Further characterization of the polyoma virus Y8e from a Rauscher leukaemia virus producing mouse cell line and detection of partial sequence homology between polyoma virus Y8eDNA and hamster papovavirus DNA.

A DNA virus of the papovavirus group spontaneously appeared in RLV-infected spleen and thymus cells of mice in vitro was further characterized as polyoma virus Y8e by haemagglutination test, banding in density gradients, sedimentation coefficients of DNA and molecular hybridization of its DNA. The latter technique showed nearly complete sequence homology to polyoma virus strain SE DNA, partial sequence homology to hamster papovavirus DNA and mouse host DNA and little or no sequence homology to SV 40 DNA. The relationship between rodent papovaviruses and primate papovaviruses is discussed.     

Polyoma and hamster papovavirus large T antigen-mediated replication of expression shuttle vectors in Chinese hamster ovary cells.

Eukaryotic expression vectors have been used successfully in viral LT-expressing cell lines (ie. COS) to clone cDNAs encoding proteins that can be detected through their bio-activity or reactivity with specific antibodies. Since Chinese hamster ovary cells (CHO) have been used extensively for the isolation and characterization of somatic cell mutants, we felt it would be an advantage to develop an expression cloning system in CHO cells. We have modified the eukaryotic expression vector CDM8 by replacing the polyoma and SV40 origins of replication with the 427bp non-coding region of the Syrian hamster papovavirus. Wild-type CHO cells and the CHO glycosylation-mutant Lec4A were transfected with plasmids bearing the early genes of either polyoma virus or hamster papovavirus in order to establish stable, LT antigen-expressing cell lines designated CHOP or CHOH, respectively. CHOP cell lines expressing polyoma LT antigen supported efficient replication of CDM8, but replicated pMH poorly. Conversely, CHOH cells expressing the hamster papovavirus LT antigen supported replication of pMH, and at a lower efficiency, CDM8. Replication of CDM8 and pMH vectors were equally efficient in selected CHOP and CHOH cell lines, respectively and comparable to that of CDM8 replication in COS-1 cells. A bacterial beta-galactosidase fusion gene inserted into the multiple cloning site of a CDM8 derivative was efficiently expressed when transiently transfected into CHOP and CHOH cells but not CHO cells since only the former supports autonomous plasmid replication. These results show that expression-cloning in CHO cells expressing either polyoma virus or hamster papovavirus LT antigens is possible using either the CDM8 or the pMH vectors, respectively.     

Characterization of the genome of the murine papovavirus K.

The DNA genome of the murine papovavirus K virus (KV) was characterized and compared with the genome of polyoma virus. A physical map of the KV genome was constructed by analysis of the size of DNA fragments generated by sequential cleavage with combinations of restriction endonucleases. By using one of the three EcoRI sites in the KV genome as the 0 map position, the KV physical map was then oriented to the polyoma virus genome. Of 42 restriction sites mapped within the KV genome, 7 were localized within 0.01 map unit of their respective sites in the polyoma virus genome; an eighth site mapped within 0.02 map unit. KV replication was examined and found to be bidirectional, initiating at approximately 0.70 map unit. This corresponds well to the origin of replication within the polyoma virus genome and further supports the orientation of the KV physical map.     

Uniform representation of the human papovavirus BK genome in transformed hamster cells.

The DNA of three cloned lines of hamster kidney cells transformed by human papovavirus BK DNA was examined by reassociation kinetics for viral sequences and found to contain 2.7 to 5.3 equivalents of viral DNA per diploid genome. In the one line examined with the four R-HindIII fragments of the human papovavirus BK genome, the entire viral genome was uniformly represented.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.     

Regions of the polyoma genome coding for T antigens.

The early region of the polyoma genome encodes three T antigens. We have analyzed the organization of the coding regions for the T antigens, using the nucleotide sequence of polyoma DNA and peptides derived from purified, radio-labeled T antigens, separated by two-dimensional electrophoresis and chromatography. We compared the peptides, predicted from the nucleotide sequence of the DNA, with those derived from the purified T antigens. We also compared chemically synthesized peptides, predicted from the DNA sequence, with observed peptides. The results show that the three polyoma T antigens are encoded in overlapping regions of the viral DNA, translated, in part, in two different reading frames.     

Sequence from early region of polyoma virus DNA containing viral replication origin and encoding small, middle and (part of) large T antigens.

