Identification of TAZ as a binding partner of the polyomavirus T antigens

Abstract

A polyomavirus mutant isolated by the tumor host range selection procedure (19) has a three-amino-acid deletion (Delta2-4) in the common N terminus of the T antigens. To search for a cellular protein bound by wild-type but not the mutant T antigen(s), a yeast two-hybrid screen of a mouse embryo cDNA library was carried out with a bait of wild-type small T antigen (sT) fused N terminally to the DNA-binding domain of Gal4. TAZ, a transcriptional coactivator with a WW domain and PDZ-binding motif (17), was identified as a binding partner. TAZ bound in vivo to all three T antigens with different apparent affinities estimated as 1:7:100 (large T antigen [lT]:middle T antigen [mT]:sT). The Delta2-4 mutant T antigens showed no detectable binding. The sT and mT of the host range transformation-defective (hr-t) mutant NG59 with an alteration in the common sT/mT region (179 D-->NI) and a normal N terminus also failed to bind TAZ, while the unaltered lT bound but with reduced affinity compared to that seen in a wild-type virus infection. The WW domain but not the PDZ-binding motif of TAZ was essential for T antigen binding. The Delta2-4 mutant was defective in viral DNA replication. Forced overexpression of TAZ blocked wild-type DNA replication in a manner dependent on the binding site for the polyomavirus enhancer-binding protein 2alpha. Wild-type polyomavirus T antigens effectively block transactivation by TAZ. The functional significance of TAZ interactions with polyomavirus T antigens is discussed.

FIG. 1.

TAZ is identified as a binding partner for all polyomavirus T antigns. (a) A “flipped” bait with wild-type sT fused through its C terminus to the Gal4 DNA-binding domain was used to perform yeast two-hybrid screening. (b) Target TAZ combines a C-terminal PDZ-binding motif, a 14-3-3-binding sequence, and a WW domain. (c) Transfections and pull-down assays with GST-TAZ. Wild-type and mutant D2N and Δ2-4 versions of T antigen cDNAs were cotransfected into NIH 3T3 cells along with GST-TAZ. Extracts of transfected cells were incubated with GST beads. Bound proteins were eluted and analyzed by Western blotting with F4 monoclonal antibody recognizing all three T antigens. Wild-type T antigens are shown to the left of the panels; mutant T antigens are shown to the right in parentheses. D2N sT bound TAZ roughly 60% as well as wild-type sT. The mutation Δ2-4 effectively abolished binding of TAZ by each of the T antigens. A total of 5% of the cell lysate was loaded in the input lanes.

FIG. 2.

Growth of thr mutant Δ2-4 is restricted to BNL cells. Monolayer cultures of BNL and primary BMK cells were uninfected (mock) or infected by wild-type virus or thr mutant Δ2-4 virus. The photographs, taken 5 days postinfection, show the cytopathic effects indicative of virus growth in all infected cultures, except for BMK infected by the mutant.

FIG. 3.

Analyses of T antigen interactions with TAZ. (a) Extracts of uninfected, wild-type, and NG59 mutant virus-infected BMK cells were immunoprecipitated by cross-linked rabbit anti-TAZ polyclonal antibody (IP) or by preimmune antiserum as a control (C). Proteins were separated and blotted with anti-T and anti-TAZ antibodies. (b) Cotransfection of NIH 3T3 cells with TAZ and lT cDNA alone or together with sT cDNA, followed by anti-TAZ IP and blotting for T antigens. (c) Cotransfection with GST-TAZ and lT alone or together with mT cDNAs followed by GST pull-down and Western blotting for T antigens. A total of 5% of cell lysates were loaded in the input lanes (a to c). (d) Schematic representation of the polyomavirus T antigens showing the two regions of interaction with TAZ, the shared amino terminal sequences (Δ) common to all three T antigens, and the hr-t (large T intron) sequences shared by mT and sT (*). (e) A GST-TAZ expression plasmid was transfected into COP cells, and extracts were immunoprecipitated by anti-T antibody or by normal rat IgG as a control (C). SDS-PAGE and Western blotting for TAZ were performed.

FIG. 4.

The WW domain but not the PDZ-binding motif in TAZ is essential for binding to polyomavirus T antigens. Wild-type GST-TAZ (TAZ), GST-TAZΔPDZ (lacking residues 386 to 395; TAZ ΔPDZ), and GST-TAZΔWW (lacking residues 125 to 156; TAZ ΔWW) constructs were transfected into NIH 3T3 cells. Transfected cells were then infected by wild-type virus. Cell extracts were prepared, and GST pull-down assays were performed 24 h postinfection. After SDS-PAGE, Western blot analyses for T antigens and TAZ were performed. A total of 5% of cell lysates were loaded in input lanes.

FIG. 5.

Expression of wild-type sT and mT leads to nuclear accumulation of TAZ. (a) BMK cells were infected by wild-type virus or NG59 mutant virus. After 24 h, cells were fixed, and immunofluorescence staining was performed with anti-lT (rat) and anti-TAZ antibody (rabbit) followed by secondary antibodies, rhodamine red and Oregon green, respectively. (b) Quantitation of results (see Materials and Methods).

FIG. 6.

Effects of TAZ on viral DNA replication. (a) BMK cells were infected by wild-type virus or Δ2-4 mutant virus. Low-molecular-weight DNA was isolated, and a Southern blot analysis was performed using ³²P-labeled polyomavirus DNA as a probe. The Δ2-4 mutant was sharply reduced in its ability to replicate its DNA.(b) High-level expression of TAZ in a Tet-repressible NIH 3T3 cell line blocks wild-type viral DNA replication. TAZ expression was induced by removing Tet 16 h before infection. TAZ-induced and uninduced cells were infected by wild-type polyomavirus, and low-molecular-weight DNA was extracted at different times. A Southern blot analysis was performed using a polyomavirus plasmid as a probe. Viral DNA replication was strongly inhibited by TAZ as seen from the minimal amplification of input viral DNA at time zero compared to 40 h. (c) The PEBP-2α-binding site is essential for TAZ inhibition of viral DNA synthesis. Wild-type polyomavirus Ori (Py-wt Ori) and an Ori with the PEBP-2α-binding site deleted (Py-Δ Ori) were transfected into TAZ-induced or uninduced cells. TAZ expression was induced by removing Tet 16 h before transfection, and cells were infected by polyomavirus immediately after transfection. Low-molecular-weight DNA was extracted after 24 h, and Southern blotting was performed with the luciferase-encoding region of the ori plasmid as a probe. The band of replicated DNA was compared to input plasmid DNA, and the replication of wild-type ori plasmid in uninduced cells was taken as 100%. (d) Quantitation shows that the inhibition of viral DNA replication by TAZ depends on retention of the PEBP-2α-binding site in the viral enhancer.

FIG. 7.

Wild-type polyomavirus infection inhibits transactivation by TAZ. NIH 3T3 cells were cotransfected with 50 ng of tk-LUC, 50 ng of pRL-EF, and 400 ng of CMV-Gal4 vector along with plasmid expressing the indicated GAL4-TAZ fusion proteins. The luciferase activities were normalized by Renilla activities.