Polyomavirus DNA replication in the pancreas and in a transformed pancreas cell line has distinct enhancer requirements.

The regulatory DNA (enhancer) of polyomavirus (Py) is a major determinant of tissue-specific DNA replication during acute infection of newborn mice. Previously, we reported that the combination of one of the two Py enhancers (A enhancer) and the repeated Moloney murine leukemia virus (Mo-MuLV) enhancer gave a chimeric Py genome (Py-MuLV) that replicates predominantly in the acinar cells of the pancreas, a tissue not permissive for wild-type PyA2 replication (R. Rochford, B. A. Campbell, and L. P. Villareal. Proc. Nat. Acad. Sci. USA 84:449-453,1987). In this report, we further examine the combined enhancer requirements for acinar cell-specific Py replication. We also compare enhancer requirements for Py replication in the acinar cells of the pancreas with those of a transformed acinar cell line (266-6 cells). The deletion of sequences within the A enhancer of Py-MuLV (nucleotides 5098 to 5132) results in a virus with 10-fold-reduced levels of pancreas-specific replication. The deletion, however, of one of the 72-bp repeated Mo-MuLV enhancer sequences from Py-MuLV results in a complete loss of pancreas-specific DNA replication. Thus, the Py A enhancer is required for efficient replication of Py in the pancreas without otherwise altering organ specificity, but both of the repeated copies of the Mo-MuLV enhancer are essential for pancreas-specific Py replication. In contrast to the enhancer requirements for in vivo pancreas replication, in transformed acinar cells (266-6), PyA2 wild-type replicated efficiently and the Py-MuLV recombinant replicated inefficiently. These data suggest that the cell-specific control of DNA replication is different between normal pancreas cells and their transformed cell line counterparts and that this difference is apparent in the enhancer requirement of cell-specific Py DNA replication.     

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.     

Interaction of nuclear factor EF-1A with the polyomavirus enhancer region.

Enhancer factor 1A (EF-1A) is a mammalian nuclear protein that previously was shown to bind cooperatively to the repeated core enhancer element I sequence in the adenovirus E1A enhancer region. We now have characterized three binding sites for EF-1A in the polyomavirus A2 (Py) enhancer region. Site 1 resides in the Py A enhancer domain, and sites 2 and 3 reside in the Py B enhancer domain. EF-1A binding to Py site 1 is independent of cooperation with other EF-1A sites or the adjacent binding sites for PEA-1 and PEA-2, two murine nuclear factors that bind in the Py A enhancer domain. EF-1A binding to Py sites 2 and 3, in contrast, is cooperative, similar to the situation previously observed with binding sites in the adenovirus E1A enhancer region. In a transient replication assay, EF-1A site 1 functions synergistically with the PEA-1 and PEA-2 sites in the A enhancer domain to enhance Py replication. The functional cooperativity observed with the EF-1A, PEA-1, and PEA-2 sites in vivo does not reflect cooperative DNA binding interactions, as detected in vitro. Py EF-1A site 1 alone is capable of weakly stimulating Py replication. EF-1A site 1 overlaps with the binding sites for the murine nuclear protein PEA-3 and the ets family of oncoproteins.     

PEA3 and AP-1 are required for constitutive IL-8 gene expression in hepatoma cells

Interleukin-8 (IL-8) mRNA was constitutively expressed in human hepatoma cell line, HepG2 and in human hepatocellular carcinoma (HCC), which often form hypervascular tumors. The sequence 5'-AGGAAG-3' at -137 to -132 bp of IL-8 promoter was shown to be polyomavirus enhancer A binding protein-3 (PEA3) binding site, which can cooperate with activator protein-1 (AP-1). Both PEA3 and AP-1 are essential for constitutive IL-8 expression in HepG2 cells, determined by promoter assays. Moreover, PEA3 and IL-8 proteins coexisted in HCC tissues, but not in uninvolved liver tissues. It is possible PEA3 may have important roles in tumor progression and in angiogenesis in HCC.     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

A region of the polyoma virus genome between the replication origin and late protein coding sequences is required in cis for both early gene expression and viral DNA replication.

Deletion mutants within the Py DNA region between the replication origin and the beginning of late protein coding sequences have been constructed and analysed for viability, early gene expression and viral DNA replication. Assay of replicative competence was facilitated by the use of Py transformed mouse cells (COP lines) which express functional large T-protein but contain no free viral DNA. Viable mutants defined three new nonessential regions of the genome. Certain deletions spanning the PvuII site at nt 5130 (67.4 mu) were unable to express early genes and had a cis-acting defect in DNA replication. Other mutants had intermediate phenotypes. Relevance of these results to eucaryotic "enhancer" elements is discussed.     

Expression of a 91-kilodalton PEA3-binding protein is down-regulated during differentiation of F9 embryonal carcinoma cells.

Proteins binding to the PEA3 enhancer motif (AGGAAG) activate the polyomavirus early promoter and help comprise the viral late mRNA initiator element (W. Yoo, M. E. Martin, and W. R. Folk, J. Virol. 65:5391-5400, 1991). Because many developmentally regulated cellular genes have PEA3 motifs near their promoter sequences, and because Ets family gene products activate the PEA3 motif, we have studied the expression of PEA3-binding proteins and Ets-related proteins during differentiation of F9 embryonal carcinoma cells. An approximately 91-kDa protein (PEA3-91) was identified in F9 cell nuclear extracts by UV cross-linking to a radiolabeled PEA3 oligonucleotide probe, and expression of PEA3-91 was down-regulated after differentiation of F9 cells to parietal endoderm. The c-ets-1 gene product binds to a sequence in the murine sarcoma virus long terminal repeat that is similar to the PEA3 motif (cGGAAG), but PEA3-91 was not cross-linked to this Ets-1-binding motif, nor did antiserum which recognizes murine c-ets-1 and c-ets-2 proteins have any effect on PEA3-binding activity in mobility shift assays. Furthermore, c-ets-1 mRNA was not detected in undifferentiated or differentiated F9 cells, and c-ets-2 mRNA levels remained high after differentiation. Antiserum against the Drosophila Ets-related E74A protein, however, recognized an approximately 92-kDa protein in F9 cells whose expression during differentiation varied in a manner identical to that of PEA3-91. These data suggest that PEA3-91 is not the product of the ets-1 or ets-2 genes but is likely to be the product of a murine homolog of the Drosophila E74 gene.