation of Friend erythroleukemia cells.

Both viral and cellular genes have been directly implicated in pathogenesis of Friend viral erythroleukemia. The virus-encoded gp55 glycoprotein binds to erythropoietin receptors to cause mitogenesis and differentiation of erythroblasts. However, if the provirus integrates adjacent to the gene for the PU.1 transcription factor, the cell loses its commitment to terminally differentiate and becomes immortal, as indicated by its transplantability and by its potential for indefinite growth in culture (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 63:4434-4437, 1989; R. Paul, S. Schuetze, S. L. Kozak, and D. Kabat, J. Virol. 65:464-467, 1991). To test the implications of these results, we produced polyclonal antiserum to bacterially synthesized PU.1, and we used it to analyze PU.1 expression throughout leukemic progression and during chemically induced differentiation of Friend erythroleukemia (F-MEL) cell lines. This antiserum identified three electrophoretically distinct PU.1 components in extracts of F-MEL cells and demonstrated their nuclear localization. Although PU.1 proteins are abundant in F-MEL cells, they are absent or present in only trace amounts in normal erythroblasts or in differentiating erythroblasts from the preleukemic stage of Friend disease. Furthermore, chemicals (dimethyl sulfoxide or N,N'-hexamethylenebisacetamide) that overcome the blocked differentiation of F-MEL cells induce rapid declines of PU.1 mRNA and PU.1 proteins. The elimination of PU.1 proteins coincides with recommitment to the program of erythroid differentiation and with loss of immortality. These results support the hypothesis that PU.1 interferes with the commitment of erythroblasts to differentiate and that chemicals that reduce PU.1 expression reinstate the erythropoietic program.

Animals↗

PubMed1991

Disulfide bonding controls the processing of retroviral envelope glycoproteins.

The mitogenic membrane glycoprotein (gp55) encoded by Friend erythroleukemia virus is inefficiently processed from the rough endoplasmic reticulum (RER) and only 3-5% reaches plasma membranes. Because this processed component (gp55P) contains larger and more complex oligosaccharides, it can be separated from RER gp55. In nonreducing conditions, gp55P is a unique disulfide-bonded dimer, whereas RER gp55 consists of monomers and dimers with diverse intrachain and interchain disulfide bonds. This suggests that gp55 folds heterogeneously and that only one homodimer is competent for export from the RER. Pulse-chase analyses of gp55 components labeled with radioactive amino acids indicated that formation of diverse disulfide-bonded components occurred within minutes of polypeptide synthesis and that malfolded components did not later isomerize to generate dimers competent for export from the RER. Chemical studies suggested that all 12 cysteines of gp55 were oxidized within 5 min after synthesis of the protein. In contrast, the envelope glycoprotein precursor (gPr90) encoded by a replication-competent murine leukemia virus folds more homogeneously, and it is then processed and cleaved to form an extracellular glycoprotein gp70 plus a transmembrane protein p15E. The fully processed glycoprotein contains an unoxidized cysteine sulfhydryl that isomerizes reversibly with a disulfide bond that links gp70 to p15E. Consequently, only a proportion of gp70 and p15E is disulfide-bonded, and dissociation occurs when the environment becomes even slightly reducing. The gp55 glycoprotein appears to be an extreme example of protein malfolding associated with imprecise and irreversible disulfide bonding. We discuss evidence that folding inefficiencies are common for retroviral proteins that have newly evolving pathogenic functions.

Blotting, Western↗

PubMed1991

Cell-surface receptor for ecotropic murine retroviruses is a basic amino-acid transporter.

The complementary DNA sequence encoding the cell-surface receptor for ecotropic host-range murine retroviruses (ecoR) shows that it contains 622 amino acids and 14 hydrophobic potentially membrane-spanning sequences. Because this receptor occurs on many or all murine cells and is probably essential for viability of cultured fibroblasts, its normal function might be to transport an essential metabolite. We expressed ecoR in Xenopus laevis oocytes by injecting RNA transcribed from the cloned cDNA. These oocytes specifically bound the gp70 envelope glycoprotein from an ecotropic murine leukaemia virus. An inward current was recorded electrophysiologically when a mixture of amino-acids was applied: this resulted from a stereoselective, saturable uptake of lysine, arginine and ornithine; it was independent of sodium and not substantially altered by gp70. Cysteine and homoserine were also taken up, but sodium was necessary for their transport. These properties of ecoR correspond to those of the y+ amino-acid transporter. Our results demonstrate the subversion of a ubiquitous cell membrane protein, in this case a basic amino acid transporter, for use as a retroviral receptor.

