Transactivation of a ribosomal gene by simian virus 40 large-T antigen requires at least three activities of the protein

Abstract

Simian virus 40 large-T antigen transactivates the ribosomal genes which are transcribed by RNA polymerase (pol I), as well as genes that are dependent on either pol II or pol III. This report identifies regions and activities of T antigen that are required to transactivate a pol I-dependent rat ribosomal gene promoter. By using the rat ribosomal gene (rDNA) promoter linked to a chloramphenicol acetyltransferase gene, we show that at least three separable T-antigen regions are necessary to achieve wild-type levels of transactivation of rDNA in transiently transfected monkey cells. One activity depends on the region of T antigen shared with small-t antigen (T/t common region). A second activity maps to amino acids 109 to 626 and is highly sensitive to mutational inactivation. Complementation analyses suggest that at least one activity in this region is independent of and must be in cis with the activity within the T/t common region. In addition, a functional nuclear localization signal is required for maximal T-antigen-mediated transactivation of rat rDNA. The three activities work in concert to override cellular species-specific controls and transactivate the rat ribosomal gene promoter. Finally, we provide evidence that although the tumor suppressor protein Rb has been shown to repress a pol I-dependent promoter, transactivation of the rat rDNA promoter does not depend on T antigen's ability to bind the tumor suppressor product Rb.

FIG. 1

Transactivation of the rat ribosomal promoter by wild-type large-T antigens and the accumulated levels of T antigens in transiently transfected cells. (A) Transactivation of the rat ribosomal promoter-CAT construct was assayed by determining the amount of butyrated chloramphenicol produced by extracts prepared from cells transfected with the reporter construct only or in conjunction with plasmids that produce wild-type large-T antigen only, small-t antigen only, or both large-T and small-t antigens (pPVU0 or Wt-2), as described in Materials and Methods. Each construct or construct combination was transfected in duplicate or quadruplicate. Replicate assays were performed on extracts from each transfection. Means were determined from the four or eight replicate samples. The level of transactivation is expressed as the mean percentage of wild-type (wt) activity in the same experiment. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%. The entire experiment was performed four times. The pattern of results was consistent; results of one representative experiment are shown. (B) Accumulated levels of T antigen produced from cells transiently transfected with pPVU0, Wt-2, or a T-antigen-only (T1-708) construct. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibody PAb901, which recognizes an epitope in the C terminus of T antigen.

FIG. 2

Transactivation by C-terminally truncated T antigens and the accumulated levels of mutant T antigens in transiently transfected cells. (A) The ability of each mutant T antigen to transactivate the rat ribosomal promoter was tested three to six times as described for Fig. 1A. The pattern of results was consistent; results of a representative experiment are shown. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%. (B) Accumulated levels of T antigen produced from cells transiently transfected with Wt-2, dl1265 (T1-699), dl1066 (T1-670), dl1263 (Tdl663-674), dl2465 (T1-626), dl1061 (T1-590), and dl2433 (Tdl586-590) constructs. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibodies PAb901, which recognizes an epitope in the C terminus of T antigen, and PAb419, which recognizes an epitope in the T/t common region.

FIG. 3

Transactivation by T antigens containing internal or N-terminal deletions and the accumulated levels of mutant T antigens in transiently transfected cells. (A) The ability of each mutant T antigen to transactivate the rat ribosomal promoter was determined as described for Fig. 1A. Each mutant was tested two to six times. The pattern of results was consistent; the results of one representative experiment are shown. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%. (B) Accumulated levels of T antigen produced from cells transiently transfected with each mutant T-antigen-producing construct. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibody PAb901, which recognizes an epitope in the C terminus of T antigen.

FIG. 4

Complementation of an N-terminally truncated T antigen. The ability of the N-terminally truncated T antigen T83-708 was tested for its ability to transactivate the rat ribosomal promoter either alone and in combination with Tdl400 or small-t antigen. Each T antigen or combination was tested at least six times. The pattern of results was consistent; results of a representative experiment for each complementation are shown. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%.

FIG. 5

Immunoblot analysis of mutant T antigens used in complementation experiments and the effect of cotransfection on protein accumulation. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibody PAb901, which recognizes an epitope in the C terminus of T antigen.

FIG. 6

Complementation between internally deleted T antigens and T83-708 or Tdl400. The abilities of the T antigens with internal deletions to transactivate the rat ribosomal promoter either alone and in combination with T83-708 or Tdl400 were examined. Each T antigen or combination was tested twice. The pattern of results was consistent; results of a representative experiment for each complementation are shown. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%.

FIG. 7

Accumulated T-antigen levels in cells coexpressing T83-708 and other mutant T antigens. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibody PAb901, which recognizes an epitope in the C terminus of T antigen.

FIG. 8

Immunoblot analysis of mutant T antigens with or without an NLS or Rb-binding capability in single transfections and cotransfections with T83-708. Immunoblot analysis of transfected cell protein extracts was performed as described in Materials and Methods with monoclonal antibody PAb901, which recognizes an epitope in the C terminus of T antigen.

FIG. 9

Involvement of specific T-antigen functions in transactivation of the ribosomal promoter. The impacts of Rb binding and nuclear localization were investigated. Each mutant was tested at least twice for the ability to transactivate the rat ribosomal promoter; results of representative experiments are shown. The graph is divided into four units for ease of comparing related functions. T-Glu107Lys does not bind the Rb family (Rb/p107/p130) of proteins. Tdl127-250NLS250 and Tdl127-250NLS650 are T antigens missing amino acids 127 to 250 and containing the SV40 NLS immediately preceding amino acid 250 and between amino acids 650 and 651, respectively. T-Lys128ThrNLS contains the SV40 NLS between amino acids 650 and 651. Each error bar represents the percent standard error of the mean; the absence of an error bar indicates that the error was less than 2%.

FIG. 10

Diagrammatic representation of mutant T antigens used in this study. The names of the plasmids encoding the mutant T antigens are given on the left. The corresponding T antigens are represented by lines in which the deleted amino acids are represented by gaps. Single amino acid substitutions and deletions of three or fewer amino acids are represented by vertical lines at the positions of their occurrence. Insertions of amino acids are represented by an inverted triangle at the position of the insertion. The deleted amino acids, substitutions, and insertions are indicated above the sites of alteration. Mutant T antigen sequences cloned in a pBR322 background are underlined; mutant T-antigen sequences cloned in a pBR328 background are not underlined; mutant T antigens that complement one another are designated by an asterisk or dot. Numbers in superscript correspond to literature citations.