Evidence of recombinant ecotropic provirus integration in thymic lymphomas induced by direct or indirect radiation effects.

Several investigators described the occurrence of ecotropic recombinant proviruses in the DNA of in-vivo or in-vitro propagated radio-induced lymphomas, but such proviruses were never detected in primary tumors. To assess their biological significance in the tumorigenic process, we reinvestigated the presence of new proviruses chiefly in primary radio-induced tumors and in models of radioleukemogenesis which could give additional support for their role. Such models included thymic lymphomas originating after (i) graft of non-irradiated thymuses in thymectomized irradiated mice and (ii) the injection of a B-ecotropic retrovirus (T1223/B) in association with a subleukemogenic dose of irradiation. We report for the first time that new ecotropic proviral sequences are encountered in a significant number (30%) of primary lymphomas induced directly by irradiation or indirectly in non-irradiated thymuses grafted in irradiated hosts. The existence of a 3.5-kbp Kpn1 restriction fragment with ecotropic sequences in the digested DNA of these tumor cells indicates that these new sequences belong to an ecotropic provirus recombinant in the gag-pol region. We observed that most of the primary radio-induced tumors in which novel recombinant provirus could be detected, displayed the integration at a single or at a few sites, demonstrating their clonality with respect to viral integration. The same was observed in thymic lymphomas arising after T1223/B virus injection and irradiation and in in-vivo or in-vitro propagated tumors. Altogether, these data bring the first evidence of the integration of ecotropic recombinant proviral genomes in a significant number of primary radiation induced thymic lymphomas and of their possible role in view of their frequent occurrence in grafted thymomas.

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.

BALB/c myeloma retroviruses: peptide mapping and immunological analysis of the pp12 structural protein.

We have compared the pp12 structural protein of the MO-21 and FL-1 BALB/c myeloma retroviruses with the pp12 of several prototype retroviruses. Chymotryptic peptide maps of 125I-labeled, immune-precipitated pp12 proteins revealed that the MO-21 and FL-1 proteins can be distinguished from one another. The MO-21 pp12 most closely resembled the NIH-xenotrophic virus pp12, and the FL-1 pp12 most closely resembled the pp12 of BV-2 and WN 1802 B. Competition radioimmunoassay studies showed that the MO-21 and FL-1 pp12 proteins are also antigenically distinct from one another and that both contain pp12 antigenic determinants of a xenotropic virus. These data support our proposal that these two BALB/c viruses contain a gag gene that was generated by recombination between endogenous eco- and xenotropic viral sequences.

Molecular relationship of the DNA and RNA of intracytoplasmic A particles to mouse and mammary tumour virus genomes.

We have directly tested the hypothesis that single-stranded cytoplasmic A particle-associated DNA (ss CAP DNA) is a murine mammary tumour virus (MMTV) proviral intermediate by hybridizing 125I-labelled ss CAP DNA to MMTV RNA or to MMTV complementary DNA (cDNA). 125I-labelled CAP DNA did not form duplexes with either MMTV RNA or MMTV cDNA. In contrast, CAP RNA hybridized readily with MMTV cDNA. CAP RNA contained all the MMTV virus sequences, but at lower concentrations than in MMTV virus particles. Single-stranded CAP DNA hybridized readily with mouse DNA from several sources. A study of the rate of hybridization of CAP DNA to cell DNA at various driver to probe ratios showed that its rate of hybridization is similar to that of tumour cell DNA reassociation. Further, in reassociation studies accelerated by using the phenol emulsion reassociation technique (PERT), CAP DNA originally isolated as single-stranded DNA was shown to reanneal (70%), to protect 125I-cell DNA to the same extent (67%) and to do so with kinetics of reassociation equivalent to that of mouse DNA. Although CAP DNA isolates were slightly enriched for MMTV specific sequences when compared to total cellular DNA, we conclude that the majority of ss CAP-associated DNA is equivalent to a random sample of total tumour cell DNA.

Characterization of the proteins of intracisternal type A and extracellular oncornavirus-like particles produced by MOPC-460 myeloma cells.