Animals↗

PubMed1991

The Sfpi-1 proviral integration site of Friend erythroleukemia encodes the ets-related transcription factor Pu.1.

Previous studies identified a common site (Sfpi-1) for proviral integration in immortalized Friend erythroleukemias. cDNAs corresponding to a 1.5-kb Sfpi-1 mRNA were isolated and sequenced. These were larger than an independently isolated Sfpi-1 cDNA described by researchers from another laboratory, and they contained common differences from that sequence, including in the coding region four extra nucleotides that altered the reading frame. The properly translated protein is identical to Pu.1, a transcription activation factor that is related to the ets oncogene family. Genetic methods were used to map Sfpi-1 with respect to other loci on mouse chromosome 2. Our results suggest that Pu.1 blocks erythroblast differentiation and thereby causes immortalization.

Amino Acid Sequence↗

PubMed1991

Plasma membrane receptors for ecotropic murine retroviruses require a limiting accessory factor.

A retroviral vector was used to express various amounts of the receptor (ecoR) for ecotropic host range murine retroviruses on naturally barren hamster, mink, and human cells. These cells and murine cells were then incubated for 2 h with dilutions of a helper-free ecotropic retrovirus that encodes human growth hormone, and the number of infected cells was later determined by growth hormone-specific immunofluorescence. For all cells under the conditions of these studies, virus adsorption was the limiting step of infection and the cellular capacities for infection were unsaturated either at cell surfaces or at intracellular sites. Thus, infections occurred at low multiplicities of infection per cell and were directly proportional to virus and cell concentrations, and only a small percentage (ca. 5%) of the infectious virions became adsorbed from the medium during the 2-h incubations. Although increasing the adsorption by raising virus or cell concentrations results in more infections in the cultures, increasing adsorption by raising the number of ecoR above a low threshold had no effect on infections. Thus, cells with a low number of ecoR were infected as efficiently as highly adsorbing cells that contained many times more ecoR. To reconcile these results, we conclude that only a small, set number of cell surface ecoR can be functional for infection and that all excess ecoR can only bind virus into an unsalvageable pool. Therefore, retroviral receptors on single cells are functionally diverse. Our results suggest that activity of ecoR in infection requires a limiting second cellular component.

3T3 Cells↗

PubMed1991

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis.

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.

Amino Acid Sequence↗

PubMed1990

Activation of erythropoietin receptors by Friend viral gp55 and by erythropoietin and down-modulation by the murine Fv-2r resistance gene.

The leukemogenic membrane glycoprotein (gp55) encoded by Friend spleen focus-forming virus appears to bind to erythropoietin receptors (EpoR) sto stimulate erythroblastosis [Li, J.-P., D'Andrea, A.D., Lodish, H.F. & Baltimore, D. (1990) Nature (London) 343, 762-764]. To directly compare the effects of gp55 with erythropoietin (Epo), we produced retrovirions that encode either gp55, Epo, or EpoR. After infection with EpoR virus, interleukin 3-dependent DA-3 cells bound 125I-labeled Epo and grew without interleukin 3 in the presence of Epo. These latter cells, but not parental DA-3 cells, became factor-independent after superinfection either with Epo virus or with Friend spleen focus-forming virus. In addition, Epo virus caused a disease in mice that mimicked Friend erythroleukemia. Although Fv-2r homozygotes are susceptible to all other retroviral diseases, they are resistant to both Epo viral and Friend viral erythroleukemias. These results indicate that both gp55 and Epo stimulate EpoR and that the Fv-2 gene encodes a protein that controls response to these ligands. However, the Fv-2 protein is not EpoR because the corresponding genes map to opposite ends of mouse chromosome 9. These results have important implications for understanding signal transduction by EpoR and the role of host genetic variation in controlling susceptibility to an oncogenic protein.

Animals↗

PubMed1990

Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production.

Retroviral vectors offer major advantages for gene transfer studies but have not been useful for producing proteins in large quantities. This deficiency has resulted in part from interference to superinfection, which limits the numbers of active proviruses in cells. Recently, we found that these vectors amplify when they are added as calcium phosphate precipitates to cocultures of cells that package retroviruses into ecotropic and amphotropic host range envelopes. Helper-free virions from either cell type can infect the other without interference, resulting in theoretically limitless back-and-forth (ping-pong) vector replication. In initial studies, however, amplifications of a vector that contained the human growth hormone gene ceased when the hormone produced was 0.3% or less of cellular protein synthesis. This limit was caused by two factors. First, recombinant shutoff viruses that are replication defective and encode envelope glycoproteins form at a low probability during any round of the vector replication cycle and these spread in cocultures, thereby establishing interference. Single cells in shutoff cocultures therefore synthesize both ecotropic and amphotropic envelope glycoproteins, and they release promiscuous (presumably hybrid) virions. The probability of forming shutoff viruses before the vector had amplified to a high multiplicity was reduced by using small cocultures. Second, cells with large numbers of proviruses are unhealthy and their proviral expression can be unstable. Stable expresser cell clones were obtained by selection. Thereby, cell lines were readily obtained that stably produce human growth hormone as 4 to 6% of the total protein synthesis. A ping-pong retroviral vector can be used for high-level protein production in vertebrate cells.