The sequence of about one third of the polyoma virus genome is presented. This sequence covers the origin of replication of two large plaque strains (A2 and A3) of polyoma virus. The two strains differ by 11 bp in the origin region. A model for replication is suggested. The sequence probably also covers the entire coding region of two of the polyoma virus early proteins--small and middle T antigens--as well as part of the coding region for large T antigen. Over a small region of the DNA, all three coding frames contain termination codons, which argues a need for spliced early messenger RNAs. In another region of the DNA, two coding frames can be used. Correlation with protein data suggests that one frame codes for part of middle T antigen and the other for part of large T antigen.     

Induction of lymphomas by the hamster papovavirus correlates with massive replication of nonrandomly deleted extrachromosomal viral genomes.

The hamster papovavirus isolated from skin epithelioma can induce lymphomas and leukemias after subcutaneous inoculation into newborn hamsters. The lymphoma cells are virus free but contain large amounts of extrachromosomal hamster papovavirus DNA. We have cloned and partly sequenced some of these DNA molecules from independent tumors. These genomes displayed overlapping deletions consistently sharing a common end within the noncoding regulatory sequences; the other end was variable but always extended into the sequence coding for the N-terminal part of the viral capsid VP2. This unique in vivo interaction between a polyomavirus and its cellular host, the genesis of these variant molecules, and their role in the lymphoma formation are discussed.     

A cloned polyoma DNA fragment representing the 5'' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.     

Polyoma virus DNA: Sequence from the late region that specifies the leader sequence for late mRNA and codes for VP2, VP3, and the N-terminus of VP1.

The DNA sequence of part of the late region of the polyoma virus genome is presented. This sequence of 1,348 nucleotide pairs encompasses the leader region for late mRNA and the coding sequence for the two minor capsid proteins VP2 and VP3. The coding sequence for the N-terminus of the major capsid protein overlaps the C-terminus of VP2/VP3 by 32 nucleotide pairs. From the DNA sequence the sizes and sequences of VP2 and VP3 could be predicted. Potential splicing signals for the processing of late mRNA's could be identified. Comparisons are made between the sequence of polyoma virus DNA and corresponding regions of simian virus 40 DNA.     

Effect of in vitro methylation at CpG sites on gene expression in a genome functioning autonomously in a vertebrate host

The effect of in vitro methylation at the HpaII sites in polyoma DNA on viral gene expression and the maintenance of the methyl groups upon replication in vivo were examined. Most of the methylatable sites are located in the early region coding for the viral large T antigen which is essential for the replication and infectivity of the viral DNA. Methylated or mock-methylated polyoma DNA produced the same number of virus plaques appearing at the same time post-transfection in either case. The lack of effect on the infectivity of the viral DNA indicates that the expression of the T antigen gene was not inhibited by methylation. Replication in vivo of the DNA also resulted in a total loss of the methyl groups introduced in vitro. These results underscore basic differences between the behavior of an autonomously functioning papovavirus DNA and the animal cell DNA vis-a-vis methylation at CpG sites. These differences might be due to subtle variations in the mechanism of regulation of gene expression and replication in the two systems.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

Characteristics of the Syrian Hamster Ribonucleic Acid Present in Cells Transformed by Polyoma, Simian Virus 40, or Adenovirus 12

Ribonucleic acids of known specific activities were separately prepared from hamster cells transformed by polyoma, simian virus 40 (SV40), and adenovirus 12. When increasing amounts of these labeled ribonucleic acids were added to nitrocellulose filters containing immobilized (14)C-hamster deoxyribonucleic acid (DNA), about 5% of the DNA sites were saturated in each case. An additive saturation-hybridization experiment indicated that a portion of the 5% of the hamster DNA transcribed in SV40-transformed cells was also transcribed in polyoma-transformed cells. In addition, a separate region of the hamster genome was also transcribed in polyoma-transformed cells.     

Rescue of silent integrated polyoma genomes suggests homologous recombination between resident and transfected DNA fragments

Two defective polyoma virus genomes, deleted in the nucleotide sequences coding the N-termini of the tumor antigens, were introduced into Fisher 3T3 rat cells by DNA-mediated gene transfer (transfection). The resulting integrated genomes were incapable of conferring a transformed phenotype to the cells. However, after transfection of these lines with small polyoma fragments overlapping the deleted sequences, transformed clones were isolated. These clones were analyzed by Southern genomic blot hybridization and by isolation in E. coli of plasmids containing viral sequences excised following fusion with mouse polyoma growth-permissive cells. In all cases at least one intact copy of the early region of the polyoma genome was found. Furthermore, restriction sites adjacent to the initial inactive insertion remained unchanged in many of the transformed lines. These results show that functional restoration of the defective polyoma early region involves homologous recombination between the deleted viral genomes integrated in the cellular DNA and the transfecting viral fragments.     

