Association of recombinant murine leukemia viruses of the class II genotype with spontaneous lymphomas in CWD mice.

We determined the phenotype and genotype of murine leukemia viruses associated with the development of spontaneous nonthymic lymphomas in the high-leukemia mouse strain CWD/J. By T1 oligonucleotide fingerprint analysis of the viral RNA, the ecotropic viruses recovered from the spleen or thymus of preleukemic CWD/J mice were found to represent the progeny of the two endogenous ecotropic proviruses present in this strain. Polytropic murine leukemia viruses were produced by tissues from one-half of the leukemic mice, and fresh tumor cells from one of the two animals tested expressed recombinant envelope glycoproteins. The genomic structure of the recombinant viruses resembled those of class II polytropic viruses of NFS X Akv mice and differed from those of class I recombinant viruses that are commonly isolated from other high-leukemia strains such as AKR and HRS. Acquired retroviral sequences with the structural features of class II recombinant proviruses were detected in the DNA from each CWD/J tumor by the Southern blot technique. Finally, the injection of a mixture of CWD/J ecotropic and class II recombinant polytropic viruses into neonatal CWD/J mice accelerated the onset of lymphoma, whereas the endogenous ecotropic virus was inactive in these assays.

A time-course study of MuLV env gene expression in the AKR thymus: qualitative and quantitative analysis of ecotropic and recombinant virus gene products.

Low levels of synthesis of two species of MuLV env gene polyprotein (PrENV protein) in thymocytes of 3-month-old AKR mice were identified. Synthesis of PrENV proteins which comigrate with those of ecotropic and recombinant, dualtropic MuLV represented, respectively, 0.03-0.05 and 0.01-0.03% of total cell protein synthesis in these animals. An increase in the rate of synthesis of both PrENV species was observed in animals at 5-6 months of age; ecotropic virus PrENV represented 0.2-0.6% of total protein synthesis and recombinant virus PrENV, 1-2.5% of total protein synthesis in thymocytes of mice of this age. This same increase in env gene expression of both the ecotropic and recombinant virus was induced in 3-month-old animals by intrathymic injection of recombinant MuLV at 4-6 weeks of age. The level of recombinant virus env gene synthesis in thymomas was similar to that observed in thymocytes of 5- to 6-month-old animals and in experimentally injected animals; elevated synthesis of ecotropic virus PrENV protein was detected in 85% of these leukemias. Partial protease digest mapping of the recombinant virus PrENV protein isolated from 23 primary thymomas revealed that the predominant type of recombinant (18/23) expressed in these cells was that of the MCF 69L1/247 type. A notable finding was the identification of expression of variant ecotropic MuLV in these thymomas. Of ten leukemias studied, eight expressed ecotropic virus PrENV proteins which were distinguishable from that of Akv virus. Four unique ecotropic virus PrENV proteins were observed.

Leukemogenesis by Gross passage A murine leukemia virus: expression of viruses with recombinant env genes in transformed cells.

Gross passage A murine leukemia virus (MuLV) derived from extracts of C3Hf/Bi mouse leukemias has been shown to be a virus complex consisting of ecotropic, xenotropic, and recombinant, dualtropic MuLV components. The three virus components were distinguished biochemically by differences in the molecular weights and peptide maps of their primary env gene products synthesized in infected cells in vivo and in vitro. Virus expression was studied in primary leukemias induced in C3Hf/Bi mice by Gross passage A virus extracts and by the individual ecotropic and recombinant MuLV components that were isolated in vitro. Our findings suggest that expression of the recombinant MuLV component of the Gross passage A virus complex is necessary and sufficient for the induction of leukemias in C3Hf/Bi mice. In contrast, induction of leukemias by the ecotropic virus component appears to involve generation of a second virus with characteristics of recombinant, dualtropic MuLV.

Env gene products of AKR dual-tropic viruses: examination of peptide maps and cell surface expression.

The env gene products of nine AKR dual-tropic murine leukemia viruses were compared by peptide mapping and were assayed for expression on the cell surface of infected fibroblasts. Seven virus isolates expressed the env gene polyprotein on the cell surface. The env gene products of six of the seven viruses had identical peptide maps. The analysis of structure and expression of env gene products carried out in this study characterizes a subset of dual-tropic murine leukemia viruses shown by others to be thymotropic.

