The Amino-Terminal Transforming Region of Simian Virus 40 Large T and Small t Antigens Functions as a J Domain

Abstract

Simian virus 40 (SV40) encodes two proteins, large T antigen and small t antigen that contribute to virus-induced tumorigenesis. Both proteins act by targeting key cellular regulatory proteins and altering their function. Known targets of the 708-amino-acid large T antigen include the three members of the retinoblastoma protein family (pRb, p107, and p130), members of the CBP family of transcriptional adapter proteins (cap-binding protein [CBP], p300, and p400), and the tumor suppressor p53. Small t antigen alters the activity of phosphatase pp2A and transactivates the cyclin A promoter. The first 82 amino acids of large T antigen and small t antigen are identical, and genetic experiments suggest that an additional target(s) important for transformation interacts with these sequences. This region contains a motif similar to the J domain, a conserved sequence found in the DnaJ family of molecular chaperones. We show here that mutations within the J domain abrogate the ability of large T antigen to transform mammalian cells. To examine whether a purified 136-amino-acid fragment from the T antigen amino terminus acts as a DnaJ-like chaperone, we investigated whether this fragment stimulates the ATPase activity of two hsc70s and discovered that ATP hydrolysis is stimulated four-to ninefold. In addition, ATPase-defective mutants of full-length T antigen, as well as wild-type small t antigen, stimulated the ATPase activity of hsc70. T antigen derivatives were also able to release an unfolded polypeptide substrate from an hsc70, an activity common to DnaJ chaperones. Because the J domain of T antigen plays essential roles in viral DNA replication, transcriptional control, virion assembly, and tumorigenesis, we conclude that this region may chaperone the rearrangement of multiprotein complexes.Simian virus 40 (SV40) encodes two proteins involved in tumorigenesis, the large and small tumor antigens. Large tumor antigen (T antigen) orchestrates many aspects of productive viral infection and is necessary and in many cases sufficient for tumorigenesis. T antigen is a 708-amino-acid multifunctional protein that elicits cellular transformation by acting on multiple targets, including members of the retinoblastoma tumor suppressor family (pRb, p107, and p130), members of the CBP family of transcriptional coactivators (CREB-binding protein [CBP], p300, and p400), and the tumor suppressor, p53. It is likely that additional T antigen targets important for transformation await discovery. T antigen sequences important for transformation map to two different regions of the molecule: the amino-terminal domain, which encompasses the first 125 amino acids, and a region located within the carboxyterminal half of the molecule (Fig. 1). Major questions that remain to be answered are the following. How does T antigen act on each of the cellular targets? How does the concerted action of T antigen on these multiple targets lead to tumorigenesis?Evidence that one or more independent transforming functions reside in the carboxy-terminal half o...

Different functions are required for initiation and maintenance of immortalization of rat embryo fibroblasts by SV40 large T antigen

“…The K1 ± JS225 cell line expresses high levels of the protein, consistent with the fact that it contains higher levels of B-myb mRNA than the other two K1-expressing lines, indicating a direct correlation between the level of T antigen and the level of B-myb mRNA. Although dl1135 was not expressed at high levels, it was present; this is consistent with the ®ndings of others that dl1135 is expressed at around 15% of the level of wild-type T antigen (Srinivasan et al, 1997). These results raise the intriguing possibility that dl1135 T antigen can interact productively with p130 and that K1 T antigen also interacts with p130.…”

“…Dl1135 was previously found to be inactive in all transformation assays Michalovitz et al, 1987;Yaciuk et al, 1991;Symonds et al, 1993;Srinivasan et al, 1997). Our results showing that it was unable to immortalize REFs directly but could maintain the growth of tsa14 cells, suggests that some of the functions initially required to immortalize REFs may not be required once the immortal state has been established.…”

“…When T antigen binds these pocket proteins, they are unable to interact with, and repress the activity of, the transcription factor E2F, so E2F-dependent transcription proceeds and the cell enters the cell cycle. It has been suggested that an intact J domain is required to disrupt a pocket protein-E2F complex (Srinivasan et al, 1997;Stubdal et al, 1997;Zalvide et al, 1998), and that this function can only be supplied in cis (Stubdal et al, 1997). Others have found that the J domain can be supplied in trans (Montano et al, 1990;Porras et al, 1996), either by another T molecule, or by the homologus region from small t antigen.…”

Retinoblastoma family proteins as key targets of the small DNA virus oncoproteins

“…SV40, JCV and BKV large T-antigens share a >70% amino-acid sequence homology and are depicted in Figure 1. All these three large T-antigens possess a J domain, the homologous of a region conserved among all cellular DnaJ/Hsp40 molecular chaperones, essential for chaperone activity and for the interaction with the DnaK homolog Hsp70/Hsc70 chaperone machinery Kelley and Georgopoulos, 1997;Srinivasan et al, 1997). Simian virus 40, JCV and BKV large T-antigen (LT-Ag) also possess the LxCxE motif, through which they physically interact with all the members of the RB family proteins (Dahiya et al, 2000), which in turn bind to the viral factors through their 'pocket' domain (Paggi et al, 1996;Helt and Galloway, 2003).…”

“…The N-terminal portion contains three important functional domains, namely CR1, J-domain and CR2 (Powell et al, 1999). The CR1, comprised within the J-domain (residues 1-82), is necessary for SV40 LT-Agmediated transformation capabilities (Srinivasan et al, 1997); CR2 physically contains the LxCxE motif (residues 103-107) and is thus responsible for the binding with pRb, p107 and pRb2/p130 (Zalvide and DeCaprio, 1995;Kim et al, 2001). From a functional point of view, while the LxCxE motif serves to the physical docking of SV40 LT-Ag to the RB family proteins, the J-domain results indispensable for the inactivation of the ability of each member of the pRb family to induce G1 arrest (Zalvide et al, 1998).…”