Mutation causing premature termination of the polyoma virus medium T antigen blocks cell transformation

We used site-specific mutagenesis to introduce a termination codon, TGA, into the reading frame for the polyoma virus medium T antigen. We induced this mutation in a region of the polyoma genome in which the overlapping coding regions for the large and medium TE antigens are translated in different reading frames. Therefore, the mutation terminated translation of the medium T antigen, but it caused only a single amino acid substitution in the large T antigen and did not affect the small T antigen. Cells infected by the mutant virus produced normal-size small and large T antigens. The infected cells produced a 28,000-dalton fragment of the 48,000-dalton medium T antigen, whose size and tryptic peptide map were consistent with its being a truncated N-terminal fragment terminating at the new termination codon of the mutant. Immunoprecipitates of mutant-infected cell extracts did not show medium-T-antigen-associated protein kinase activity. The mutant virus replicated normally in mouse 3T6 cells and induced cellular DNA synthesis in resting mouse 3T3 cells, but it failed to transform rat or hamster cells, as judged by focus formation and growth in agar. The mutant complemented a tsA mutant which affects the large T antigen for transformation, implying that the mutant defect for transformation was in the medium T antigen. These results imply that the small T antigen and the large T antigen together are insufficient to cause transformation and support the conclusion that the medium T antigen is essential for cell transformation by polyoma virus.     

Human papillomavirus 1a complete DNA sequence: a novel type of genome organization among papovaviridae

The complete nucleotide sequence of human papillomavirus type 1a (7811 nucleotides) has been established. The overall organization of the viral genome is different from that of other related papovaviruses (SV40, BKV, polyoma). Firstly, genetic information seems to be coded by one strand. Secondly, no significant homology is found with SV40 or polyoma coding sequence for either DNA or deducted protein sequences. The relatedness of human and bovine papillomaviruses is revealed by a conserved coding sequence in the two species. Two regions can be defined on the viral genome: the putative early region contains two large open reading frames of 1446 and 966 nucleotides, together with several split ones, and corresponds to the transforming part of the bovine papillomavirus type 1 genome, and the remaining sequences, which include two open reading frames likely to encode structural polypeptide(s). The DNA sequence is analysed and putative signals for regulation of gene expression, and homologies with the Alu family of human ubiquitous repeats and the SV40 72-bp repeat are outlines.     

Mutation affecting late gene expression in polyoma virus maps in the late region.

A mutation in polyoma virus strain 3049 which results in the overproduction of capsid proteins has been mapped to the late region of the genome between the HindIII site at 45.0 map units and the BamHI site at 58.6 map units. This region contains the coding sequence for VP3 and a portion of VP2, but does not include the late promoters or the coding sequence for the late leaders. The possible role of VP2 or VP3 in the regulation of genetic expression in polyoma virus is discussed.     

A mathematical model of homologous recombination in cultured cells.

This work presents a model describing the rate of recombination between homologous segments of DNA stably integrated into the genome of cultured cells. The model has been applied to rat cell lines carrying the polyomavirus middle T oncogene and a functional origin of viral DNA replication. Introduction of the gene coding for the polyoma large T antigen or the SV40 large T antigen into cells by DNA transfection promotes homologous recombination in the resident viral inserts with rates varying between 0.1 x 10(-3) and 3.7 x 10(-1) per cell generation.     

Episomal amplification or chromosomal integration of the viral genome: alternative pathways in hamster polyomavirus-induced lymphomas.

The state and expression of the hamster polyomavirus genome in a large panel of virus-induced lymphomas have been investigated. The viral genome is present within tumor cells either as abundant nonrandomly deleted extrachromosomal copies or as a single copy integrated into cellular DNA. We show that these two physical states are likely to be functionally equivalent: first, deletion and integration of the viral genome both inactivate the late coding region; second, the amount of viral early RNAs yielded by a single integrated copy appears to be very similar to that associated with several thousands of extrachromosomal copies of the viral genome. These data underline two essential requisites for hamster polyomavirus to become lymphomagenous: suppression of the late coding functions of the viral genome and expression of the viral oncogenes above a threshold level.