Simian Virus 40 T Antigens and J Domains: Analysis of Hsp40 Cochaperone Functions in
            <i>Escherichia coli</i>

Genevaux, Pierre; Lang, F.; Schwager, Françoise; Vartikar, Jai V.; Rundell, Kathleen; Pipas, James M.; Georgopoulos, Costa; Kelley, William L.

doi:10.1128/jvi.77.19.10706-10713.2003

Cited by 20 publications

(20 citation statements)

References 46 publications

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“…ST contains an N-terminal J domain, which is an ϳ70-amino-acid, four-helix structure found in DNA J-type molecular chaperones (11,41). J domains function as cochaperones by recruiting and activating the ATPase activity of Hsp70 (heat shock protein 70) proteins (21).…”

Section: Resultsmentioning

confidence: 99%

Simian Virus 40 Small t Antigen Mediates Conformation-Dependent Transfer of Protein Phosphatase 2A onto the Androgen Receptor

Yang

Vitto

Busby

et al. 2005

Molecular and Cellular Biology

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The tumor antigens simian virus 40 small t antigen (ST) and polyomavirus small and medium T antigens mediate cell transformation in part by binding to the structural A subunit of protein phosphatase 2A (PP2A). The replacement of B subunits by tumor antigens inhibits PP2A activity and prolongs phosphorylationdependent signaling. Here we show that ST mediates PP2A A/C heterodimer transfer onto the ligand-activated androgen receptor (AR). Transfer by ST is strictly dependent on the agonist-activated conformation of AR, occurs within minutes of the addition of androgen to cells, and can occur in either the cytoplasm or the nucleus. The binding of ST changes the conformation of the A subunit, and ST rapidly dissociates from the complex upon PP2A A/C heterodimer binding to AR. PP2A is transferred onto the carboxyl-terminal half of AR, and the phosphatase activity is directed to five phosphoserines in the amino-terminal activation function region 1, with a corresponding reduction in transactivation. Thus, ST functions as a transfer factor to specify PP2A targeting in the cell and modulates the transcriptional activity of AR.The nuclear receptor (NR) superfamily of ligand-activated transcription factors directs the expression of genes that control a wide range of processes, including development, metabolism, and reproductive biology (24). NRs such as the androgen receptor (AR) reside initially in the cytoplasm, where they bind their cognate ligand, undergo chaperone-assisted conformational changes, engage with the nuclear import machinery, and translocate to the nucleus (8). NRs undergo sequencespecific binding to response element DNAs and recruit regulatory proteins to enhancers and promoters of target genes en route to assembling preinitiation complexes, attracting RNA polymerase II, and initiating transcription (14). There is recent evidence that NR recruitment of regulatory proteins needed for transcription occurs in an ordered and cyclical fashion (25).Members of the NR superfamily have a modular structure consisting of an N-terminal domain, a central DNA binding domain (DBD), a flexible hinge region, and a C-terminal ligand-binding domain (LBD) (10). The AR makes contact with regulatory proteins through subdomains with activation functions in the N-terminal domain activation function region 1 (AF-1) and the C-terminal LBD (AF-2). The AR DBD mediates binding to androgen response element DNA, which contains two palindromic hexanucleotide half-sites separated by a three-nucleotide spacer. The AR LBD forms the ligand-binding pocket, provides binding sites for heat shock proteins, and mediates intramolecular interactions with the N-terminal domain (17, 31).Ligand binding is the primary mechanism for controlling the transactivation of NRs, and for some steroid hormone receptors, including AR, ligand binding controls subcellular localization as well (2). There is, however, a growing body of evidence that NR activity can be regulated by posttranslational modification. The phosphorylation of NRs can regulate ligand sensiti...

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Section: Resultsmentioning

confidence: 99%

Simian Virus 40 Small t Antigen Mediates Conformation-Dependent Transfer of Protein Phosphatase 2A onto the Androgen Receptor

Yang

Vitto

Busby

et al. 2005

Molecular and Cellular Biology

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“…There has been little systematic genetic analysis of J domains, even for E. coli DnaJ (17,24,25). To map the DnaJ residues necessary for polyomavirus large T function, we carried out extensive mutagenesis of the J domain.…”

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confidence: 99%

“…Less is known about the role of the J domain in polyomavirus middle T and small T. Although some mutations in the middle T J domain have been shown to affect transforming ability (11), mutation of the HPD loop was found not to alter the ability of middle T to transform cells (4,26). This suggests that activation of DnaK, the normal role for DnaJ, is not required for transformation.There has been little systematic genetic analysis of J domains, even for E. coli DnaJ (17,24,25). To map the DnaJ…”

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Genetic Analysis of the Polyomavirus DnaJ Domain

Whalen

Jesus

Kean

et al. 2005

J Virol

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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 ...

