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E Fanning

Publications and source records attributed to E Fanning.

At least 55 records · Page 3Linked to original sources
PubMed1992

Adenovirus helper function activity of simian virus 40 T antigen mutants.

The SV40 large T antigen provides a helper function that permits human adenovirus yields in monkey cells to approach those obtained in human cells. The carboxy-terminus of large T antigen is involved in providing this activity. The ability of a large number of SV40 mutants affecting T antigen to enhance the growth of adenovirus type 2 in the CV-1 line of African green monkey kidney cells was examined. Mutation of those serines and threonines at the carboxy terminus which are normally phosphorylated had no effect on adenovirus helper function. A cytoplasmic T antigen was very effective in providing adenovirus helper function. Mutants that produce unstable T antigens provided helper function, but to a reduced degree. Finally, mutations in T antigen which permit it to interfere trans-dominantly with replication catalyzed by wildtype T antigen provided adenovirus helper function at wildtype levels.

Adenoviruses, Human↗
PubMed1992

Intracistronic complementation reveals a new function of SV40 T antigen that co-operates with Rb and p53 binding to stimulate DNA synthesis in quiescent cells.

The ability of the oncogene products of DNA tumor viruses to induce DNA synthesis in quiescent cells is thought to depend on their capacity to bind to cellular proteins such as the retinoblastoma-suppressor protein Rb and the tumor suppressor p53, thereby abolishing the growth-arresting properties of these proteins. We have tested this hypothesis using SV40 T antigens carrying lesions that affect Rb binding, p53 binding or other functions involved in cell transformation. The results demonstrate that Rb binding is not essential for growth stimulation by T antigen. However, detailed analysis, including intracistronic complementation, suggests that at least three functions, Rb binding, a novel second activity localized to the DNA-binding domain and a function residing in the carboxy terminus, probably p53 binding, cooperate to generate the full growth induction potential of T antigen.

Animals↗
PubMed1991

Biochemical characterization of phosphorylation site mutants of simian virus 40 large T antigen: evidence for interaction between amino- and carboxy-terminal domains.

The simian virus 40 large T antigen is phosphorylated at eight or more sites that are clustered in an amino-terminal region and a carboxy-terminal region of the protein. Mutants carrying exchanges at these phosphorylation sites have been generated in vitro by bisulfite or oligonucleotide-directed mutagenesis and analyzed for their phosphorylation patterns. Two-dimensional phosphopeptide analyses of the mutant large T antigens confirmed most of the previously identified phosphorylation sites, namely, serine residues 106, 112, 123, 639, 677, and 679 and threonine residues 124 and 701. In addition, serine residue 120 was identified as a new site, whereas serines residues 111 and 676 were excluded. Interestingly, several of the mutants exhibited secondary effects in that a mutation in the amino-terminal region affected phosphorylation at distant and even carboxy-terminal sites and vice versa. Thus, the amino- and carboxy-terminal domains appear to be in close proximity in the three-dimensional structure of large T antigen. The possible consequences of the above findings and the role of phosphorylation are discussed.

Amino Acid Sequence↗
PubMed1991

A hydrophobic protein sequence can override a nuclear localization signal independently of protein context.

Simian virus 40 T antigen is specifically targeted to the nucleus by the signal Pro-Lys-Lys-128-Lys-Arg-Lys-Val. We have previously described the isolation of a simian virus 40 T-antigen mutant, 676FS, which retains a wild-type nuclear localization signal but fails to accumulate properly in the nucleus and interferes with the nuclear localization of heterologous proteins. Here we report that the hydrophobic carboxy-terminal sequence novel to 676FS T antigen overrides the nuclear localization signal if fused to other proteins, thereby anchoring the proteins in the cytoplasm. We discuss possible mechanisms by which missorting of such a fusion protein could interfere with the nuclear transport of heterologous proteins.

Amino Acid Sequence↗
PubMed1990

SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha.

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.

Animals↗
PubMed1990

Simian virus 40 small tumor antigen and an amino-terminal domain of large tumor antigen share a common transforming function.

The 82-residue amino-terminal sequences of simian virus 40 large tumor antigen (TAg) and small tumor antigen (tAg) are identical. Genetic analysis of TAg lacking amino acids 1-82 revealed that it was transformation-defective, as revealed by the agar growth assay, except when introduced in the presence of tAg. Since the latter, alone, lacks overt transforming activity, it would appear that the function of the sequence common to TAg and tAg is necessary, but not sufficient, for TAg transforming activity and that tAg can provide that function or its equivalent in trans. Thus, tAg may, in part, be viewed as a "portable" copy of a TAg functional domain.

Animals↗
PubMed1990

Altered phosphorylation pattern of simian virus 40 T antigen expressed in insect cells by using a baculovirus vector.

The phosphorylation pattern of simian virus 40 (SV40) large tumor (T) antigen purified from insect cells infected with a recombinant baculovirus was compared with that reported previously for T antigen from SV40-infected monkey cells. The specific activity of metabolic phosphate labeling of baculovirus T antigen was reduced, and the phosphopeptide map of the baculovirus protein, while qualitatively similar to that of lytic T, revealed several quantitative differences. The most striking difference was the prominence in the baculovirus map of peptides containing phosphothreonine 124. These peptides are known to arise from other phosphopeptides upon dephosphorylation of neighboring serines, suggesting that baculovirus T may be underphosphorylated at these serines and perhaps other sites. Functional assays used to further investigate the phosphorylation state of the baculovirus protein included SV40 DNA binding after enzymatic dephosphorylation with alkaline phosphatase and after phosphorylation by a murine homolog of cdc2 protein kinase. The results imply that baculovirus T antigen is underphosphorylated, in particular at those serine residues whose phosphorylation is responsible for down regulation of DNA-binding activity at site II in the core origin of DNA replication. In contrast, no evidence for a functionally significant underphosphorylation at threonine 124 could be found.

