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Journal of Virology, May 2002, p. 4621-4624, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4621-4624.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

The Carboxy Terminus of Simian Virus 40 Large T Antigen Is Required To Disrupt the Yeast Cell Cycle

Sheara W. Fewell, Dena M. Markle, and Jeffrey L. Brodsky*

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Received 1 November 2001/ Accepted 25 January 2002


   ABSTRACT
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Wild-type and J domain mutant simian virus 40 large T antigens alter the cell cycle and bud morphology of Saccharomyces cerevisiae. In contrast, yeast cells expressing mutant T antigen lacking the carboxy-terminal 150 aa exhibit normal morphology, indicating that this region of T antigen is required for cell cycle disruption.


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Simian virus 40 (SV40) large T antigen is expressed early in infection and interacts with multiple host factors to coordinate the expression of viral coat protein-encoding genes, the replication of viral DNA, and the assembly of virions (15). Large T antigen is also capable of driving quiescent cells into S phase, an event that is required for viral DNA replication in permissive cells and that culminates in transformation of nonpermissive mammalian cells.

Large T antigen's oncogenic potential requires at least three regions of the protein (21). Two of these include binding sites for p53 and the retinoblastoma family of tumor suppressors, respectively (5, 10). The third encodes an amino-terminal J domain (Fig. 1A), the defining motif of the Hsp40 family of molecular chaperones (8). Hsp40s bind to and regulate Hsc70s via their J domains, and together, these cochaperones may modulate the conformations of bound proteins or protein complexes (4). We and others demonstrated that the T antigen J domain is functional in vivo (3, 6, 9) and in vitro (17) and that it is required for viral DNA replication and to fully promote cellular transformation (3, 6, 17, 19). Based on these results, we predicted that the T antigen J domain recruits and stimulates cellular Hsc70 to rearrange protein complexes required to initiate DNA replication and to inactivate p53- and pRb-mediated cell cycle inhibition (2).



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  		FIG. 1. Wild-type and mutant constructs of SV40 large T antigen. (A) Domain map of large T antigen (reproduced from reference 17). (B) The dl1135 mutant T antigen is missing aa 17 to 27 of the J domain. (C) The last 150 aa of T antigen are deleted in the {Delta}C T antigen construct. (D) The dl1135-{Delta}C double mutant T antigen lacks aa 17 to 27 and 559 to 708. (E) The locations of the Y34N, H42R, and K53R point mutations in the J domain of T antigen are shown. The four alpha helices of the T antigen J domain, as determined by Kim et al. (11), are depicted in red. The positions of the mutations in the folded structure of the T antigen J domain are depicted elsewhere (6). Details regarding construction of the plasmids used in these experiments are available upon request.

 
Interestingly, when overexpressed from an inducible promoter, large T antigen disrupts the cell cycle of Saccharomyces cerevisiae, resulting in an elongated-bud morphology due to a delay in the G1 to S phase transition (14). T antigen binds and appears to alter the activity of p34CDC28, a kinase that governs the G1 to S phase transition in yeast (14). An amino-terminal fragment (amino acids [aa] 5 to 383), which includes the J domain, and a carboxy-terminal 150-aa region were both shown to bind independently and directly to p34CDC28 (14).

To determine specifically whether the T antigen J domain is required to disrupt the yeast cell cycle, we compared the morphology of yeast cells expressing wild-type T antigen with that of yeast cells expressing one of three chaperone-defective J domain mutant forms of T antigen (6), Y34N, H42R, or K53R (Fig. 1E), from a galactose-inducible promoter. As reported previously (14), approximately 4 to 12% of the wild-type yeast (W303) transformed with an intronless T antigen construct exhibited an elongated-bud morphology 4 to 6 h after induction of T antigen expression (Fig. 2A; Table 1), which corresponds to the time when T antigen accumulates to significant levels (Fig. 3A). In contrast, normal bud morphology was exclusively observed in cells containing a vector lacking the T antigen insert (Fig. 2B; Table 1). The chaperone-defective Y34N, H42R, and K53R mutant T antigens formed altered bud morphologies (Table 1) and were expressed in similar amounts and with similar kinetics compared to the wild-type protein (Fig. 3B to D).



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  		FIG. 2. Overexpression of large T antigen in yeast cells induces elongated-bud morphology. Wild-type yeast (W303) cells transformed with pYes2-TAg (A), containing an intronless version of large T antigen fused to a galactose promoter, or the empty pYes2 vector (B) were grown in SD medium (1) lacking uracil and containing 2% galactose and analyzed by light microscopy.

 

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  		TABLE 1. Wild-type and J domain mutant T antigens disrupt the yeast cell cyclea

 


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  		FIG. 3. Galactose-induced expression of wild-type and mutant T antigens in yeast cells. Immunoblot analysis with MAb416 was performed on total yeast cell lysates to compare the steady-state levels of wild-type and mutant T antigens. Yeast cells were grown as described in Table 1, footnote a. Lanes 0 to 8 of panels A to E represent samples taken before induction with galactose (lane 0) or taken every hour after addition of galactose to the medium (lanes 1 to 8); lane 9 is a negative control containing lysate from cells transformed with the pYes2 vector. Levels of Sec61p, a constitutive membrane protein in the endoplasmic reticulum, were examined as a loading control by using a specific anti-Sec61p polyclonal antibody (18).

