DNA sequence alterations in Hr-t deletion mutants of polyoma virus.

We have investigated the DNA sequence alterations in several hr-t mutants of polyoma virus. These mutants are defective in one of the two known viral functions essential for transformation and are altered with respect to several minor T antigen species. The lesions in some of these mutants have been mapped previously by marker rescue experiments to Hpa II fragment 4 (Hpa II-4, 78.4--91.7 map units) in the proximal part of the early region of the viral DNA. Thirteen of sixteen hr-t mutants examined carry deletions 2 to 5 map units (100--250 bp) long in Hpa 11-4. Three mutants carry either point mutations or very small deletions/insertions. Eight of the deletion mutants were mapped closely with restriction enzymes. Seven of them have deletions located entirely within the Hae III subfragment A of Hpa II-4 (the Hae A subfragment, 78.4--85.2 map units), and one extends just beyond this subfragment, ending at 85.5 map units. The complete sequence of the wild-type Hae A subfragment was determined and compared with those of four deletion mutants, NG-18, A-8, 6B5 and B-2. The deletion in each of these mutants is out-of-phase: NG-18, 187 bp; A-8, 127 bp; 6B-5, 179 bp; B-2, 241 bp. All are expected to remove protein sequences in the C terminal part of the small t antigen.     

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.     

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.     

Efficient oligodeoxyribonucleotide-directed deletion mutagenesis using pEMBL vectors: removal of early region introns from polyoma virus mutants

We have used oligodeoxyribonucleotide-directed deletion mutagenesis to remove early region introns from polyoma virus mutants. To this end we compared single priming, double priming, and gapped duplex approaches using either priming at 37 degrees C or at the critical temperature. The gapped duplex approach, coupled with priming at the critical temperature, resulted in up to 70% yield of the desired product. In conjunction with the use of the pEMBL vector system this method was simplified to yield specific deletions from cloned large DNA fragments with high efficiency. The resulting mutant plasmids could be used directly for biological assays without retransformation or recloning. RNA and protein analyses showed that removal of the large T- or middle T-antigen introns from polyoma early region mutants dl23 and dl8 was specific and resulted in DNA competent for the synthesis of only one T antigen.     

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.     

DNA replication origin of polyoma virus: early proximal boundary.

We constructed a series of deleted polyoma genomes by Bal 31 nuclease digestion from the unique Bg/I site at nucleotide 86 on the "early" side of the origin of DNA replication. The ability of the cloned deleted genomes to replicate was tested after transfection into mouse 3T6 fibroblasts or into the polyomatransformed C127 (COP5) mouse cell line (Tyndall et al., Nucleic Acids Res. 9:6231-6251, 1981). Deletions up to nucleotide 64-had no effect on the amount of replicated DNA accumulated, but larger deletions, extending up to nucleotide 42, decreased this amount 7- to 10-fold. By nucleotide 38, the quantity of detected DNA was down 100-fold, and by nucleotide 20, no replication could be detected. The minimum origin segment does not contain any known high-affinity, large tumor antigen binding site.     

Viable deletion mutant in the medium and large T-antigen-coding sequences of the polyoma virus genome.

A polyoma virus mutant that maps in the early region between the known hr-t and ts-a mutants has been isolated. Its 66-base-pair deletion results in structural changes in both medium and large T-antigens but causes no substantial alterations in viral replication or cell transformation.     

Isolation and characterization of simian virus 40 early region deletion mutants.

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.     

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.     

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.     

Studies of polyoma virus DNA: cleavage map of the polyoma virus genome.

A small-plaque polyoma virus, MPC-1, was isolated from a mouse plasmacytoma. The DNA of this polyoma virus was cleaved with a restriction enzyme from Haemophilus influenzae (Hin d), and the molecular weights of the limit products were analyzed by electrophoresis and electron microscopy. The fragments produced by this enzyme have been ordered by analysis of partial digest products. A physical map of the polyoma virus genome was then constructed.     

Tumor antigens induced by nontransforming mutants of polyoma virus.

We have studied the tumor (T) antigens induced by wild-type polyoma virus and several nontransforming mutants using immunoprecipitation with antisera from animals bearing polyomya-induced tumors followed by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. In a variety of mouse cells, wild-type virus induces a major T antigen species with apparent molecular weight of 100,000 daltons, and four minor T antigen species with apparent molecular weights of 63,000, 56,000, 36,000 and 22,000 daltons. Hr-t mutants, which have an absolute defect in transformation, induce a normal 100,000 dalton T antigen but are altered in the minor T antigen species. Hr-t deletion mutants induce none of the minor T antigen species seen in wild-type virus. In their place, these mutants induce T antigen species with molecular weights in the range of 6,000--9,000 daltons. The size of the very small T antigen products does not correlate in any simple way with the size or location of the deletions in the viral DNA. Point hr-t mutants induce two of the four minor T antigen species; they make apparently normal amounts of the 56,000 dalton product and reduced amounts of the 22,000 dalton product, but none of the 63,000 or 36,000 dalton species. Ts-a mutants, which have a temperature-sensitive defect in the ability to induce stable transformation, and which complement hr-t mutants, induce T antigens with the same mobility as wild-type; however, the 100,000 dalton T antigen of ts-a mutants is thermolabile compared to wild-type. A double mutant virus carrying both a ts-a mutation and a deletion hr-t mutation induces a thermolabile 100,000 dalton product and none of the minor T antigen species. Cell fractionation studies with productively infected cells have been carried out to localize the T antigen species.     

Hepatitis B virus: DNA polymerase activity of deletion mutants

The hepadnavirus P gene product is a multifunctional protein with priming, DNA- and RNA-dependent DNA polymerase, and RNase H activities. Nested N- or C-terminal deletion mutations and deletions of domain(s) in human HBV polymerase have been made. Wild-type and deletion forms of MBP-fused HBV polymerase were expressed in E. coli, purified by amylose column chromatography, and the DNA-dependent DNA polymerase activities of the purified proteins were compared. Deletion of the terminal protein or spacer regions reduced enzyme activity to 70%, respectively. However, deletion of the RNase H domain affected polymerase activity more than that of the terminal protein or spacer region. The polymerase domain alone or the N-terminal deletion of the polymerase domain still exhibited enzymatic activity. In this report, it is demonstrated that the minimal domain for the polymerizing activity of the HBV polymerase is smaller than the polymerase domain.     

Nucleotide sequence changes in polyoma virus A gene mutants.

The mutational alterations in polyoma virus mutants ts-a and ts-25E which cause their large T-antigens to be thermolabile have been identified. In ts-a, a G leads to A transition at nucleotide 2193 causes the replacement of Ala (GCT) by Thr (ACT). In ts-25E, a G leads to T transversion at nucleotide 2883 causes the replacement of Gly (GGC) by Cys (TGC). Revertants of both mutants have been isolated and shown to have the original nucleotides restored at these positions.     

Hybridization method for quantitation of replicating polyoma DNA sequences.

Polyoma DNA was cleaved with restriction endonuclease HpaII, the fragments were separated by gel electrophoresis and transferred in good yield to separate nitrocellulose filters by a modification of the procedure of E. M. Southern (1975, J. Mol. Biol.98, 503–517). The filters were then used in hybridization experiments to localize the isotope in different parts of the polyoma genome after in vitro incorporation of labeled deoxyribonucleoside triphosphates into the DNA.