scholarly journals Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein.

1989 ◽  
Vol 9 (12) ◽  
pp. 5563-5572 ◽  
Author(s):  
J Settleman ◽  
A Fazeli ◽  
J Malicki ◽  
B H Horwitz ◽  
D DiMaio
Keyword(s):  
Dna Synthesis ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Serum Starvation ◽  
Bovine Papillomavirus ◽  
Focus Formation ◽  
E5 Protein ◽  
Cellular Dna

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.

Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein

1989 ◽  
Vol 9 (12) ◽  
pp. 5563-5572
Author(s):  
J Settleman ◽  
A Fazeli ◽  
J Malicki ◽  
B H Horwitz ◽  
D DiMaio
Keyword(s):  
Dna Synthesis ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Serum Starvation ◽  
Bovine Papillomavirus ◽  
Focus Formation ◽  
E5 Protein ◽  
Cellular Dna

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.

scholarly journals Interactions of the Papovavirus DNA Replication Initiator Proteins, Bovine Papillomavirus Type 1 E1 and Simian Virus 40 Large T Antigen, with Human Replication Protein A

1999 ◽  
Vol 73 (6) ◽  
pp. 4899-4907 ◽  
Author(s):  
YuFeng Han ◽  
Yueh-Ming Loo ◽  
Kevin T. Militello ◽  
Thomas Melendy
Keyword(s):  
Dna Replication ◽  
Protein A ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Bovine Papillomavirus ◽  
Replication Proteins ◽  
E1 Protein ◽  
Cellular Dna

ABSTRACT Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia colias a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene

1983 ◽  
Vol 3 (2) ◽  
pp. 214-219
Author(s):  
K J Soprano ◽  
N Galanti ◽  
G J Jonak ◽  
S McKercher ◽  
J M Pipas ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rrna Genes ◽  
Hybrid Cells ◽  
The Common ◽  
Cellular Dna ◽  
Stimulation Of

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).

scholarly journals Mutational analysis of simian virus 40 T antigen: stimulation of cellular DNA synthesis and activation of rRNA genes by mutants with deletions in the T-antigen gene.

1983 ◽  
Vol 3 (2) ◽  
pp. 214-219 ◽  
Author(s):  
K J Soprano ◽  
N Galanti ◽  
G J Jonak ◽  
S McKercher ◽  
J M Pipas ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Mutational Analysis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rrna Genes ◽  
Hybrid Cells ◽  
The Common ◽  
Cellular Dna ◽  
Stimulation Of

The biological activity of several deletion mutants of simian virus 40, cloned in pBR322, was determined. Three functions of the simian virus 40 A gene were studied: (i) the ability to express T antigen; (ii) the ability to induce cell DNA replication; and (iii) the ability to reactivate silent rRNA genes in hybrid cells. Recombinant plasmid DNA was introduced into cells by manual microinjection or by transfection. The results (together with previous reports) indicate that the critical sequences for these three functions are located separately on the simian virus 40 A gene, as follows: (i) the sequences necessary for the detection of the common antigenic determinant of T antigen extend from nucleotide 4147 to nucleotide 4001 (map units 0.45 to 0.42); (ii) the sequences critical for the stimulation of cell DNA synthesis extend from nucleotide 4327 to nucleotide 4001 (map units 0.49 to 0.42); and (iii) those critical for the reactivation of rRNA genes extend approximately from nucleotide 3827 to nucleotide 3526 (map units 0.39 to 0.33).

scholarly journals Phosphate and the regulation of DNA replication in normal and virus-transformed 3T3 cells

1983 ◽  
Vol 214 (3) ◽  
pp. 695-702 ◽  
Author(s):  
W Engström ◽  
A Zetterberg
Keyword(s):  
Dna Synthesis ◽  
Time Course ◽  
Simian Virus 40 ◽  
Complete Removal ◽  
Simian Virus ◽  
Phosphate Concentration ◽  
Serum Starvation ◽  
Cell Multiplication ◽  
Inhibitory Effects ◽  
3T3 Cells

3T3 cells were cultured in media with different phosphate concentrations and the effects on DNA synthesis were examined. Even a modest phosphate depletion markedly inhibited DNA synthesis and cell multiplication in proliferating cultures. Furthermore, the decrease in the proportion of DNA-synthesizing cells observed after phosphate starvation followed the same time-course as the decrease seen after serum starvation. Cells starved to quiescence in a medium with a 100-fold decrease in phosphate concentration remained viable but non-proliferating for up to 3 weeks, i.e. they had entered a state of quiescence comparable with that seen after serum starvation. Addition of phosphate to phosphate-depleted cultures restored DNA synthesis within 24h. Furthermore, the kinetics of [3H]thymidine labelling after phosphate addition were nearly identical with the labelling kinetics following addition of serum to serum-depleted cultures. In contrast, phosphate deprivation had no inhibitory effects on DNA synthesis in simian-virus-40-transformed 3T3 cells. Furthermore, the inhibitory effects on DNA synthesis in such cells caused by a complete removal of serum could not be further enhanced by decreasing the phosphate concentration in the culture medium.

Functional simian virus 40 T antigen is expressed in hybrid cells having finite proliferative potential

1987 ◽  
Vol 7 (4) ◽  
pp. 1541-1544
Author(s):  
O M Pereira-Smith ◽  
J R Smith
Keyword(s):  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Transformed Cells ◽  
Proliferative Potential ◽  
Hybrid Cells ◽  
Ras Oncogenes ◽  
Immortal Cells ◽  
Cellular Dna

Simian virus 40-transformed human cells fused with other independently derived simian virus 40-transformed cells and tumor-derived cells containing activated H-ras and N-ras oncogenes yielded hybrids capable of indefinite division. Fusions with various other immortal cells yielded hybrids that had limited division potential. T antigen expressed in limited-division hybrids was functional for the induction of cellular DNA synthesis.

scholarly journals Induction of cellular DNA synthesis by a simian virus 40 mutant defective in nuclear transport of T antigen.

1985 ◽  
Vol 5 (6) ◽  
pp. 1531-1533 ◽  
Author(s):  
R E Lanford ◽  
J K Hyland ◽  
R Baserga ◽  
J S Butel
Keyword(s):  
Dna Synthesis ◽  
Limit Of Detection ◽  
Nuclear Transport ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Quiescent Cells ◽  
Cellular Dna

The simian virus 40 (SV40) (cT)-3 mutant [SV40(cT)-3], which is defective in nuclear transport of T antigen, was utilized to determine whether cellular DNA synthesis can be stimulated by SV40 in the absence of detectable nuclear T antigen. Cellular DNA synthesis was examined in the temperature-sensitive cell cycle mutants, BHK ts13 and BHK tsAF8, after microinjection of quiescent cells with plasmid DNA containing cloned copies of wild-type SV40 or SV40(cT)-3. The efficiency of induction of cellular DNA synthesis was identical for both wild-type SV40 and SV40(cT)-3 in both cell lines. The results suggest that cell surface-associated T antigen, either alone or possibly in combination with minimal amounts of nuclear T antigen below our limit of detection, is able to stimulate cellular DNA synthesis.

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