Amplified env and gag products on AKR cells. Origin from different murine leukemia virus genomes.

Thymocytes of AKR mice express two species of gp70, the envelope glycoprotein of murine leukemia virus (MuLV), encoded by the env gene. One is denoted Ec+ gp70 in reference to the type-antigen Ec and association with ecotropic virus. The other, Ec- gp70, resembles gp70 found also on thymocytes of mouse strains that are not overt producers of MuLV, and has no evident relation to ecotropic virus. Expression of Ec- gp70 type, but not of Ec+ gp70 type, is amplified with age on AKR thymocytes. In contrast, viral core polyproteins, encoded by the gag gene and simultaneously amplified with age, appear to be related to ecotropic virus. These observations imply selective amplification of products of env and gag genes from two sorts of provirus, a phenomenon which may be connected to the dual genetic origin of recombinant mink-cell-focus inducing viruses in AKR mice.

X-gp70: a third molecular species of the envelope protein gp70 of murine leukemia virus, expressed on mouse lymphoid cells.

Three variants of the gp70 envelope component of MuLV are now recognizable serologically: GIX-gp70, 0-gp70, and X-gp70. The last of these, X-gp70, has so far been found only in mice or cells producing abundant C-type virus. This distinguishes X-gp70, provisionally, from the GIX-gp70 and 0-gp70 variants, each of which can be expressed on normal thymocytes without accompanying virus production, as exemplified by mouse strains 129 and B6, respectively. The X-gp70 genotype, however, is not limited to strains of mice-producing abundant virus, because X-gp70+ leukemias occur in strains of mice which do not produce a great deal of virus and whose thymocytes and other tissues are X-gp70-; this is analogous to the appearance of GIX+ leukemias in GIX- mouse strains.

Expression of murine leukemia virus envelope glycoprotein gp69/71 on mouse thymocytes. Evidence for two structural variants distinguished by presence vs. absence of GIX antigen.

Thymocytes of several mouse strains were tested for expression of the gp69/71 envelope component of murine leukemia virus by surface iodination, followed by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Theses strains included two congenic lines differing from their partner stocks with respect to expression of GIX antigen demonstrable in the cytoxicity assay. We conclude that:(a) two structural variants of gp69/71 can be expressed on mouse thymocytes, (b) these are distinguishable by a small difference in mobility in SDS gels, (c) one carries GIX antigen and the other not, (d) they are coded, or their expression is regulated, by different chromosomal loci that are not closely linked, and (e) both can be expressed together on the thymocytes of inbred mice. In the intact thymocyte plasma membrane, the sites of group-specific antigen shared by the two gp69/71 variants, unlike the GIX type specificity carried by only one of them, are probably inaccessible to antibody.

Biochemical evidence linking the GIX thymocyte surface antigen to the gp69/71 envelope glycoprotein of murine leukemia virus.

It is known that the thymocyte surface antigen GIX is found in some strains of mice and not others, and that its expression in mice of strain 129, in which most extensive genetic studies have been made, is controlled by two unlinked cellular chromosomal loci. We have now isolated a protein with a mol wt of approximately 70,000 daltons from the surface of thymocytes from 129 mice, which have antigenic and biochemical properties characteristic of the gp69/71 envelope component of murine leukemia virus. Our evidence is compatible with the conclusion that it carries the GIX antigen.

Common cell surface antigen associated with mammalian C-type RNA viruses. Cell membrane-bound gs antigen.

The indirect membrane immunofluorescence test and the absorption analysis of rabbit anti-FeLV, rabbit anti-FeLVp 30, and rabbit anti-MuLVp 30 antisera yielded the following conclusions. An antigen shared by mammalian (murine and feline) C-type RNA leukemia and sarcoma viruses was detected on the surface of cells infected or transformed by C-type viruses. The antigen was characterized as membrane-bound gs antigen bearing two determinants, membrane-bound gs-1, intraspecies-specific antigenic determinant, and membrane-bound gs-3, interspecies-specific antigenic determinant. Membrane-bound gs antigen was located on the cell surface, frequently near the site of virus budding but not on the envelope of murine C-type RNA virus.

Relationships of gp70 of MuLV envelopes to gp70 components of mouse lymphocyte plasma membranes.

