Genetic Analysis of the Polyomavirus DnaJ Domain

Abstract

Polyomavirus T antigens share a common N-terminal sequence that comprises a DnaJ domain. DnaJ domains activate DnaK molecular chaperones. The functions of J domains have primarily been tested by mutation of their conserved HPD residues. Here, we report detailed mutagenesis of the polyomavirus J domain in both large T (63 mutants) and middle T (51 mutants) backgrounds. As expected, some J mutants were defective in binding DnaK (Hsc70); other mutants retained the ability to bind Hsc70 but were defective in stimulating its ATPase activity. Moreover, the J domain behaves differently in large T and middle T. A given mutation was twice as likely to render large T unstable as it was to affect middle T stability. This apparently arose from middle T's ability to bind stabilizing proteins such as protein phosphatase 2A (PP2A), since introduction of a second mutation preventing PP2A binding rendered some middle T J-domain mutants unstable. In large T, the HPD residues are critical for Rb-dependent effects on the host cell. Residues Q32, A33, Y34, H49, M52, and N56 within helix 2 and helix 3 of the large T J domain were also found to be required for Rb-dependent transactivation. Cyclin A promoter assays showed that J domain function also contributes to large T transactivation that is independent of Rb. Single point mutations in middle T were generally without effect. However, residue Q37 is critical for middle T's ability to form active signaling complexes. The Q37A middle T mutant was defective in association with pp60 c-src and in transformation.Polyomavirus T antigens function both in replication of the virus and in transformation of the host cell. Large T is central to virus production as the initiator of viral DNA replication (20). Middle T and small T also play important roles in different aspects of polyomavirus infection (21,23,55). Defects in viral DNA replication and transcription, as well as defects in viral assembly, have been observed in different mutants of middle T and small T (1,7,8,22,38). Each of the viral early proteins also contributes to regulation of host cell function. Large T is able to immortalize primary cells (44), to block differentiation (37), and to provoke apoptosis (18, 48). These activities are mediated via association with the retinoblastoma susceptibility (Rb) family of tumor suppressors. Middle T, the major transforming protein, works through activation of cellular signaling pathways that are regulated by src-family tyrosine phosphorylation (15). Small T is able to promote cell cycle progression via association with protein phosphatase 2A (PP2A) (39).All three T antigens are produced by differential splicing of common primary transcripts (56). As a result, they have the identical N-terminal sequence of 79 amino acids that encompasses a DnaJ domain. DnaJ domains, consisting of approximately 70 amino acids, have a helical structure in which a conserved HPD motif is found between helix 2 and helix 3 (2, 13, 32, 43, 54). DnaJ domains, found in a broad range of proteins, function to stimulate ...

Lessons in Signaling and Tumorigenesis from Polyomavirus Middle T Antigen

“…One specific example of an MT-induced diffusible product is osteopontin, encoded by an Ap-1-and Pea3/Ets-dependent gene (100). Its overexpression has been demonstrated in invasive nMTV-PyMT 634 tumors and cell lines derived from them, as well as in tissue culture cells transformed by MT (325). Osteopontin is in turn able to induce Ap-1-plus Ets-mediated secretion of a urokinase-type plasminogen activator through EGF activation (75).…”

“…However, other sequences also contribute to the assembly of the MT-PTK complex. Mutations near the N terminus of MT (59, 116, 289), in the J domain (325), and in the PP2A binding site (26,253) also prevent association of MT with PTKs. Most of these mutants fail to bind PP2A as well, supporting PP2A's role discussed above in forming MT complexes.…”

“…There are two unexplained exceptions. The PTK-defective Q37A mutant (325) has been reported to bind PP2A, and L5E (116) binds Src but apparently binds a phosphatase different from PP2A.…”

Association of Simian Virus 40 Vp1 with 70-Kilodalton Heat Shock Proteins and Viral Tumor Antigens

“…The ATPase activity and substrate selection of these chaperones are regulated by the Hsp40 cochaperone via their J domains (20,40,56). SV40 oncoproteins LT and ST are viral cochaperones and contain an N-terminal J domain (5,15,17,24,46) that interacts with Hsc70 (44,45,47,50,55). The J domain is necessary for viral DNA replication, transformation, transcriptional activation, and virion assembly (14, 51).…”

Formation of Covalently Modified Folding Intermediates of Simian Virus 40 Vp1 in Large T Antigen-Expressing Cells

“…Cochaperones of the Hsp40 family, which interact with HSP70 via their J domains, regulate the ATPase activity and substrate selection of HSP70 (16,22,23). The SV40-encoded oncoproteins, the large T (LT) and small t (ST) antigens, are also J domain proteins (24)(25)(26)(27)(28) and interact with Hsc70 (29)(30)(31)(32)(33). The roles of LT/ST in viral DNA replication, transcriptional regulation, transformation, and virion maturation are well known (reviewed in references 34 and 35).…”