Marcel Bastin

Role of the Three Polyoma Virus Early Proteins in Tumorigenesis

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

Similarity in structure and function of the 3′-terminal region of the four brome mosaic viral RNAs☆

Abstract A 3′-terminal fragment, about 160 nucleotides long, was cleaved by limited nuclease digestion from each of the four RNA components of brome mosaic virus, and purified by two cycles of gel electrophoresis. These fragments accepted tyrosine in reactions catalyzed by wheat germ aminoacyl-tRNA synthetase. Analyses of nuclease digests suggested that the sequences of the fragments from brome mosaic virus RNA 3 and 4 were identical and that the fragments from RNA 1 and 2 differed from that of RNA 4 only in the positions of two and one nucleotides, respectively. A fragment isolated in a similar way from cowpea chlorotic mottle virus was similar in size to the brome mosaic virus RNA fragments, accepted tyrosine in the presence of wheat germ aminoacyl-tRNA synthetase, but had a substantially different nucleotide sequence.

Tumorigenic activity of polyoma virus and SV40 DNAs in newborn rodents.

A procedure has been developed whereby the oncogenicity of the DNA from polyoma (Py) virus and Simian virus 40 (SV40) can be tested directly by injecting recombinant DNA into newborn rodents. Injection of 0.2-2.0 micrograms of linear DNA induced the development of subcutaneous liposarcomas and fibrosarcomas at the site of inoculation. Coinjection of high-molecular-weight rat DNA as carrier had little or no effect on tumor formation but plasmids pBR322, pAT153 , and pML2 behaved as strong inhibitors. Tumor induction by injecting DNA into newborn rodents provides an in vivo equivalent to a transformation assay but appears to be a more stringent and rigorous criterion of oncogenic transformation. The oncogenic potential of Py virus in newborn hamsters could be expressed by a recombinant encoding only the middle T protein, although with average tumor latencies 5-10 times longer than those observed with wild-type Py DNA. Py middle T required the cooperation from small T to induce tumors in newborn rats. SV40 DNA was tumorigenic only in newborn hamsters. delta 2005 DNA which is unable to produce the SV40 small T antigen was much less active and required a latent period about twice that of wild-type SV40 DNA. However, its tumorigenic potential was restored by addition of the Py small T antigen gene. This indicates that Py and SV40 small T antigens are interchangeable and that they probably play an identical role in malignant transformation. Finally, evidence was provided that intermolecular recombination or recombination between DNA fragments can occur in vivo.

Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.

Tumorigenic activity of cloned polyoma virus DNA in newborn rats

The proximal portion of the polyoma virus early region as well as the complete viral genome were cloned in pBR322. Recombinant plasmids induced tumors in newborn rats but only after linearization of the DNA by various restriction endonucleases.

Biological Properties of Polyoma DNA Fragments Cloned in Plasmid pBR322

The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, and the biological activity of the recombinant plasmids was tested in tissue culture cells. A mixture of recombinant plasmids containing the HindIII-A and HindIII-B fragments was infectious, but only after cleavage with HindIII. Recombinant plasmids containing the HindIII-A fragment, but not those containing the HindIII-B fragment, induced the transformation of Fischer rat 3T3 cells. These findings indicate that about half of the early region of polyoma virus DNA is not essential for the initiation of the maintenance of transformation.

A possible replicative form of brome mosaic virus RNA 4

Abstract A double-stranded RNA of a size expected for the replicative form of BMV RNA 4, the smallest of the four RNAs of brome mosaic virus, was isolated from infected leaves by a procedure involving removal of single-stranded RNA by precipitation in 2 M LiCl, followed by cellulose chromatography and polyacrylamide gel electrophoresis. Like the other double-stranded RNAs of BMV, it had characteristics of a replicative form, such as distinctive elution patterns from cellulose and hydroxyapatite columns, and purple staining with toluidine blue O. It coelectrophoresed in 2.5% polyacrylamide gels with double-stranded RNA synthesized in vitro by Qβ replicase with BMV RNA 4 as template. The origin and mode of replication of BMV RNA 4 is discussed.

Mutations in polyomavirus large T affecting immortalization of primary rat embryo fibroblasts.

To clarify the relationship between various functions of the polyomavirus large T antigen and the contribution of this oncogene toward neoplastic transformation, we have analyzed the properties of mutants with in-frame deletions in the second large T exon. dl45, dl96, and dl97 have retained the ability to immortalize primary rat embryo fibroblasts and to trans-activate viral promoters. dl8, dl23, and dl300, which are deficient immortalization, are also deficient in transactivation. However, a newly constructed mutant, designated dl141, which is deficient in immortalization, is still able to trans-activate both the polyoma and SV40 late promoters. This indicates that the ability to trans-activate promoters is not sufficient to confer on the large T antigen the ability to immortalize primary cells.

Site-directed mutagenesis of the polyomavirus genome: Replication-defective large T mutants with increased immortalization potential

We used the deletion loop mutagenesis procedure to direct point mutations into a small region of the polyomavirus genome, at 0.97 map units, affecting the structure of both the middle and large T antigens. We describe the construction of six middle T mutants which have retained the ability to transform rat cells in culture and four large T mutants, three of which are deficient in viral DNA replication and capable of immortalizing primary rat embryo fibroblasts very efficiently.

Intrachromosomal recombination mediated by the polyomavirus large T antigen

We used a spleen necrosis virus-based retroviral vector to introduce the polyomavirus replication origin into rat cells and developed a system to analyze homologous recombination events that do not reconstitute a selectable marker. Introduction of the gene coding for the polyomavirus large T antigen into the cell lines by DNA transfection promoted high-frequency recombination between the two retroviral LTRs, leading to amplification and excision of DNA sequences. To analyze homology requirements, we constructed cell lines carrying only the replication origin without exogenous repeats. Most of the cell lines sustained high-frequency recombination, presumably by undergoing homologous recombination between repetitive DNA lying in the vicinity of the integrated origin. Our results indicate that homologous recombination promoted by large T antigen does not require recombination hot spots in the viral genome other than the replication origin and they explain the cytotoxicity observed in some cell types when large T antigen is expressed in the presence of a functional origin.