Biotechnology↗

PubMed1989

Molecular biology of Friend viral erythroleukemia.

Friend virus clearly provides an important model for understanding the molecular biology of cancer. Moreover, the most important aspects of the erythroleukemia can be caused by a single SFFV infection in the absence of any helper virus. The SFFV env gene encodes a membrane glycoprotein, gp55. This glycoprotein, when expressed on erythroblast surfaces, causes a constitutive mitogenesis. However, SFFV infections only rarely increase the cell's self-renewal capability or abrogate its commitment to differentiate. Therefore, the consequence of infection is initially a polyclonal erythroblastosis. This polyclonal proliferation usually leads to cell differentiation and to recovery unless helper virus is present to cause continuing infection of new erythroblasts. Extremely rare SFFV proviral integrations, however, result in abrogation of the cell's commitment to differentiate and in the concomitant acquisition of cell immortality. These immortalizing proviral integrations occur at only a small number of sites in the mouse genome. Therefore, the mitogenic and immortalizing stages of erythroleukemia are now known to be caused by discrete genetic events--the first involving the SFFV env gene and the second involving the rare proviral integration sites. In early investigations of Friend virus, the first stage always preceded the second stage by at least several weeks. Now it is known that this delay in onset of the second stage is caused solely by statistics. Every SFFV-infected erythroblast is mitogenically activated, yet only rarely does the SFFV proviral integration produce immortality. Both steps in leukemogenesis can be caused simultaneously in an erythroblast by a rare single SFFV proviral integration. There has been an explosion of interest in retroviral env gene-mediated pathogenesis. Such pathogenesis has been recently associated with most of the naturally transmitted retroviral diseases including AIDS. Such pathogenesis involves in different viruses immunosuppression, anemia, neuropathy, and leukemia (Mathes et al. 1978; Simon et al. 1984, 1987; Weiss et al. 1985; Lifson et al. 1986; Riedel et al. 1986; Sitbon et al. 1986; Sodroski et al. 1986; Mitani et al. 1987; Schmidt et al. 1987; Klase et al. 1988; Overbaugh et al. 1988a, b). The shuffling and dynamic env gene rearrangements that have been associated with murine retroviral leukemogenesis have also now been seen in FeLV-FAIDS and HIV (Fisher et al. 1988; Overbaugh et al. 1 t88b; Saag et al. 1988; Tersmette et al. 1988). Friend virus provides an important established example of such env gene pathogenesis. Although we still do not understand precisely how gp55 causes erythroblast mitosis, workers in this field have discovered important clues that may lead to answers.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1989

Cell surface receptors for ecotropic murine retroviruses: mobile membrane proteins that mediate binding and slow endocytosis of the viral envelope glycoprotein.

The gp70 envelope glycoproteins of ecotropic murine leukemia viruses bind to receptors that occur only on mouse and rat cells and on interspecies hybrid cells that contain mouse chromosome 5. A substantial fraction of the gp70 that was bound specifically by these criteria remained undegraded and accessible to extracellular labeling reagents for many hours. Accordingly, cells with ecotropic receptors could be labeled specifically. As seen by immunofluorescence microscopy, the gp70-receptor complexes were uniformly dispersed on mouse fibroblast plasma membranes. These complexes were mobile, and they aggregated into patches when crosslinked by antibodies at 37 degrees, but not when membrane lipid fluidity was frozen at 0 degrees. Ecotropic receptors still bound gp70 specifically after cells were fixed with 3.7% formaldehyde, but these receptors could not be patched, indicating that they were nondiffusible. Viable cells slowly endocytosed gp70-receptor complexes at 37 degrees (approximate half-life 5-7 hr) and the gp70 was then proteolytically degraded in lysosomes. In the presence of 20 microM chloroquine, a lysosomal inhibitor, undegraded gp70 was seen to slowly accumulate in these intracellular organelles. These results suggest that ecotropic receptors mediate a slow internalization of attached ligand. Long-lived binding of gp70 onto surfaces of uninfected cells may explain important features of viral-induced leukemia, the host immune response, and immunosuppression.

