New class of polyomavirus mutant that can persist as free copies in F9 embryonal carcinoma cells.

A small circular DNA was found extrachromosomally in a clone of F9 embryonal carcinoma (EC) cells at high copy numbers per cell. The DNA was cloned in plasmid pUC19. Restriction endonuclease analyses of the DNA indicated that the DNA (fPyF9) was a mutant of polyomavirus (Py) DNA and had a mutation in a noncoding regulatory region. There have been many reports on the isolation of Py mutants capable of replication in undifferentiated cells. However, fPyF9 was different from other Py mutants in the following aspects: it was harbored stably as a free copy at 1 X 10(4) to 5 X 10(4) copies per cell in EC cells; it replicated in undifferentiated cells better than in differentiated cells; it was extremely rearranged in the sequences of the enhancer B domain; and it carried in the enhancer B domain three copies of an exogenous sequence which does not exist in Py strain A2. From these observations, we propose a new class of Py EC mutant which has an autonomous state similar to that of plasmid and small circular DNA in host cells.     

Genetic analysis of the enhancer requirements for polyomavirus DNA replication in mice.

In this report, we describe the first systematic analysis of the genetic requirements for polyomavirus (Py) enhancer-activated viral DNA replication during the acute phase of infection in mice. Four mutants were made which substituted XhoI sites for conserved enhancer consensus sequences (adenovirus type 5 E1A, c-fos, simian virus 40, and a glucocorticoidlike consensus sequence). Viral DNA replication in infected mouse organs was measured by DNA blot analysis. Only the loss of the glucocorticoidlike consensus sequence element significantly reduced Py DNA replication in the kidneys, the primary target organ for viral replication. The loss of the c-fos, adenovirus type 5 E1A, or simian virus 40 consensus sequences, however, expanded organ-specific viral DNA replication, relative to wild-type Py, by allowing high-level replication in the pancreas or heart or both. Analysis of Py variants selected for replication in undifferentiated embryonal carcinoma cell lines (PyF441, PyF111) showed that there was little change in levels of viral DNA replication in kidneys and other organs as compared with those in the wild-type virus. If the entire B enhancer is deleted, only low overall levels of viral replication are observed. Wild-type levels of replication in the kidneys can be reconstituted by addition of a single domain from within the A enhancer (nucleotides 5094 to 5132) to the B enhancer deletion virus, suggesting that a single domain from the A enhancer can functionally substitute for the entire B enhancer. This also indicates that the determinants for kidney-specific replication are not found in the B enhancer.     

Transient Gene Expression by SV40 Promoter Characterizes Sequential Differentiation of Embryohal Carcinoma F9 Cells into Primitive and Visceral Endoderm

Transient expression of the chloramphenicol acetyl-transferase (CAT) gene under the control of simian virus 40 (SV40), Moloney murine leukemia virus, human T cell leukemia virus, and cytomegalovirus promoters was stimulated by the differentiation of F9 stem cells into primitive endoderm, but repressed again by further differentiation into visceral endoderm. Deletion mutants of the SV40 enhancer showed that a similar set of motifs is critical for CAT expression at all stages of F9 differentiation, but differentiation dependency was observed even in their absence. The stability of transient gene expression under the control of the SV40 promoter was markedly dependent on F9 differentiation. Appreciable expression was detected even in undifferentiated F9 cells immediately after gene transfection, was maximal at 12 h and declined rapidly thereafter. On the other hand, expression in primitive endoderm increased until 72 h. The decline was accelerated again in visceral endoderm. This shift was somewhat specific to the virus promoter since CAT expression in undifferentiated F9 cells under the control of the elongation factor 1α promoter was more stable than for virus promoters tested. Thus, the change in stability of expression is important for differentiation-dependent virus promoter activity.     

Structural diversity and nuclear protein binding sites in the long terminal repeats of feline leukemia virus.

The long terminal repeat U3 sequences were determined for multiple feline leukemia virus proviruses isolated from naturally occurring T-cell tumors. Heterogeneity was evident, even among proviruses cloned from individual tumors. Proviruses with one, two, or three repeats of the long terminal repeat enhancer sequences coexisted in one tumor, while two proviruses with distinct direct repeats were found in another. The enhancer repeats are characteristic of retrovirus variants with accelerated leukemogenic potential and occur between -155 and -244 base pairs relative to the RNA cap site. The termini of the repeats occur at or near sequence features which have been recognized at other retrovirus recombinational junctions. In vitro footprint analysis of the feline leukemia virus enhancer revealed three major nuclear protein binding sites, located at consensus sequences for the simian virus 40 core enhancer, the nuclear factor 1 binding site, and an indirect repeat which is homologous to the PEA2 binding site in the polyomavirus enhancer. Only the simian virus 40 core enhancer sequence is present in all of the enhancer repeats. Cell type differences in binding activities to the three motifs may underlie the selective process which leads to outgrowth of viruses with specific sequence duplications.     

Multiple domains in the polyomavirus B enhancer are required for productive infection of F9 embryonal carcinoma cells.

A point mutation at nucleotide 5258 in the B enhancer of the polyomavirus host range mutant F441 leads to productive infection of F9 embryonal carcinoma cells, which are refractory to infection by wild-type polyomavirus. Specific oligonucleotides were used to construct mutations in two other potentially important domains within the B enhancer of F441 DNA. One of these domains is the binding site for a factor present in nuclear extracts of F9 cells, and the other is a region that has sequence similarity to putative core sequences observed in a number of different viral enhancers. Mutation within either of these two domains, even in the presence of the F441 mutation, was detrimental to polyomavirus enhancer activity in F9 cells, as determined by both transfection and infection assays.