Polyoma virus defective DNAs. I. Physical maps of a related set of defective molecules (D76, D91, D92).

Three related polyoma virus species, designated D92 (92% the size of full-length polyoma virus DNA), D91 (91%) and D76 (76%) have been analysed and their structures compared with that of polyoma virus A2 DNA. Three independent methods (restriction endonuclease cleavage, depurination fingerprinting and DNA-DNA hybridization) were used in the analysis.The defective DNAs appear to be: (1) entirely composed of viral sequences (no host DNA sequences were detected): (2) made up in part of long continuous sequences of DNA which appear identical to sequences of A2 DNA (D92 contains continuous sequences from 1 to 72 map units on the physical map of A2 DNA; that is, it contains the entire late region and part of the early region of the viral DNA. D91 and D76 contain those same sequences except for a 1% deletion around 18 map units): (3) made up in part of rearranged viral sequences.Several interesting features were noted about the rearranged sequences present in the defective DNAs. Sequences from the region around 67 map units were found linked to other (non-contiguous) regions of the DNA. Sequences from about 72 map units were linked to sequences from about 1 map unit. Multiple copies of sequences from 67 to 72 map units (from around the origin of DNA replication) were found (4 copies in D91 and D92, and 2 copies in D76).     

New classes of viable deletion mutants in the early region of polyoma virus.

Viable mutants of polyoma virus have been isolated which have deletions in defined parts of the early region of the genome. One class of mutants has deletions (less than 1% of viral genome length) located between 71.5 and 73.5 on the physical map of polyoma virus DNA, near the origin of replication. These mutants appear to grow and to transform cells in a manner indistinguishable from wild-type virus. A second type of mutant with deletions (about 2% of viral genome length) located between about 88 and 94.5 units on the physical map of polyoma virus DNA have altered transformation properties. One of the latter (which maps between 88 and 91.5 units) also has altered growth characteristics, whereas another (which maps between 91.5 and 94.5 units) resembles wild-type virus in its growth properties. The regions with deleted sequences have been defined by cleaving mutant DNAs with restriction endonucleases and analyzing pyrimidine tracts.     

Fine structure of polyoma virus DNA.

A fine structure map of polyoma DNA has been made based on cleavage with a number of restriction endonucleases (including HaeII and III, BamI, HindII and III, BumI, HpaII, and in part, HphI) and depurination of wild-type DNA, the eight HpaII restriction fragments and some HaeIII fragments. This analysis has made possible some correlation with simian virus 40 DNA, and has facilitated detailed examination of various polyoma strains and variants. Sequences from the region of the origin of DNA replication have been examined.     

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.     

DNA sequences of polyoma virus early deletion mutants.

The DNA sequences of four "early" viable deletion mutants of polyoma virus have been determined. Two of these (dl-8 and dl-23) are mutants with deletions in the region of the genome that codes for parts of both large and middle T-antigens, and two (dl-6 and dl-28) are mutants with deletions around the viral origin of replication. The former mutants have altered transformation properties relative to wild-type virus, and dl-8 appears to be replication deficient (B. E. Griffin and C. Maddock, J. Virol. 31:645-656, 1979). Sequences are discussed in terms of the altered phenotypes observed for the various mutants, the DNA structures and protein sequences that are affected by the deletions, and how these might affect the biological properties of the mutants.     

Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.