The family of glycoproteins called gp70 includes molecules that are the main constituent of murine C-type viral envelopes, and some that are expressed as mendelian constituents of thymocyte plasma membranes in the absence of virions. To investigate further the relation of viral gp70s to plasma- membrane gp70s we compared peptide maps of gp70s derived by immunoprecipitation from cells infected with chosen viruses and from various thymocytes and leukemiacells known to express one or more of three immunogenetically defined gp70 types: Glx-gp70, X-gp70, and O-gp70. Maps of gp70 from cultured cells infected with ecotropic and xenotropic viruses were distinguishable from one another, and in general resembled gp70 maps prepared directly from ecotropic and xenotropic virions respectively. Maps of gp70s immunoprecipitated from thymocytes of five mouse strains and from two A strain T-cell leukemias also fell into two distinguishable and generally corresponding patterns. Thus peptide-mapping substantiates earlier conclusions that viral gp70s and plasma-membrane gp70s inherited independently of virus-production are highly related or identical molecules. The gp70 maps of thymocytes from B6, B6-G(+IX), 129, and A mice formed a group resembling the map from cultured cells infected with xenotropic virus. Thymocytes from AKR mice, and the two A strain leukemias, gave gp70 maps conforming more to the second pattern, that of cultured cells infected with ecotropic virus. This second pattern probably comprises at least two gp70 types, one of which is X-gp70. Our data indicate that the G(IX)-gp70 and O-gp70 sub-species of gp70 expressed in the cell populations we have studied are coded by xenotropic viral genomes, and X-gp70 by ecotropic viral genomes.

Deregulation of the c-myc oncogene in virus-induced thymic lymphomas of AKR/J mice.

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.

Abelson murine leukemia virus-induced thymic lymphomas: transformation of a primitive lymphoid precursor.

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.

Constitutive c-myc expression enhances the response of murine mast cells to IL-3, but does not eliminate their requirement for growth factors.

An interleukin-3 (IL-3) dependent mast cell line (MC) was infected with a recombinant retrovirus expressing the proto-oncogene c-myc and the drug selectable marker neo. Cells containing the transcriptionally activated c-myc gene displayed an increased growth rate in liquid culture and a higher cloning efficiency in soft agar when compared to control virus infected cells. All infected cells remained absolutely dependent on IL-3 for growth and were not tumorigenic in nude mice. Similar results were obtained with two additional IL-3 dependent cell lines, the mast cell 32D and the pre-B-cell Ea3. Thus, while constitutive expression of c-myc potentiates the response of mast cells to IL-3, it is not sufficient to eliminate their requirement for growth factors.

Virus-specific antigens in hamster cells transformed by Rous sarcoma virus.

Virus-specific antigens were studied in hamster cells transformed by Rous sarcoma virus (RSV). Antigens were localized in the cytoplasm, as demonstrated by fluorescent antibody staining of fixed cells as well as by complement fixation (CF) following subcellular fractionation. Cytoplasmic extracts were analyzed by velocity and isopycnic centrifugation. CF antigens were found in a soluble form and in association with membranes and polyribosomes. Isolated plasma membranes had no CF antigen. Both soluble and particulate fractions with CF activity contained the same antigenic determinants by Ouchterlony analysis. These antigenic determinants were identical to those released by ether treatment of RSV.

Chromatographic separation and antigenic analysis of proteins of the oncornaviruses. I. Avian leukemia-sarcoma viruses.

Gel filtration of avian tumor virus proteins in 6 m guanidine hydrochloride clearly resolved seven major protein species. The antigenic activity of these proteins was recovered in good yield after removal of the denaturing solvent, permitting a correlation of specific polypeptides with the principal antigens of the virion. Two of the proteins, of molecular weights 70,000 and 32,000, contain carbohydrate and are situated on the viral membrane, as shown by their being accessible in the intact virus to specific antibodies. Four proteins, with molecular weights (in guanidine) of 27,000, 19,000, 15,000, and 12,000, have different group-specific (gs) antigens and are enclosed within the viral membrane. The smallest protein, with a molecular weight of 10,000, has not previously been described; it is not detectable with antisera and possesses a mobility identical to that of one gs protein when subjected to electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate. Of the proteins lacking carbohydrate, three are present in the virion in a molecular ratio of 2:2:1, and the two others, present in almost equal amount, are rich in lysine and arginine.

Structural studies of retroviruses: characterization of oligomeric complexes of murine and feline leukemia virus envelope and core components formed upon cross-linking.

