Viral Myc oncoproteins in infected fibroblasts down-modulate thrombospondin-1, a possible tumor suppressor gene.

We are interested in identifying the transcriptional targets of the Myc oncoproteins. To this end, we have fused Myc of the MC29 retrovirus with the rat glucocorticoid receptor. This chimeric protein requires dexamethasone to undergo nuclear translocation and achieve an active conformation. We employed a differential hybridization approach to identify mRNAs that are induced or repressed in infected avian fibroblasts in response to dexamethasone. This screen yielded one mRNA underrepresented in the dexamethasone-treated cells. In Myc-transformed cell clones, its level decreases 6-fold as early as 4 h and more than 30-fold after 32 h of exposure to the hormone. This mRNA was also down-regulated by recombinant Myc retroviruses in rodent fibroblasts, including those refractory to transformation. Sequence analysis revealed that it is homologous to the 3' untranslated regions of the mammalian thrombospondin-1 genes. Using an anti-thrombospondin antibody, we confirmed that rodent cells overexpressing Myc produce very small amounts of this protein. Also, they do not support efficient expression of a reporter gene driven by the thrombospondin-1 promoter. Thus, thrombospondin-1 is a bona fide target of Myc. Moreover, its silencing might pertain to the transforming activity of Myc, since in several systems thrombospondin-1 exhibits tumor suppressor properties, presumably due to its negative effect on neovascularization.

v-Myc is invariably required to sustain rapid proliferation of infected cells but in stable cell lines becomes dispensable for other traits of the transformed phenotype.

The v-myc-containing retrovirus MC29 induces neoplastic transformation of avian embryo cells. To determine which traits of the transformed phenotype are directly controlled by v-Myc, we engineered a conditional MC29 mutant (GRIM) expressing v-Myc as a fusion protein with the glucocorticoid receptor and the retroviral Gag polyprotein. Only in the presence of glucocorticoids such as dexamethasone is GRIM capable of transforming embryo cells, from which six stable GRIM-lines have been derived. Although their survival in culture no longer requires functional v-Myc, hormone deprivation causes all six GRIM clones as well as acutely infected fibroblast cultures to either withdraw from cell cycle completely or to grow much more slowly and to much lower densities. However, removal of dexamethasone does not allow GRIM-transformed mass cultures and most of the clones to revert to normal shapes or to reconstruct actin cables. Furthermore, most clones do not require the hormone sustain anchorage-independent growth. We propose that certain secondary events have let the GRIM-clones sustain immortality, transformed morphology, and anchorage-independent growth independently of v-Myc. None of these events, however, has obliterated the requirement for v-Myc in cell division control. We thus conclude that enhanced proliferation is the primary effect of v-Myc expression.

Transforming variants of the avian myc-containing retrovirus FH3 arise prior to phenotypic selection.

The avian retrovirus FH3, which encodes a Gag-Myc fusion protein, transforms chicken macrophages but not fibroblasts. However, passage of FH3 viral stock in fibroblasts leads to emergence of a virus capable of fibroblast transformation. This virus has not acquired myc mutations; instead, it carries internal gag deletions which confer the ability to transform fibroblasts. We now demonstrate that this and similar deletion variants emerge repeatedly during selection. Sequence analysis reveals direct repeats at or near deletion junctions, suggesting that errors during reverse transcription may be involved in genesis of these viruses, which are then positively selected in fibroblast culture. By using the polymerase chain reaction, we found that such variants preexisted in original stocks even before selection, although they could not be detected by focus assay.

Overproduction of v-Myc in the nucleus and its excess over Max are not required for avian fibroblast transformation.

The cellular proto-oncogene c-myc can acquire transforming potential by a number of different means, including retroviral transduction. The transduced allele generally contains point mutations relative to c-myc and is overexpressed in infected cells, usually as a v-Gag-Myc fusion protein. Upon synthesis, v-Gag-Myc enters the nucleus, forms complexes with its heterodimeric partner Max, and in this complex binds to DNA in a sequence-specific manner. To delineate the role for each of these events in fibroblast transformation, we introduced several mutations into the myc gene of the avian retrovirus MC29. We observed that Gag-Myc with a mutated nuclear localization signal is confined predominantly in the cytoplasm and only about 5% of the protein could be detected in the nucleus (less than the amount of endogenous c-Myc). Consequently, only a small fraction of Max is associated with Myc. However, cells infected with this mutant exhibit a completely transformed phenotype in vitro, suggesting that production of enough v-Gag-Myc to tie up all cellular Max is not needed for transformation. While the nuclear localization signal is dispensable for transformation, minimal changes in the v-Gag-Myc DNA-binding domain completely abolish its transforming potential, consistent with a role of Myc as a transcriptional regulator. One of its potential targets might be the endogenous c-myc, which is repressed in wild-type MC29-infected cells. Our experiments with MC29 mutants demonstrate that c-myc down-regulation depends on the integrity of the v-Myc DNA-binding domain and occurs at the RNA level. Hence, it is conceivable that v-Gag-Myc, either directly or circuitously, regulates c-myc transcription.

gag as well as myc sequences contribute to the transforming phenotype of the avian retrovirus FH3.