The genome of equine herpesvirus 1 (EHV-1) defective interfering (DI) particle DNA originates from discrete regions within the standard (STD) EHV-1 genome: the left terminus (0.0 to 0.04 map units) and the inverted repeats (0.78 to 0.79 and 0.83 to 0.87 map units of the internal inverted repeat; 0.91 to 0.95 and 0.99 to 1.00 map units of the terminal inverted repeat). Since DI DNA must contain cis-acting DNA sequences, such as replication origins, which cannot be supplied in trans by the STD EHV-1 virus, regions of the EHV-1 genome shown to be in DI DNA were assayed for the presence of a viral origin of DNA replication. Specifically, STD EHV-1 DNA fragments encompassing the genomic regions present in DI particle DNA were inserted into the vector pAT153, and individual clones were tested by transfection assays for the ability to support the amplification and replication of plasmid DNA in EHV-1-infected cells. The Sma-1 subfragment of the internal inverted repeat sequence (0.83 to 0.85 map units) was shown to contain origin of replication activity. Subcloning and BAL 31 deletion analysis of the 2.35-kilobase-pair (kbp) Sma-1 fragment delineated a 200-bp fragment that contained origin activity. The origin activities of all EHV-1 clones which were positive by the transfection assay were confirmed by methylation analysis by using the methylation-sensitive restriction enzymes DpnI and MboI. DNA sequencing of the 200-bp fragment which contained an EHV-1 origin of replication indicated that this region has significant homology to previously characterized origins of replication of human herpesviruses. Furthermore, comparison of known origin sequences demonstrated that a 9-bp sequence, CGTTCGCAC, which is conserved among all origins of replication of human lytic herpesviruses and which is contained within the 18-bp region in herpes simplex virus type 1 origins shown by others to be protected by an origin-binding protein (P. Elias, M. E. O'Donnell, E. S. Mocarski, and I. R. Lehman, Proc. Natl. Acad. Sci. USA 83:6322-6326) is also conserved across species in the EHV-1 origin of replication.     

Encapsidation and expression of the herpes thymidine kinase gene in polyoma virus.

A recombinant DNA of 5,150 base pairs was prepared containing the intact early region of polyoma virus, including the viral origin of replication and the structural sequences of the herpes simplex virus type 1 thymidine kinase gene. Although no thymidine kinase activity was detected when herpes structural sequences alone were transfected into cells, activity was produced when the structural gene followed the polyoma early region. The recombinant DNA was encapsidated into polyoma virions when cotransfected into mouse 3T6 cells with helper DNA from an early polyoma virus mutant. Herpes thymidine kinase activity was detected by a rapid in situ autoradiographic assay in which [125]iododeoxycytidine was utilized as a substrate for the viral but not the cellular enzyme.     

Regulatory mutants of polyoma virus defective in DNA replication and the synthesis of early proteins

In polyoma virus the origin of replication, the 5′ ends of early mRNAs, and the initiation codon for early protein synthesis map within an approximately 200 bp region of the genome. We have previously reported the isolation and partial characterization of viable mutants of polyoma virus with deletions in this important regulatory region of the genome. Three of the mutants with large deletions, one of which had significantly altered growth properties, have been further characterized with respect to their nucleotide sequence alterations and their levels of viral DNA replication and of early protein synthesis. The nearly coincident deletions in mutants 17 and 2–19 reduce the capacity of these viruses to replicate, even in the presence of a coinfecting virus; thus they help define one boundary of the origin of DNA replication. The deletion in mutant 75 appears to remove sequences that are essential for efficient expression of early genes, but has little or no effect upon DNA replication. Its defect is complemented in trans by wild-type virus. All three mutants eliminate sequences which are candidates for RNA polymerase and ribosome binding sites near the initiation codon for early proteins.     

Discrete regions of simian virus 40 large T antigen are required for nonspecific and viral origin-specific DNA binding.

The nondefective adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses, Ad2+ND2 and Ad2+ND4, have been used to determine which regions of the SV40 genome coding for the large tumor (T) antigen are involved in specific and nonspecific DNA binding. Ad2+ND2 encodes 45,000 M4 (45K) and 56,000 Mr (56K) T antigen-related polypeptides. The 45K polypeptide did not bind to DNA, but the 56K polypeptide bound nonspecifically to calf thymus DNA, Ad2+ND4 encodes 50,000 Mr (60K), 66,000 Mr (66K), 70,000 Mr (70K), 74,000 Mr (74K), and 90,000 Mr (90K) T antigen-related polypeptides, all of which bound nonspecifically to calf thymus DNA. However, in more stringent assays, where tight binding to viral origin sequences was tested, only the 90K protein specified by Ad2A+ND4 showed specific high affinity for sequences at the viral origin of replication. From these results and previously published experiments describing the SV40 DNA integrated into these hybrid viruses, it was concluded that SV40 early gene sequences located between 0.39 and 0.44 SV40 map units contribute to nonspecific DNA binding, whereas sequences located between 0.50 and 0.63 SV40 map units are necessary for specific binding to the viral origin of replication.     