 
To confirm the J domain independence of the altered bud phenotype, we examined cells expressing a more severe J domain mutant T antigen, dl1135, which has an 11-aa deletion in the J domain ({Delta}17-27; 16). Because the dl1135 mutant T antigen was poorly expressed from the galactose-inducible plasmid (data not shown), we drove its expression from the stronger, copper-inducible promoter (12) (Table 2; Fig. 4B). Under these conditions, ~2.6% of the cells exhibited the altered bud morphology 2 h after induction with copper (Table 2). Because these mutants are defective in vitro for chaperone activity and in vivo for viral DNA replication and the ability to elicit maximal levels of cellular transformation in mammalian cells (6, 16, 17), we concluded that the J domain is dispensable for T antigen's disruption of the yeast cell cycle.


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  		TABLE 2. The carboxy-terminal 150 aa of T antigen are required for disruption of the yeast cell cyclea

 


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  		FIG. 4. Copper-induced expression of mutant T antigens in yeast cells. An immunoblot analysis with MAb416 was performed on total lysates from yeast cells grown in the absence (lanes 2, 4, 6, 8, and 10) or presence (lanes 1, 3, 5, 7, and 9) of 0.2% CuSO4. Cultures were grown as described in Table 2, footnote a, for 0 h (lanes 1 and 2), 1 h (lanes 3 and 4), 2 h (lanes 5 and 6), 3 h (lanes 7 and 8), or 4 h (lanes 9 and 10).

 
Because both an amino-terminal J domain-containing fragment (aa 5 to 383) and a carboxy-terminal fragment (aa 557 to 707) of large T antigen bind p34CDC28 independently (14), we next tested whether the last 150 aa of T antigen are required for the altered bud phenotype. Only normal cells were observed in yeast cultures containing TAg-{Delta}C (deletion of the last 150 aa of T antigen; Fig. 1C) expressed from either a galactose-inducible (Table 1) or a copper-inducible (Table 2) plasmid, although the truncated protein was expressed efficiently (Fig. 3E and 4A). Consistent with these observations, cells expressing a TAgdl1135-{Delta}C double mutant T antigen (Fig. 1D) also exhibited exclusively normal bud morphology (Table 2).

Although these data indicate that the last 150 aa of T antigen, which bind p34CDC28 (14), are required for disruption of the G1 to S phase transition in yeast, one explanation for these results is that the carboxyl terminus of T antigen is required for nuclear residency. To exclude this possibility, we examined the cellular distribution of wild-type, H42R, dl1135, {Delta}C, and dl1135-{Delta}C T antigens in cells by a indirect immunofluorescence assay using monoclonal antibody 416 (MAb416) and found that each of the proteins localize to the nucleus (Fig. 5).



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  		FIG. 5. Wild-type and mutant T antigens localize to the yeast nucleus. Cells expressing wild-type T antigen (A) or H42R T antigen (B) were grown in galactose-containing medium for 6 h, and cells expressing {Delta}C T antigen (C), dl1135 T antigen (D), or dl1135-{Delta}C double mutant T antigen (E) were grown in medium supplemented with CuSO4 for 3 h before fixation in 3.7% formaldehyde for 20 min at room temperature. Indirect immunofluorescence assays using MAb416 and 4',6'-diamidino-2-phenylindole (DAPI) staining were performed as described previously (7).

 
In sum, the ability of chaperone-defective J domain mutant T antigens to perturb the yeast cell cycle suggests that recruitment of cellular Hsc70 is not required for this phenotype. In contrast, the carboxy-terminal 150 aa, which bind p34CDC28, are required for the cell cycle arrest. As depicted in Fig. 1A, this C-terminal region specifies the host range and contains portions of the ATPase and p53-binding domains; thus, how this domain of T antigen modulates the activity of p34CDC28 is not completely clear. However, in contrast to our data suggesting that the J domain is dispensable for altering the cell cycle, Ydj1p, a yeast cytosolic Hsp40, is required for the p34CDC28-dependent phosphorylation of the Cln3 G1 cyclin (20), indicating that there is precedence for the alteration of p34CDC28 kinase activity by molecular chaperones.

As reported previously, we have also been able to identify immune complexes containing both T antigen and p34CDC28 (14; data not shown)). Interestingly, T antigen, by directly binding to p34CDC28, delays the G1 to S component of the cell cycle in yeast whereas T antigen facilitates this step in mammals. It is intriguing that T antigen also binds p34CDC2, the human homolog of yeast p34CDC28 (14). Moreover, phosphorylation of T antigen at threonine 124 by p34CDC2 stimulates replication of SV40 DNA in vitro (13), suggesting a functional role for this interaction in mammalian cells. Dissecting the significance of the T antigen-p34CDC28 interaction in yeast may further our understanding of T antigen's role in mediating viral DNA replication and cellular transformation.


   ACKNOWLEDGMENTS
 
We thank J. M. Pipas for critical reading of the manuscript and for providing MAb416 and Daniel Lew for advice and materials. We thank Thomas Harper for assistance with the figures.

This work was supported by grant RPG-99-267-01-MBC to J.L.B. from the American Cancer Society. S.W.F was supported by a National Research Service Award (1 F32 CA 83270-01) from the National Institutes of Health.


   FOOTNOTES
 
* Corresponding author. Mailing address: 267 Crawford Hall, University of Pittsburgh, Pittsburgh, PA 15260. Phone: (412) 624-4831. Fax: (412) 624-4759. E-mail: jbrodsky{at}pitt.edu. Back


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Journal of Virology, May 2002, p. 4621-4624, Vol. 76, No. 9
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.9.4621-4624.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.




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