The avian retrovirus FH3, like MC29 and CMII, encodes a Gag-Myc fusion protein. However, the FH3-encoded protein is larger, about 145 kDa, and contains almost the entire retroviral gag gene. In contrast to the other gag-myc avian retroviruses, FH3 fails to transform fibroblasts in vitro, although macrophages are transformed both in vitro and in vivo (C. Chen, B. J. Biegalke, R. N. Eisenman, and M. L. Linial, J. Virol. 63:5092-5100, 1989). We have used the polymerase chain reaction technique to obtain a molecular clone of FH3. Sequence analysis of the FH3 myc oncogene revealed a single proline----histidine change (position 223) relative to c-myc. However, substitution of the FH3 myc sequence with the chicken c-myc sequence did not alter the transformation potential of the virus. Hence, overexpression of the proto-oncogene as a Gag-Myc retroviral protein is sufficient for macrophage, but not fibroblast, transformation. After passage of FH3 in fibroblast cultures, a virus (FH3L) that is capable of rapidly transforming fibroblasts appears. The Gag-Myc protein encoded by FH3L is smaller (ca. 130 kDa) than that encoded by the original viral stock (FH3E). Sequencing of an FH3L molecular clone revealed a 212-amino-acid deletion within the Gag portion. Using FH3E/FH3L recombinants, we have demonstrated that the ability of encoded viruses to transform fibroblasts directly correlates with the presence of this deletion. Moreover, the addition of the Gag sequence deleted from FH3L to the MC29 oncoprotein significantly reduces its transforming activity as measured by focus assay. These data suggest that the C-terminal segment of Gag attenuates the oncogenic potential of Gag-Myc fusion proteins.

Long terminal repeats of dwarf hamster endogenous retrovirus are highly diverged and do not maintain efficient transcription.

Recently we described a new endogenous proretrovirus of dwarf hamster Phodopus sungorus (MRS-Ps). Its sequence possesses evident homology with the endonuclease domain of the mouse mammary tumor virus pol gene. Here we present nucleotide sequence data on three clones of retroviral long terminal repeats. As many as 15% of substituted, deleted, and inserted base pairs were found while comparing these sequences. Hence, MRS-Ps seems to be rather an old genetic element which originated about 30 million years ago. One LTR is 877 bp long and contains numerous elements that control its transcriptional activity: TATA-box, glucocorticoid responsive element, NF1-binding site, etc. Nevertheless, this LTR does not govern efficient transcription of adjacent genes in a transient expression assay. In addition, we failed to find MRS-specific mRNA in adult, embryonic, and mammary tumor cells.

Distribution of mouse mammary tumor virus-related sequences does not correlate with the taxonomic position of their hosts.

Sequences (MRS) distantly related to mouse mammary tumor virus (MMTV) were found in genomes of a wide range of mammalian species using blot hybridization. The number of MRS copies and the degree of their homology with the hybridization probe varied and did not correlate with the taxonomic position of the species. Nevertheless, within a genus the set of MRS was species specific and reflected the taxonomic relation between the species. MRS were also found in avian genomes and the degree of their homology did not correlate with the taxonomic position of the species either. The origin and distribution of MRS is discussed on the basis of the authors' and published data.

Avian endogenous provirus (ev-3) env gene sequencing: implication for pathogenic retrovirus origination.

The avian endogenous env gene product blocks the surface receptor and, as a result, cells become immune to related exogenous retroviruses. On the other hand, the same sequence can be included in the pathogenic retrovirus genome, as shown by oligonucleotide mapping. However, since the complete env gene sequence was not known, the comparison of genomic nucleotide sequences was not possible. Therefore an avian endogenous provirus with an intact env gene was cloned from a chicken gene bank and the regions coding for the C terminus of the gp85 and gp37 proteins were sequenced. Comparison of this sequence with those of other retroviruses proved that one of the pathogenic viruses associated with osteopetrosis is a cross between avian endogenous virus and Rous sarcoma virus. Retroviruses and, especially, endogenous retroviruses are traditionally of the most developed models of viral carcinogenesis. Many endogenous retroviruses are implicated in neoplastic transformation of the cell. For instance, endogenous mouse mammary tumor virus of some inbred lines appears to be the only causative agent in these mammary cancers. Other even nonpathogenic murine endogenous retroviruses are involved in the origination of MCF-type recombinant acute leukosis viruses. Some endogenous retroviruses are implicated in the transduction or activation of cellular protooncogenes. Our interest in endogenous viruses is based on their ability to make cells resistant to exogenous retroviruses. Expression of their major envelope glycoprotein leads to cellular surface receptor blockage and imparts immunity to infection by the related leukemia retroviruses. This problem is quite elaborated for chicken endogenous virus RAV-O (7-9).(ABSTRACT TRUNCATED AT 250 WORDS)

[Sequences homologous to mouse mammary cancer virus are common in mammalian and avian genomes]. / Posledovatel'nosti, gomologichnye virusu raka molochnoi zhelezy myshei, shiroko predstavleny v genomakh mlekopitaiushchikh i ptits.