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.     

Construction and analysis of viable deletion mutants of polyoma virus.

Viable mutants of polyoma with small deletions ranging in size from 2 to 75 base pairs were obtained by infecting 3T3 cells with polyoma DNA that had been cleaved once with HaeII endonuclease or with DNase-Mn2+ digestion. The HaeII endonuclease-cleaved DNA yielded mutants with deletions at map position 72--73, whereas the mutants generated by DNase I-Mn2+ digestion had deletions either at map position 72--73 or within the map coordinates 92 and 99. Both groups of mutants appeared to grow as well as wild-type virus in 3T3 cells. The deletions at map position 72--73 did not alter the virus's ability to transform rat cells. Hence, the region just to the early side of the origin of DNA replication is not essential for vegetative growth or transformation. But the mutants with deletions in the region between map coordinates 92 and 99, a segment thought to code for polyoma large and middle T antigens (Hutchinson et al., Cell 15:65--77, 1978; Smart and Ito, Cell 15:1427--1437, 1978; Soeda et al., Cell 17:357--370, 1979), transformed rat cells at 0.2 to 0.05 the efficiency of wild-type virus.     

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.     

Simian agent 12 is a BK virus-like papovavirus which replicates in monkey cells.

We have begun to characterize the genomic structure and replication of the baboon papovavirus simian agent 12 (SA12). We have defined a wild-type clone of SA12 (SA12 wt100) by plaque purification from a heterogeneous stock. The functional map of SA12 wt100 can be aligned with those of the other primate papovaviruses by assigning one of the two EcoRI sites as 0/1.0 map units. The origin of bidirectional viral DNA replication maps near 0.67 map units, consistent with the limits of sequences homologous to origin sequences in the other papovaviruses. DNA sequence analysis shows that the organization of the SA12 genome is similar to that of the other primate papovaviruses studied. The arrangement and sequence of functional elements in the origin of replication region, as well as the sequences of the N-terminal regions of early protein products, indicate that SA12 is most closely related to the human virus BK, next most closely related to JC virus, and less closely related to simian virus 40. Unlike BK virus, SA12 is capable of productive infection of African green monkey kidney cells.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Structural and biological analysis of integrated polyoma virus DNA and its adjacent host sequences cloned from transformed rat cells.

EcoRI fragments containing integrated viral and adjacent host sequences were cloned from two polyoma virus-transformed cell lines (7axT and 7axB) which each contain a single insert of polyoma virus DNA. Cloned DNA fragments which contained a complete coding capacity for the polyoma virus middle and small T-antigens were capable of transforming rat cells in vitro. Analysis of the flanking sequences indicated that rat DNA had been reorganized or deleted at the sites of polyoma virus integration, but none of the hallmarks of retroviral integration, such as the duplication of host DNA, were apparent. There was no obvious similarity of DNA sequences in the four virus-host joins. In one case the virus-host junction sequence predicted the virus-host fusion protein which was detected in the transformed cell line. DNA homologous to the flanking sequences of three out of four of the joins was present in single copy in untransformed cells. One copy of the flanking host sequences existed in an unaltered form in the two transformed cell lines, indicating that a haploid copy of the viral transforming sequences is sufficient to maintain transformation. The flanking sequences from one cell line were further used as a probe to isolate a target site (unoccupied site) for polyoma virus integration from uninfected cellular DNA. The restriction map of this DNA was in agreement with that of the flanking sequences, but the sequence of the unoccupied site indicated that viral integration did not involve a simple recombination event between viral and cellular sequences. Instead, sequence rearrangements or alterations occurred immediately adjacent to the viral insert, possibly as a consequence of the integration of viral DNA.