Animals↗

PubMed1989

Splenic accumulation of interleukin-3-dependent hematopoietic cells in Friend erythroleukemia.

Interleukin-3-dependent hematopoietic stem cells commonly accumulate in spleens of mice infected with leukemia viruses. To study their origins, a molecularly tagged helper-free Friend spleen focus-forming virus was used to produce erythroleukemias. Uninfected interleukin-3-dependent basophil-mast cell progenitors coproliferated amidst the spleen focus-forming virus-infected leukemic cells. Splenic proliferation of normal stem cells is apparently a host response to leukemogenesis, and we propose that it may contribute to certain retroviral diseases.

Animals↗

PubMed1989

A common site for immortalizing proviral integrations in Friend erythroleukemia: molecular cloning and characterization.

By using a tagged derivative of Friend spleen focus-forming virus, we previously obtained evidence that proviral integration(s) in the host genome can cause erythroblast immortality by abrogating the commitment of the cell to differentiate (C. Spiro, B. Gliniak, and D. Kabat, J. Virol. 62:4129-4135, 1988). Exploiting the fact that each leukemia was a single clone that contained one tagged provirus, we have now molecularly cloned and characterized one common genomic site for immortalizing proviral integrations.

Animals↗

PubMed1989

Leukemogenic membrane glycoprotein encoded by Friend spleen focus-forming virus: transport to cell surfaces and shedding are controlled by disulfide-bonded dimerization and by cleavage of a hydrophobic membrane anchor.

The leukemogenic glycoprotein (gp55) encoded by Friend spleen focus-forming virus is predominantly retained in the rough endoplasmic reticulum (RER). However, a small proportion (ca. 5%) is processed to form a derivative that occurs on plasma membranes and causes mitosis of infected erythroblasts. We have now found that gp55 folds heterogeneously in the RER to form components with different disulfide bonds and that this difference may determine their processing fates. RER gp55 consists predominantly of monomers with intrachain disulfide bonds. In contrast, the processed molecules are disulfide-bonded dimers. These dimers are extensively modified in transit to cell surfaces by conversion of four N-linked high-mannose oligosaccharides to complex derivatives and by attachment of a sialylated O-linked oligosaccharide. The plasma membrane dimers are then slowly shed into the medium by a mechanism that involves proteolytic cleavage of approximately 25 membrane-anchoring hydrophobic amino acids from the carboxyl termini of the glycoproteins. Consequently, shed molecules have shorter polypeptide chains than cell-associated gp55. We conclude that gp55 folds into different disulfide-bonded components that do not substantially isomerize, and that only one specific dimer is competent for export from the RER. Mitogenic activity of gp55 could be caused by the cell surface dimers, by the shed derivative, or by the carboxyl-terminal hydrophobic anchors that remain in the membranes after the shedding reaction.

Biological Transport↗

PubMed1988

An enhancer sequence instability that diversifies the cell repertoire for expression of a murine leukemia virus.

Studies of recombinants between murine leukemia viruses (MuLVs) that cause thymic or erythroid leukemias have shown that enhancer sequences in the long-terminal repeats (LTRs) can determine the target tissues for pathogenesis. It has been inferred that the enhancers may specifically target viral expression into the cells that then become neoplastic. However, the neoplasms in those studies formed after latencies and contained ultimate viruses (called MCFs) that differed from the injected viruses in their enhancer sequences and envelope (env) genes. Transcriptional activities of LTRs from these proximal and ultimate viruses have not been thoroughly analyzed in different hematopoietic lineages. We present evidence that the enhancer of Friend spleen focus-forming virus (SFFV), an ultimate erythroleukemogenic retrovirus, contains an unstable 42-nucleotide direct repeat. Other ultimate erythroleukemogenic MuLVs (Friend MCFs) contain an enhancer nearly identical to that of SFFV both in its sequence and in its specific instability. The instability occurs in sequences that contain inverted repeats and we propose that it occurs by a simple reverse transcriptase hop mechanism. We constructed plasmids that contain the two forms of the SFFV LTR linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, and we compared these in transient transfection assays with LTR-CAT plasmids constructed from Friend and Moloney MuLVs. The assays employed erythroleukemia cells, thymic lymphoma cells, and fibroblasts. The tropisms of expression correlated only weakly with tissue specificities of pathogenesis and each LTR was active in all cells. The SFFV 42-nucleotide duplication reduced expression in erythroid cells and increased expression in fibroblasts. We conclude that retroviral enhancers do not stringently direct gene expression into specific cell lineages, but on the contrary they are leaky and contain replicative instabilities that also may facilitate viral entrenchment throughout the host. These results have important implications for understanding murine retroviral evolution and the multi-step process of leukemogenesis.