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“…The particles formed in vitro with VP1 pentamers and HF-VP2 are morphologically irregular, indicating that some other component(s) is required to form completely spherical virions. It has been suggested that proper virus assembly requires chaperones in vivo (35,36). Furthermore, the SV40 minichromosome is also 4 M. Kawano, unpublished observations.…”

Section: Discussionmentioning

confidence: 99%

The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

Kawano

Inoue

Tsukamoto

et al. 2006

Journal of Biological Chemistry

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The SV40 capsid is composed primarily of 72 pentamers of the VP1 major capsid protein. Although the capsid also contains the minor capsid protein VP2 and its amino-terminally truncated form VP3, their roles in capsid assembly remain unknown. An in vitro assembly system was used to investigate the role of VP2 in the assembly of recombinant VP1 pentamers. Under physiological salt and pH conditions, VP1 alone remained dissociated, and at pH 5.0, it assembled into tubular structures. A stoichiometric amount of VP2 allowed the assembly of VP1 pentamers into spherical particles in a pH range of 7.0 to 4.0. Electron microscopy observation, sucrose gradient sedimentation analysis, and antibody accessibility tests showed that VP2 is incorporated into VP1 particles. The functional domains of VP2 important for VP1 binding and for enhancing VP1 assembly were further explored with a series of VP2 deletion mutants. VP3 also enhanced VP1 assembly, and a region common to VP2 and VP3 (amino acids 119 -272) was required to promote VP1 pentamer assembly. These results are relevant for controlling recombinant capsid formation in vitro, which is potentially useful for the in vitro development of SV40 virus vectors. Viral capsids are highly organized protein complexes that assemble and disassemble depending on environmental conditions during the viral life cycle. An elucidation of the mechanisms of assembly and disassembly of viral capsids may greatly help in understanding these highly organized protein complexes.SV40 is a small, nonenveloped DNA tumor virus that belongs to the family Polyomaviridae. Its capsid is formed by 72 copies of pentamers of the VP1 major capsid protein (360 molecules in total) and 72 molecules of the VP2 or VP3 minor capsid proteins. VP1 pentamers are arranged in a T ϭ 7d icosahedral lattice, and the carboxyl-terminal arm of VP1 mediates inter-pentameric contacts that hold the capsid together (1-4). VP3 is an amino-terminally truncated form of VP2. One molecule of either minor capsid protein binds to the center of a VP1 pentamer through their common carboxyl-terminal region inside the virion (5-7).However, the precise molecular mechanism for virion assembly is not known.We and others have shown that the VP1 proteins of SV40 (8 -12) and of closely related viruses such as JC virus (13, 14), murine polyomavirus (8,(15)(16)(17)(18)(19), and papillomavirus (20, 21) form virus-like particles (VLPs) 2 when expressed in insect Sf9 cells from baculoviral vectors. The properties of VP1-VLPs are very similar to those of wild type virions in that they dissociate into pentamers following treatment with a calcium-chelating agent (EGTA) under reducing conditions in vitro (22-24). Previously, we prepared highly purified SV40 VP1 pentamers (9, 25, 26), which assembled into morphologically heterogeneous particles under various nonphysiological conditions (26). Such particles include T ϭ 7d (triangulation number 7 dextro) spherical particles, small T ϭ 1 (triangulation number 1) particle composed of 12 pentamers, and long tubu...

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Simian Virus 40 T Antigens and J Domains: Analysis of Hsp40 Cochaperone Functions in Escherichia coli

Cited by 20 publications

References 46 publications

Simian Virus 40 Small t Antigen Mediates Conformation-Dependent Transfer of Protein Phosphatase 2A onto the Androgen Receptor

Simian Virus 40 Small t Antigen Mediates Conformation-Dependent Transfer of Protein Phosphatase 2A onto the Androgen Receptor

Genetic Analysis of the Polyomavirus DnaJ Domain

The VP2/VP3 Minor Capsid Protein of Simian Virus 40 Promotes the in Vitro Assembly of the Major Capsid Protein VP1 into Particles

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