Sequences distantly related to mouse mammary tumor virus (MRS) were found in a wide range of mammalian genomes using blot hybridization. The number of MRS copies and the degree of their relationship with the hybridization probe varied and did not correlate with the evolutionary similarity of the species. Nevertheless, within a genus the set of MRS was species-specific and reflected the degree of relationship between species. MRS were also found in avian genomes and the degree of their relationship also did not correlate with the evolutionary similarity of the species. The origin and distribution of MRS are discussed.

[Cloning and analysis of the primary structure of an element, related to the murine mammary cancer virus, from the genome of the Djungarian hamster]. / Konirovanie i analiz pervichnoi struktury élementa, rodstvennogo virusu raka molochnoi zhelaezy myshei, iz genoma dzhungarskogo khomiachka.

11 recombinant bacteriophages from the genomic library of Djungarian hamster genome, that carry MMTV-related sequences (MRS-Ps), have been cloned with the murine mammary cancer virus (MMTV) as a hybridization probe. The sequences are repeated 50 times in the genome. MRS-Ps contain the tracts of homology with the long end repeat MMTV, the genes pol and, possibly, env, but not with the gag gene. Sequencing of the 0,87 kb restrict, common to all EcoRI-BamHI clones, and the analysis of the sequence have demonstrated the high level of homology with the pol gene of MMTV and its origination from the somewhat functioning gene.

[Endogenous proretroviruses and other autonomous retroposons: traces of viral origin or decay?]. / Endogennye proretrovirusy i drugye avtonomnye retropozony: sledy zarozhdeniia ili raspada virusov?

Origin of viruses is considered to be a fundamental trend in biology. In 1970 H. Temin proposed that some of them (namely, retroviruses) could originate from preexisting transcriptional units of cell genome. This "protovirus hypothesis" was supported recently by new findings. It is clear now that eukaryotic genomes contain sequences related to retroviral genes and long terminal repeats. Some endogenous retrovirus-related sequences can be considered to be interstitial stages in retrovirus origin. We suppose that not only retroviruses but all the autonomous retroposons could arise from cellular sequences.

[The genome of chickens free of endogenous avian leukosis-sarcoma proviruses contains sequences distantly related to Rous sarcoma virus]. / V genome kur, svobodnykh ot éndogennykh provirusov leikozo-sarkomnogo kompleksa ptits, prisutstvuiu posledovatel'nosti, otdalenno rodstvennye virusu sarcomy Rausa.

Line of Brown leghorn chickens free of RAV-O-type endogenous proviruses was obtained by selection under blot hybridization control. A set of dispersed sequences distantly related to avian leukosis virus genome was found in DNA of these chickens by means of hybridization in non-stringent conditions. Different restriction fragments were detected by gag, pol and env hybridization probes.

Genetic structure of the endogenous proviruses and expression of the gag gene in Brown Leghorn chickens.

Seven loci of endogenous proviruses were detected in the genome of Brown Leghorn chickens. Sets of endogenous proviruses in DNA of the chicken embryos examined were identified by blot hybridization with 32P-labelled DNA of RSV and EcoRI restriction endonuclease digestion. Comparison of the results showed that only one locus (A) of endogeneous provirus was associated with a gs+ phenotype as determined by the immunoperoxidase reaction and antibodies against gag gene products of RSV. Restriction endonuclease analysis with HindIII, BamHI and SacI revealed that proviruses A and F in Brown Leghorn chickens correspond to loci ev-3 and ev-6, respectively, in White Leghorn chickens. Other loci (B, C, D, E, and X) were designated ev-22, ev-23, ev-24, ev-25, ev-26, respectively. None of these loci expressed infectious virions. The structure of most of the endogenous proviruses examined is considerably different from the genome of the endogenous chicken virus RAV-O. The difference in structure may be one possible cause of the absence of endogenous provirus expression.

Methylation of endogenous proviruses of Brown Leghorn chickens.

Among the 7 endogenous proviruses we have detected in Brown Leghorn chickens none encodes the production of virions and only one, ev-3, expresses the gag gene. To study the possible role of DNA methylation in the inhibition of provirus expression, we performed blot hybridization and restriction endonuclease analysis with EcoRI and SmaI, is sensitive to methylation. Of the six endogenous proviral loci examined (ev-3, ev-6,, ev-22, ev-23, ev-24, ev-25), two loci, ev-23 and ev-24, were methylated at all SmaI restriction sites, in both the DNA from erythrocytes of adult chickens and the DNA from 10-day embryos. Since both these viruses are closely related to the genome of RAV-O, DNA methylation might be the cause of the absence of gene expression.