Glycoproteins of murine leukemia viruses. III. Glycosylation of env precursor glycoproteins.

We have compared the glycopeptides obtained after extensive pronase digestion of the env precursors (PrENV proteins) of ecotropic, xenotropic, and dual-tropic murine leukemia viruses. Two glycopeptide size classes, having molecular weights of approximately 2,200 and 1,500, were shown to be associated with the PrENV proteins of all murine leukemia viruses studied. Glycopeptides associated with the env precursors were totally susceptible to endo-beta-N-acetyglucosaminidase H. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of partial endo-beta-N-acetylglucosaminidase H digestion products of the env precursor of dual-tropic mink cell focus-forming virus (MCF 247) revealed the presence of seven bands, suggesting that six glycosylation sites were present on the precursor molecule. The MCF 247 PrENV protein had been previously shown to be accessible to lactoperoxidase-catalyzed radioiodination on the surface of infected cells. The cell surface PrENV molecules had the same electrophoretic mobility as pulse-labeled PrENV protein, and after endo-beta-N-acetylglucosaminidase H treatment a similar shift in electrophoretic mobility was observed for the cell surface PrENV protein and the pulse-labeled precursors, a finding which indicated that the PrENV protein located on the cell surface also possessed only mannose-rich oligosaccharides. These results indicated that the env precursor glycoproteins of dual-tropic viruses had the unusual property of migrating to the cell surface without undergoing the normal oligosaccharide processing and proteolytic cleavage events that had been observed for ecotropic and xenotropic murine leukemia virus glycoproteins.

Glycopeptides of murine leukemia viruses. II. Comparison of xenotropic and dual-tropic viruses.

The glycosylation patterns of the gp70 glycoproteins of xenotropic and dualtropic murine leukemia virus (MuLV) were compared with those of ecotropic viruses. Ecotropic viruses contain a large glycopeptide size class designated G1 (molecular weight, approximately 5100), and such glycopeptides were not detected in xenotropic viruses grown in mink cells nor in dual-tropic viruses grown in mouse or mink lung cells. Both xenotropic and dual-tropic MuLV had glycopeptide size classes designated G2, G3, and G4 (molecular weights, approximately 2900, 2,200, and 1,500, respectively). G2 glycopeptides of xenotropic and dual-tropic MuLV were shown to be resistant to endo-beta-N-acetylglucosaminidase H, whereas G3 and G4 glycopeptides were susceptible. The relative abudance of glycopeptide G3 was increased in xenotropic and dual-tropic viruses as compared with ecotropic viruses, whereas the relative amount of G4 was decreased in xenotropic viruses. The similarity in the glycosylation patterns of a number of xenotropic and dual-tropic viruses suggests that glycosylation sites are highly conserved within the env gene products of each of these classes of viruses.

Cell surface expression of the env gene polyprotein of dual-tropic mink cell focus-forming murine leukemia virus.

Differences have been observed in the kinetics of processing of the env gene polyprotein of ecotropic, xenotropic, and dual-tropic mink cell focus-forming (MCF) murine leukemia virus. In pulse-chase experiments, the env gene polyprotein of the dural-tropic MCF virus exhibits a marked increase in stability relative to that of either ecotropic or xenotropic virus. A comparison of cell surface expression of env gene products of ecotropic, xenotropic, and dual-tropic MCF murine leukemia virus has been made. Only gp70 is accessible to lactoperoxidase-catalyzed radioiodination of fibroblasts infected by ecotropic or xenotropic virus, whereas both gp70 and the env gene polyprotein are expressed on the surface of dual-tropic MCF virus-infected cells.

Presence of murine leukemia virus envelope proteins gp70 and p15(E) in a common polyprotein of infected cells.

The murine leukemia virus envelope proteins, p15(E) and gp70, exhibit a mode of processing distinct from that of virion core proteins according to three criteria. First, the incorporation of both p15(E) and gp70 into virions is more sensitive to the metabolic analogue 2-deoxy-D-glucose than the incorporation of core proteins. Second, the kinetics with which the newly synthesized envelope proteins appear in the released virions is delayed relative to the appearance of core proteins. Third, immunoprecipitation of large polypeptides from infected cells reveals the presence of gp70 and p15(E) in a common precursor distinct from the core polyprotein.

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.