Human Hsp70 and Hsp40 Chaperone Proteins Facilitate Human Papillomavirus-11 E1 Protein Binding to the Origin and Stimulate Cell-free DNA Replication

Abstract

Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.Molecular chaperones regulate many cellular processes such as protein folding and translocation and the assembly and disassembly of multiprotein complexes (reviewed in Ref. 1). Two major Escherichia coli chaperones DnaK and DnaJ were originally identified as genes required for the initiation of bacterial or bacteriophage DNA replication. Mutations in DnaK and DnaJ lead to defects in DNA and RNA synthesis, cell division, and proteolysis (for reviews, see Refs. 2-4). DnaK is a weak ATPase with the ability to bind unfolded polypeptides (5, 6). DnaJ functions as a dimer (7, 8) and is considered a cochaperone, since it dramatically stimulates the ATPase activity of DnaK in the presence of GrpE (6, 9). Together, these proteins facilitate the binding of the replication initiator protein to the origin (ori) and the initiation of DNA replication (Ref. 7; for reviews, see .The families of eukaryotic heat shock protein 70 (Hsp70/ Hsc70) and heat shock protein 40 (Hsp40) have a high degree of homology to DnaK and DnaJ, respectively. Hsp70 and Hsp40 proteins are co-localized to the cytosol and also function in the nucleus (15, 16). As in prokaryotes, the Hsp40 proteins function as co-chaperones of Hsp70, but they also have weak, independent activity (5,7,(17)(18)(19)(20). All of the DnaJ homologues, such as HDJ-1, HDJ-2, and YDJ-1, members of the human and yeast Hsp40 family, have a conserved J domain at the amino terminus. Truncated E. coli or YDJ-1 containing only the J domain is sufficient to modulate the ATPase activity of DnaK/Hsp70 (20, 21). The corresponding J domain of the human Hsp40 proteins is also thought to mediate interactions with Hsp70 and regulate its ATPase activity (for a review, see Ref. 8). Within the J domain, there is a highly conserved HPD tripeptide loop flanked by two ␣-helices, designat...

Human Papillomavirus DNA Replication

“…The elution profile of the p180⅐E1 protein complex suggests that the interaction between E1 and p180 is not stoichiometric and perhaps more than one molecule of E1 interacts with one molecule of p180, in agreement with previous observations that E1 can exist as a multimer in solution (14,20,21,23). Finding that only a portion of the p180 associated with E1 during gel filtration and a small percentage of the total polymerase ␣ activity co-immunoprecipitated from lysates of co-infected insect cells (data not shown) leads us to the hypothesis that E1 may exist as a multimer in solution.…”

“…It has been proposed that the replication competent form of BPV-1 E1 is a multimeric complex of 10 -12 E1 molecules (20). Recently, the HPV-11 E1 protein has been shown to bind to the human chaperone protein Hsp40, and in its presence, a dihexameric E1 complex forms on the ori (23), mirroring the structure of SV40 T antigen on the SV40 ori (24) as well as other known Escherichia coli helicases (25). In addition to its role in initiation, HPV-11 E1 is also required during elongation in vitro, suggesting its helicase activity may be critical at the replicating forks (26).…”

Similarities between the DNA replication initiators of Gram-negative bacteria plasmids (RepA) and eukaryotes (Orc4p)/archaea (Cdc6p)

“…Hsp70, the eukaryotic homologue of DnaK, binds to the human papilloma virus initiator E1, stimulating origin recognition and replication (36).…”

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori s , an origin of viral DNA replication

“…The SV40 Tag forms a dihexamer that surrounds the DNA at the SV40 origin (33,34). HPV E1 protein binds to DNA mainly as a hexamer (35). However, in the presence of Hsp40 (a homologue of bacterial DnaJ protein), the E1 protein also bound to DNA as a dihexamer.…”

“…As shown in Fig. 1A, the 35 S-labeled hTid-1 was co-immunoprecipitated with the UL9 protein by the polyclonal UL9 protein antibody.…”