To examine the protein proximity and subunit organization of type C retroviruses, preparations of AKR murine leukemia virus were treated with bifunctional cross-linking reagents and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The cross-linked components obtained were characterized by immunoprecipitation with monospecific antisera against purified viral proteins, followed by SDS-PAGE analysis both before and after cleavage of the cross-links. With these procedures, complexes of both viral envelope and core components were identified. The major envelope subunit obtained was a large (apparent molecular weight of 450,000 to 500,000), glycosylated complex, composed of four to six gp70-p15(E) subunits. This complex was detected over a 100-fold range of cross-linker concentration and thus seems to represent a particularly stable viral substructure. The cross-linked complexes of the core proteins consisted of oligomers of p30 dimers, suggesting that the p30 dimer is a basic structural unit of the viral core. When virion preparations, which had previously been disrupted with the nonionic detergent Nonidet P-40, were cross-linked, the envelope complex was still observed, indicating that this structure is stable in the presence of Nonidet P-40. A similar envelope structure was observed for feline leukemia virus, suggesting that such a complex may be a conserved feature of oncornavirus structure.

Identification by transfection of transforming sequences in DNA of human T-cell leukemias.

DNA from human T-cell leukemia cell lines was tested for focus-inducing activity on cultures of NIH 3T3 cells. Three leukemias yielded DNA active in this assay; restriction enzyme sensitivity of this activity indicated that similar, relatively large DNA sequences were involved. Southern blot analysis revealed conserved size classes of restriction fragments containing human repetitive (Alu) sequences in serially transfected foci derived from the active DNAs. Similar blot hybridizations with a probe specific for the human N-ras oncogene detected a 9-kilobase EcoRI fragment in all cases. DNA containing this fragment from one of the leukemias, molecularly cloned in bacteriophage lambda, displayed highly amplified focus-inducing activity in transfection assays. Thus, the N-ras oncogene appears to be active in these three human leukemias of T-cell origin.

Chromatographic and electrophoretic analysis of viral proteins from hamster and chicken cells transformed by Rous sarcoma virus.

Several methods have been explored for the detection and characterization of viral proteins from soluble extracts of cells transformed by Rous sarcoma virus (RSV). Viral antigens have been analyzed after gel filtration in several solvents. In addition, immune complexes formed with virus-specific sera have been isolated by agarose gel filtration and by high- or low-speed centrifugation through sucrose solutions. Radioactive proteins from these immune complexes have been analyzed by gel filtration in 6 m guanidine hydrochloride or by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Comparison with proteins from purified virus indicates the presence of two viral core proteins (gs1 and gs2) in the soluble fraction from virus-producing chicken cells. In the same fraction from RSV-transformed hamster cells (which do not produce virus), three gs proteins (gs1, gs2, and gs3) could be identified. The soluble viral gs proteins are strongly bound to at least two larger polypeptides in cell extracts. These polypeptides do not appear to be viral in origin and have the property of undergoing a time-dependent aggregation in the extracts. One of these cell-derived proteins, which is present in a variety of uninfected cell types, closely resembles actin.

Isolation of a ribonucleoprotein structure from oncornaviruses.

Detergent treatment releases from both avian and mammalian oncornaviruses a structure containing the viral RNA and a major arginine-rich protein, as well as reverse transcriptase activity.

High-titer replication of nondefective Sendai virus in MDBK cells.

Egg-grown Sendai virus was adapted to growth in a bovine kidney cell line (MDBK cells) by serial passage under defined conditions. The adapted virus contained only 50S RNA and was highly infectious for MDBK cells. Infection of these cells with a high multiplicity of adapted virus resulted in a yield of 10(8) MDBK-infectious units/ml by 18 h, accompanied by severe cytopathic changes in the host. Cell fusion did not occur. Examination of the proteins of the adapted virus revealed that despite the high infectivity of this virus for MDBK cells the virions contained considerable quantities of Fo, the precursor to the F glycoprotein that is responsible for cell fusion and high infectivity in other systems.

Kinetics of utilization of Sendai virus RNA and protein in the process of virion assembly.

The synthesis of the 50S genomic RNA and strucural proteins of Sendai virus was examined with respect to their utilization in virus assembly. It was found that during a single cycle of infection, 50S RNA was synthesized before the structural proteins and that both RNA and protein were synthesized 2 to 4 h before their appearance in released virions. Pulse-chase labeling indicated that the NP and P proteins synthesized early and the M and F proteins synthesized late were preferentially incorporated into virus relative to the other viral proteins. The kinetics of incorporation of pulse-labeled NP protein suggested that it was withdrawn from a relatively large pool whereas the M protein appeared to be present in a relatively small pool in the cytoplasm. Further, it was possible to chase pulse-labeled M protein, but not NP protein, from the cell during an 8-h time period.