The mouse plasmacytoma cell line, MOPC-460, produces both intracisternal and intracytoplasmic A-type particles when grown as a solid tumor. When these cells are grown either as an ascites tumor or in tissue culture, a third type of particle is produced extracellularly. This particle, the "myeloma-associated virus," is closely related to, and probably an alternate form of, the intracisternal A-type particle. The proteins present in these two types of particles were compared by tryptic peptide mapping. Both types of particles were found to contain essentially the same major proteins of 76,000 (p76), 68,000 to 70,000 (p68-70), and 45,000 (p45) daltons, in addition to varying amounts of smaller proteins. The relative proportions of all these proteins varied from preparation to preparation in an unpredictable way. The p45, p68, and p70 proteins all contained sequences found in p76, suggesting precursor-product relationships of p76 leads to p70 leads to p45 for solid tumor A-type particles and p76 leads to p68 leads to p45 for extracellular myeloma-associated virus. In addition, immune precipitation experiments have established that p76 contains at least some of the antigenic determinants characteristic of murine leukemia virus p30. This confirms earlier nucleic acid hybridization studies which indicated a moderate degree of relatedness between MOPC-460 A-type particles and several standard murine leukemia and sarcoma viruses. Taken together, our results provide evidence supporting the concept that MOPC-460 A-type particles may represent aberrant forms of C-type murine viruses.

Characterization of infectious oncornaviruses from MOPC-460 plasmacytomas: their relation to A-type particles.

MOPC-460 mouse plasmacytoma cells produce intracellular A-type particles and extracellular oncornavirus-like particles ("myeloma-associated virus," abbreviated MAV). The genomes of these two particles are closely related. During attempts to establish infections with MOPC-460 extracellular particles, we isolated ecotropic and xenotropic infectious forms of murine leukemia virus. We have investigated the relation of these isolates to A-type particles and to MAV by nucleic acid hybridization. Using complementary DNA probes prepared from the two isolates, we found that these infectious murine leukemia viruses differ from A-type particles and from MAV. Moreover, we found that MAV is the predominant extracellular component: the ecotropic and xenotropic forms of murine leukemia virus were present at only low levels (less than 5%) in MAV preparations. Neither the SC-1 cells infected with ectropic murine leukemia virus nor the mink cells infected with xenotropic murine leukemia virus showed any A-type particles in their cytoplasm when examined by electron microscopy. Our inability to demonstrate infection by the A-type particle-related component, MAV, suggests that these may be defective.

Presence of virus-specific DNA sequences in murine type C viruses.

Total nucleic acids prepared from a number of murine retroviruses have been shown to contain virus-specific DNA in addition to genomic RNA. This virus-specific DNA has been shown to be at least partially double stranded and to be present within the virus core particle. The DNA isolated from the virus is greatly enriched in virus-specific DNA relative to that from virus infected cells.

Selective transcription of genomic sequences common to both N- and X-tropic endogenous retroviruses in BALB/c mouse tissues.

Total RNAs from four BALB/c mouse tissues, containing mostly non-dividing cells (liver and kidney) or variable proportions of dividing cells (uterus and embryo) were analysed for sequences complementary to 3H-DNA transcripts synthesized from BALB/c endogenous. N- and X-tropic retroviruses. Extensive transcription of virogene information was detected in the tissues examined, but such transcription was found to be mostly limited to the homologous regions of the two virus genomes. No additivity of hybridization values could be detected when RNAs from two different tissues were mixed, which suggests that BALB/c mouse liver, kidney, uterus and embryo transcribe a common set of nucleic acid sequences of the homologous regions of the N- and X-tropic viral genomes, in addition to other sequences of the same region that are specific for indiviual tissues.

Genetic individuality of intracisternal A-particles of Mus musculus.

