T antigens of simian virus 40: molecular chaperones for viral replication and tumorigenesis

Abstract

Simian virus 40 (SV40) is a small DNA tumor virus that has been extensively characterized due to its relatively simple genetic organization and the ease with which its genome is manipulated. The large and small tumor antigens (T antigens) are the major regulatory proteins encoded by SV40. Large T antigen is responsible for both viral and cellular transcriptional regulation, virion assembly, viral DNA replication, and alteration of the cell cycle. Deciphering how a single protein can perform such numerous and diverse functions has remained elusive. Recently it was established that the SV40 T antigens, including large T antigen, are molecular chaperones, each with a functioning DnaJ domain. The molecular chaperones were originally identified as bacterial genes essential for bacteriophage growth and have since been shown to be conserved in eukaryotes, participating in an array of both viral and cellular processes. This review discusses the mechanisms of DnaJ/Hsc70 interactions and how they are used by T antigen to control viral replication and tumorigenesis. The use of the DnaJ/Hsc70 system by SV40 and other viruses suggests an important role for these molecular chaperones in the regulation of the mammalian cell cycle and sheds light on the enigmatic SV40 T antigen-a most amazing molecule.

FIG. 1.

SV40 genomic organization and early gene products. (A) SV40 genomic organization of the alternatively spliced early (large T antigen [LT], small t antigen [St], and 17k T antigen) and late (VP1 to -3) proteins. The early (PE) and late (PL) promoters exist in opposite orientations that flank the SV40 origin (Ori) of replication. (B) T antigen is a large 708-amino-acid multidomain protein. It consists of an amino-terminal J domain (that directly contacts Hsc70), followed by an pRB family binding motif (LXCXE) which binds to all three pRB family members, pRB, p107, and p130; a nuclear localization signal (NLS); a specific DNA binding domain (ori binding); a zinc finger motif (Zn); ATPase domain; a bipartite p53 binding domain that is also essential for mediating interaction with the transcriptional adapter protein p300; and an HR specificity region. 17kT antigen is comprised of the first 131 amino acids of large T antigen (including the J domain [J]) and an pRB-binding (LXCXE) motif, plus an additional four unique amino acids. The amino terminus of small t antigen is comprised of the J domain (amino acids 1 to 82), plus an additional carboxyl-terminal domain that binds to the multimeric protein phosphatase 2A (pp2A) comprised of a catalytic (c) and a regulatory (a) peptide.

FIG. 2.

T antigen inhibits both the pRB and p53 tumor suppressor pathways. Mitogenic stimulation triggers phosphorylation of pRB by cyclin D/CDK4,6 complexes. This releases pRB-mediated repression of E2F transactivation, thus allowing the synthesis of the enzymes necessary for cell cycle progression and DNA replication. T antigen induces free E2F. p53, “the guardian of the genome,” inhibits cell cycle progression; one way this is accomplished is via p21, which inhibits phosphorylation of pRB. Additionally, p53 induces apoptosis when activated by genotoxic stresses. T antigen inhibits multiple activities of p53. Hsc70 inhibits apoptosis and may play an additional role in regulating the activities of both pRB and p53. See the text for more details.

FIG. 3.

pRB binding to T antigen and chaperones. (A) The crystal structure of pRB bound to an E7 peptide (from papillomavirus) containing an LXCXE motif (PDB code 1Gux [131]). Note that pRB binds to LXCXE entirely through its B domain, even though the A domain is required for efficient complex formation with LXCXE motif-containing proteins. For comparison, the conserved amino acids of the T antigen and E7 RB binding (LXCXE) motifs are shown in black. (B) The crystal structure of pRB bound to the first 117 amino acids of T antigen (PDB code 1GH6 [122]). Notice the LXCXE motif of T antigen binds to pRB in a manner similar to the LXCXE E7 peptide. Additionally, the J domain of T antigen is depicted as four multicolored helices: helix 1 (yellow), helix 2 (dark blue), the highly conserved HPD loop in red connecting helices 2 and 3, helix 3 (green), and helix 4 (light green). (C) Domain map demonstrating that the A and B domains of pRB are highly conserved among the other pRB family members, p130 and p107. The essential regions of pRB required for various activities, such as binding to Hsp70 or T antigen, are diagrammed with black lines corresponding to particular regions of pRB (35, 106, 131).

FIG. 4.

pRB repression of the E2F transcription factors. In growth-arrested cells the pRB family of proteins (pRB, p107, and p130) can mediate transcriptional repression in at least two ways, via direct repression domains and by recruiting the activity of the protein RBP1 which directly represses transcription and indirectly represses transcription by recruiting HDAC (126). In dividing cells, pRB family members are no longer bound to E2F, thus allowing for transcriptional activation of promoters containing E2F binding sites.