Animals↗
PubMed1990

The finger domain of simian virus 40 large T antigen controls DNA-binding specificity.

The specificity and regulation of protein-DNA interactions play a crucial role in all aspects of communication between genotype and phenotype in a cell. The large T antigen of simian virus 40 binds to identical, yet quite differently arranged, pentanucleotide motifs in the simian virus 40 control region, sites I and II. Wild-type T antigen preferentially binds site I. We demonstrate that a bacterial peptide encoding residues 1 to 259 (T260) includes the essential amino acids required for binding to both DNA sites but predominantly binds site II. However, a longer peptide (residues 1 to 369; T370) binds almost exclusively to site I. Thus, the addition of amino acids 260 to 369 to the T260 peptide results in the loss of site II binding. This region includes a single putative metal-binding region, and mutation of T370 at either conserved cysteine of the finger results in equal but inefficient binding to both sites. While no metal binding has been shown to be directly associated with this sequence, these results suggest a novel, perhaps structural, function for such a finger motif, since this domain of T antigen appears to play a crucial role in modulating the DNA-binding behavior of T-antigen peptides.

Antigens, Polyomavirus Transforming↗
PubMed1990

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

Animals↗
PubMed1988

Partial purification and characterization of a cellular protein that binds to the SV40 core origin of DNA replication.

A monkey cell factor that interacts specifically with double- and single-stranded DNA sequences in the early domain of the simian virus 40 (SV40) core origin of replication was identified using gel-retention assays. The protein was enriched over 1200-fold using ion-exchange and affinity chromatography on single-strand DNA cellulose. Binding of protein to mutant origin DNA restriction fragments was correlated with replication activity of the mutant DNAs. Exonuclease footprint experiments on single-stranded DNA revealed prominent pause sites in the early domain of the core origin. The results suggest that this cellular protein may be involved in SV40 DNA replication.

Base Sequence↗
PubMed1988

A mutant SV40 large T antigen interferes with nuclear localization of a heterologous protein.

A mutant SV40 genome carrying a frameshift at the carboxyl terminus of the large T antigen failed to replicate SV40 DNA and to transform rat2 cells, although the altered region is known to be dispensable for these functions. The mutant T antigen also failed to localize normally in the nucleus and interfered with nuclear localization of at least one other nuclear protein, adenovirus fiber. A double mutant carrying an additional lesion in the nuclear localization signal was also localized in the cytoplasm, but regained the ability to transform rat2 cells and no longer affected the nuclear localization of fiber protein. We suggest that the frameshift T antigen may disrupt a mechanism required for nuclear localization of proteins.

Adenoviruses, Human↗
PubMed1988

Mutations in the phosphorylation sites of simian virus 40 (SV40) T antigen alter its origin DNA-binding specificity for sites I or II and affect SV40 DNA replication activity.

A series of mutants of simian virus 40 was constructed by oligonucleotide-directed mutagenesis to study the role of phosphorylation in the functions of large T antigen. Each of the previously mapped phosphorylated serine and threonine residues in large T antigen was replaced by an alanine or cysteine residue or, in one case, by glutamic acid. Mutant DNAs were assayed for plaque-forming activity, viral DNA replication, expression of T antigen, and morphological transformation of rat cells. Viable mutants were isolated, suggesting that modification of some residues is not essential for the biological functions of T antigen. Two of these mutants replicated more efficiently than did the wild type. Seven mutants were partially or completely deficient in viral DNA replication but retained cell transformation activity comparable with that of the wild-type protein. Biochemical analysis of the mutant T antigens demonstrated novel origin DNA-binding properties of several mutant proteins. The results are consistent with the idea that differential phosphorylation defines several functional subclasses of T-antigen molecules.

Animals↗
PubMed1988

Expression of simian virus 40 T antigen in Escherichia coli: localization of T-antigen origin DNA-binding domain to within 129 amino acids.

The genomic coding sequence of the large T antigen of simian virus 40 (SV40) was cloned into an Escherichia coli expression vector by joining new restriction sites, BglII and BamHI, introduced at the intron boundaries of the gene. Full-length large T antigen, as well as deletion and amino acid substitution mutants, were inducibly expressed from the lac promoter of pUC9, albeit with different efficiencies and protein stabilities. Specific interaction with SV40 origin DNA was detected for full-length T antigen and certain mutants. Deletion mutants lacking T-antigen residues 1 to 130 and 260 to 708 retained specific origin-binding activity, demonstrating that the region between residues 131 and 259 must carry the essential binding domain for DNA-binding sites I and II. A sequence between residues 302 and 320 homologous to a metal-binding "finger" motif is therefore not required for origin-specific binding. However, substitution of serine for either of two cysteine residues in this motif caused a dramatic decrease in origin DNA-binding activity. This region, as well as other regions of the full-length protein, may thus be involved in stabilizing the DNA-binding domain and altering its preference for binding to site I or site II DNA.

Antigens, Viral, Tumor↗