Role of p50/CDC37 in Hepadnavirus Assembly and Replication

Abstract

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pulldown assays showed that p50 or p50␦C, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50␦C interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of proteinprimed initiation of reverse transcription by p50␦C in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50␦C in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.Reverse transcription in hepadnaviruses (hepatitis B viruses) is carried out by a novel virally encoded reverse transcriptase (RT) 1 (1, 2). The RT is able to initiate DNA synthesis de novo using a specific tyrosine residue located within its N-terminal domain (the terminal protein (TP)) as a protein primer (Refs. 3-6; for a recent review, see Ref. 7). This protein priming reaction requires the interaction between the RT and a specific RNA signal (termed ⑀) located on the viral pregenomic RNA (pgRNA; the template for reverse transcription) (8, 9). The ⑀ RNA is used as a specific template for protein priming (and thus, the origin of reverse transcription) (Refs. 9 -11; for a recent review, see Ref. 12). In addition, ⑀ serves as the RNA packaging signal (13, 14) and directs, through its interaction with the RT, the selective encapsidation of both the pgRNA and the RT into viral nucleocapsids (8,15). Therefore, the specific interaction between the RT and ⑀ triggers two essential early steps in hepadnavirus assembly and replication, i.e. the protein-primed initiation of reverse transcription and the assembly of replication-competent nucleocapsids.Using the duck hepatitis B virus (DHBV) as a model system, we have recently found that the RT requires the assistance of host cell factors to carry out specific ⑀ binding and protein priming functions (16,17). One such cellular factor is the 90-kDa heat shock protein (Hsp90). Hsp90 associates with the DHBV RT and is required for RT-⑀ interaction and protein priming in vitro and for pgRNA packaging and DNA synthesis in vivo. Hsp90 is thought to facilitate the functions of a specific subset of cellular proteins (the target or substrate proteins) by helping to establish and maintain certain poised but labile conformations of these target proteins through a dynamic...

Novel Interaction between the Co-chaperone Cdc37 and Rho GTPase Exchange Factor Vav3 Promotes Androgen Receptor Activity and Prostate Cancer Growth*

“…Cdc37 binding to Hsp90 inhibits the intrinsic ATPase activity of Hsp90 and facilitates the loading of client proteins (48 -50). In addition to serving as a co-chaperone of Hsp90 with specificity for protein kinases, Cdc37 exerts effects that may be independent of Hsp90 (21)(22)(23)(24)(25)(26)(27). Interestingly, the hsp90 inhibitor geldanamycin did not block Cdc37-Vav3 interaction, suggestive of a new Hsp90 independent action of Cdc37.…”

“…Cdc37 confers Hsp90 specificity for client protein kinases (18 -20). In addition to serving as an Hsp90 co-chaperone, Cdc37 appears to also function as a chaperone independent of Hsp90 with client proteins ranging from protein kinases to steroid hormone receptors (21)(22)(23)(24)(25)(26)(27). Analysis of publicly available databases and published data reveals that Cdc37 is up-regulated in localized human prostate cancer compared with benign prostate tissues (28).…”

Sti1 and Cdc37 Can Stabilize Hsp90 in Chaperone Complexes with a Protein Kinase

“…Folding of protein kinases, for example, requires a cochaperone called Cdc37, that does not associate with GR or PR, although it does function in activation of androgen receptor and a viral reverse transcriptase (Fliss et al, 1997;Rao et al, 2001;Wang et al, 2002). Cdc37 interacts with many different protein kinases and is an essential gene (Hunter and Poon, 1997).…”

Identification of a Conserved Sequence Motif That Promotes Cdc37 and Cyclin D1 Binding to Cdk4

“…In this paper, we address the question experimentally by dissecting the association of Cdc37 with Cdk4, a well characterized Cdc37 and Hsp90 client. Previous studies showed that Cdc37 interacts directly with Cdk4 and stabilizes the kinase (12,21). The population of Cdk4 molecules that interact with Cdc37 are largely devoid of cyclin D1, suggesting that the chaperone complex functions to prepare Cdk4 for cyclin D1 interaction.…”

“…A truncated form of Cdc37 that no longer binds Hsp90 is still capable of partially rescuing the catalytic activity of a temperature-sensitive mutant of Hck and promoting its association with Hsp90 (28). A similar Cdc37 mutant defective in Hsp90 binding maintains the ability to interact specifically with the reverse transcriptase of the duck hepatitis B virus both in vitro and in vivo (21).…”