FIG. 5.

Domain structure of Hsc70. (A) The peptide binding domain of the E. coli Hsc70 protein DnaK (PDB code 1DKZ [275]). The substrate binding domain is shown in purple, the lid (or “variable”) domain is shown in blue, and the bound peptide substrate is shown in yellow. (B) NMR structure of the J domain of E. coli DnaJ (PDB code 1BQZ [100]). The D35 residue is modeled with space filling in red to highlight its important role in directly contacting the ATPase domain of DnaK (see the text for more details). (C) The crystal structure of the ATPase domain of DnaK is shown as four alpha-helical regions in purple (PDB code 1DKG [89]). Residue R167 is modeled with red space filling to underscore the importance of the surrounding region in directly binding to residue D35 of the DnaJ J domain. (D) Domain map of Hsc70, the ATPase (amino acids 1 to 386), and the substrate binding (P [for peptide]) domains are shown in purple. The lid (L) domain is shown in blue.

FIG. 6.

The ATPase cycle of Hsc70. When bound to ADP, Hsc70 has a high substrate affinity; conversely when bound to ATP, Hsc70 displays a weak affinity for peptide substrates. BAG-1 and GrpE are nucleotide exchange factors that promote the exchange of ATP for ADP, increasing the steady-state ATPase activity of Hsc70. J domain-containing proteins (J proteins) stimulate the ATPase activity of Hsc70, which is inhibited by CHIP. Hip promotes the stabilization of the ADP-bound form of Hsc70.

FIG. 7.

J domain structure. (A) NMR structures of the J domains of E. coli DnaJ (PDB code 1BQZ [100]), human HDJ1 (PDB code 1HDJ [184]), polyomavirus T antigen (PYV) (PDB code 1Faf [9]) and crystal structure of SV40 T antigen (SV40) (PDB code 1GH6 [122]). Alpha-helix I is shown in yellow, alpha-helix II is shown in blue, alpha-helix III is shown in dark green, and alpha-helix IV (DnaJ and HDJ1 only) is shown in light green. The absolutely conserved HPD tripeptide comprising the loop between helices II and II is shown in red. (B) Amino acid alignment of DnaJ, HDJ1, PYV, and SV40 is shown. The amino acids that make up the particular helices are indicated by colored boxes. The absolutely conserved HPD tripeptide is shown in red. (C) Amino acids of key SV40 T antigen mutants. Alpha-helix 4 of the SV40 J domain is omitted to better show the location of three distinct mutants of SV40 T antigen, representing three different phenotypes (Table 1). The locations of the D44N point mutant and the L19F,P28S double point mutant are indicated by highlighting these residues and their side chains in cyan. The region that corresponds to the small deletion mutant, Δ17-27, is shown in black.

FIG. 7.

J domain structure. (A) NMR structures of the J domains of E. coli DnaJ (PDB code 1BQZ [100]), human HDJ1 (PDB code 1HDJ [184]), polyomavirus T antigen (PYV) (PDB code 1Faf [9]) and crystal structure of SV40 T antigen (SV40) (PDB code 1GH6 [122]). Alpha-helix I is shown in yellow, alpha-helix II is shown in blue, alpha-helix III is shown in dark green, and alpha-helix IV (DnaJ and HDJ1 only) is shown in light green. The absolutely conserved HPD tripeptide comprising the loop between helices II and II is shown in red. (B) Amino acid alignment of DnaJ, HDJ1, PYV, and SV40 is shown. The amino acids that make up the particular helices are indicated by colored boxes. The absolutely conserved HPD tripeptide is shown in red. (C) Amino acids of key SV40 T antigen mutants. Alpha-helix 4 of the SV40 J domain is omitted to better show the location of three distinct mutants of SV40 T antigen, representing three different phenotypes (Table 1). The locations of the D44N point mutant and the L19F,P28S double point mutant are indicated by highlighting these residues and their side chains in cyan. The region that corresponds to the small deletion mutant, Δ17-27, is shown in black.

FIG. 8.

Chaperone models for disruption of pRB-E2F family complexes. The multiple pRB and E2F family members are represented by “RB” and “E2F.” (A) In this model, T antigen recruits Hsc70 to pRB to directly act as a molecular machine that pries apart the pRB-E2F multiprotein complexes. (B) In this model, T antigen recruits Hsc70 to a multiprotein complex that requires the action of an additional unknown cellular protein (C [for cellular factor]) to disrupt pRB-E2F complexes (see the text for details). Factor C may posttranslationally modify pRB or E2F (denoted with asterisks) after they are separated from each other by the action of Hsc70. An alternative explanation is that factor C may enhance the activity of Hsc70 directly.