Base Sequence↗

PubMed1988

Overcoming interference to retroviral superinfection results in amplified expression and transmission of cloned genes.

A procedure is described for stably expressing cloned genes at high levels in vertebrate cells and for obtaining these genes in high-titer virus preparations. The process uses retroviral vectors and mixtures of two "packaging cell lines" that incorporate retroviral genomes into virions with different host-range envelopes. In these cocultures, interference barriers to superinfection are overcome, retroviral vectors can replicate in the absence of a transmissible helper virus, and the cells become infected with multiple copies of the provirus that contains the cloned gene. This procedure was used to amplify expression of the membrane glycoprotein that is encoded by Friend spleen focus-forming virus, a retrovirus that is replication defective in other cell cultures. Amplifications were measured at the DNA provirus, RNA, and protein levels. In addition, the human growth hormone gene was inserted into retroviral vectors and we observed amplifications of growth hormone synthesis and secretion. The amplified growth hormone was properly processed as indicated by immunoblot analyses. A vector is described (pSFF) that is exceptionally active in coculture amplification.

Animals↗

PubMed1988

A tagged helper-free Friend virus causes clonal erythroblast immortality by specific proviral integration in the cellular genome.

A colinear molecular clone of the Lilly-Steeves polycythemia strain of Friend spleen focus-forming virus (SFFV) was modified by inserting a 215-base-pair tag of simian virus 40 DNA into its nonfunctional pol gene region. The DNA was then transfected into psi-2 packaging cells, and helper-free tagged SFFV was recovered in the culture medium. Injection of this helper-free virus into NIH/Swiss mice caused transient mild splenomegaly and formation of spleen foci at 9 to 10 days. Although the vast majority of infected erythroblast clones then differentiated and died out, rare cell clones that were present in only 20 to 30% of the mice grew extensively by 26 to 33 days to form transplantable leukemias. The clonality of these leukemias was established by Southern blot analysis of their DNAs by using several restriction endonucleases and the simian virus 40 tag as a hybridization probe. All transplantable leukemias lacked helper virus contamination and contained a single tagged SFFV provirus that expressed the mitogenic env gene product gp55. The SFFV proviruses in these leukemias also appeared to be integrated into a few tightly clustered sites in the cellular genome. Although the tagged SFFV caused polycythemia during the polyclonal early stage of erythroblastosis, growth of the helper-free clonal erythroleukemias caused severe anemia. These results suggest that a single SFFV can cause mitosis of erythroblasts, and that cell immortalization also occurs when the provirus integrates into a critical site in the host genome. We propose that mice with clonal-stage leukemia become anemic because the immortalizing proviral integrations interfere with the cellular commitment to differentiate.

Animals↗

PubMed1987

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.

Animals↗

PubMed1986

Role of a membrane glycoprotein in Friend virus erythroleukemia: nucleotide sequences of nonleukemogenic mutant and spontaneous revertant viruses.

We previously isolated spontaneous env gene mutants of Friend spleen focus-forming virus that are nonleukemogenic in adult mice but form leukemogenic revertants in newborns; we found that the revertants contain secondary env mutations. To identify sites in the encoded membrane glycoprotein that are important for its pathogenic function, we molecularly cloned and partially sequenced the env genes of two mutant viruses (clone 63 and clone 4) and one revertant (clone 4REV). Clone 63 contained three noncontiguous point mutations that caused nonconservative amino acid substitutions of Gly-119----Arg-119, Cys-180----Tyr-180, and Gly-203----Arg-203 in the xenotropic-related domain of the env glycoprotein. These substitutions were presumably responsible for the altered electrophoretic and pathogenic properties of the mutant glycoprotein. The presence of these and several other G-A nucleotide substitutions at different sites in one spontaneous mutant provided striking evidence that error-rich proviruses can form during retroviral replication. Clone 4 contained a point mutation that generated a premature termination condon at amino acid residue 304 (Gln-304----Ochre-304). This termination codon was located immediately after the proposed xenotropic-ecotropic recombination site and eliminated the ecotropic-related domain, including the putative membrane anchor of the glycoprotein. Clone 4REV was a true revertant derived from clone 4 in which the premature termination codon had back-mutated to re-form the wild-type sequence. These results confirm an essential role for the env gene in Friend spleen focus-forming virus pathogenesis and suggest that the encoded membrane glycoprotein contains different domains that contribute to its pathogenic function.

Amino Acid Sequence↗