We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli . We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of ∼260 kDa and ∼130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV ɛ RNA), the ∼260-kDa active band was gradually converted to ∼130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV ɛ RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
Archives of Virology – Springer Journals
Published: Jul 1, 2000
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