The nucleic acid sequence relationship between mouse intracisternal type A-particles and type C and B viruses was examined by reciprocal complementary DNA-RNA hybridization; complementary DNAs prepared from the RNAs of intracisternal A-particles were hybridized with high-molecular-weight RNAs from a variety of murine tumor viruses, and complementary DNAs representing a variety of RNA tumor virus genomes were hybridized with the high-molecular-weight RNAs from A-particles. The criterion for homology between two types of virus was that the heterologous hybridization reaction occurs over the same RNA concentration range as the homologous reacton. The results of these hybridizations indicate that there are no major sequence homologies between the RNA of intracisternal A-particles and the RNA of representative members of type B and C viruses of Mus musculus.

Nucleotide sequence relationship between intracisternal type A particles of Mus musculus and an endogenous retrovirus (M432) of Mus cervicolor.

Intracisternal type A particles are retrovirus-like structures found in embryonic cells and many tumors of Mus musculus but having no clear relationship with other retroviruses of this mouse species. We have observed a partial nucleotide sequence homology between the high-molecular-weight (32S and 35S) RNA components of intracisternal A-particles from a neuroblastoma cell line and the 70S RNA fraction from M432, a type of retrovirus endogenous to the Asian mouse Mus cervicolor. M432 complementary DNA (cDNA) was hybridized to the extent of 30% by the A-particle RNAs. The hybrids showed a lower thermal stability (DeltaT(m), 7 degrees C) than those formed with homologous RNA. The reaction was commensurate with that found between M432 cDNA and divergent sequences in the M. musculus genome. The capacity to hybridize M432 cDNA was closely correlated with the concentration of A-particle sequences in the cytoplasmic RNA of several M. musculus cell types. The major RNA fraction of M432 virus showed a reciprocal partial reaction with the A-particle cDNA's; the virus, which was grown in NIH/3T3 (M. musculus) cells, also contained a small proportion of apparently authentic A-particle nucleotide sequences. A subset of A-particle sequences seemed to be almost totally lacking in the main M432 RNA. The A-particle cDNA's hybridized extensively with divergent sequences in M. cervicolor cellular DNA, indicating that this mouse species may contain not only the partially homologous M432 virogene, but also a more complete genetic equivalent of the intracisternal A-particle.

5'-terminal nucleotide sequences of mammalian type C helper viruses are conserved in the genomes of replication-defective mammalian transforming viruses.

The RNAs of replication-defective murine and primate type C transforming viruses were analyzed for the presence of nucleotide sequences homologous to the genomes of their respective helper type C viruses by using DNAs complementary (cDNA) to either the 5'-terminal (cDNA5') or total (cDNAtotal) nucleotide sequences of the helper virus RNA. The defective viruses examined have previously been shown to vary in their ability to express helper viral gag gene proteins. With cDNAtotal as a probe, these transforming viruses were shown to vary in their representation of helper sequences (15 to 60% hybridization of cDNAtotal). In striking contrast, 5'-terminal-specific sequences of the helper virus were conserved in the RNAs of every transforming virus tested (is greater than 80% hybridization of cDNA5'). These findings suggest a critical role for these sequences in the life cycle of the defective transforming virus.

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques.

The isoelectric point of the p30 polypeptide as a marker of mouse endogenous viruses.

The isoelectric point (PI) of the p30 polypeptide of members of the three known classes of mouse C-type endogenous viruses was determined both by column and by thin-layer gel isoelectric focusing. Each class was found to be characterized by a particular variant of p30 (isop30), with pI values of 6.1 for class I (ecotropic), 5.7 for class II (xenotropic), and 5.5 for class III (NZB, NIH, ATS124, also xenotropic). The 6.1-isop30 was found as a minor component of rat-grown NZB virus and of a number of laboratory strains of mouse C-type viruses.

Identification of a 30S RNA with properties of a defective type C virus in murine cells.

A novel species of 30S RNA has been detected in a variety of mouse cell lines. The 30S RNA is specifically packaged by helper-independent type C viruses propagated in such cells. Nucleic acid hybridization detects no homology between the 30SRNA and the genomic RNA of helper-independent mouse type C viruses. The properties of the 30S RNA suggest that it is a defective endogenous mouse type C virus and that it is analogous to a previously described class of defective endogenous rat type C virus, which has been shown previously to be the progenitor of Kirsten and Harvey murine sarcoma viruses.