Phosphorylated Delta Antigens in the HDV Life Cycle

Protein conformation is directly correlated with its biological function. One must therefore be very careful when using mutant proteins to investigate function, because mutations can interfere with function indirectly by affecting protein conformation. More reliable approaches to investigate the biological function of a phosphorylated protein include abolishing kinase activity by using dominant negative mutants or RNA interference (RNAi) technology. However, because most of the phosphorylation sites and corresponding ki-nases for HDAg phosphorylation have still not been identified, site-directed mutagenesis and kinase inhibitor(s) were used to investigate the influence of HDAg phosphorylation on HDV replication.

Some of the serine and threonine residues have been mutated in S-HDAg (Table 1). Mutation at Serine 2 diminished the activity of SHDAg in assisting HDV replication. Mutations at Threonine/Serine 95 of SHDAg also influenced HDV RNA replication. Serine 177 has been identified as a phosphorylation site in vivo and is located within a conserved motif, Pro-Glu-Ser-Pro-Phe (PESPF). Mutation of serine 177 to alanine interfered with synthesis of HDV genomic RNA from the antigenomic template (Mu et al. 2001). Furthermore, mutation at serine 177 also reduced the phosphorylation level of S-HDAg, the degree of RNA editing and production of L-HDAg. Serine to alanine mutations at positions 4 and 123 seemed not to exert any effect on the HDV life cycle.

DRB (5,6-dichloro-1-p-D-ribofuranosylbenzimidazole), an inhibitor ofthe CKII, diminished phosphorylation levels of both HDAgs and suppressed HDV replication. This observation is in accordance with a S-HDAg mutant assay that implies serine 2 is phosphorylated by CK II (Yeh et al. 1996). Nevertheless, as CKIIalso regulatesthe phosphorylationofother cellular proteins, the effect of kinase inhibitors on cells is global rather than HDAg-specific. Therefore, it was not clear whether the effect of kinase inhibitor on HDV replication was due to the direct inhibition of HDAg phosphorylation or to the modulation of cellular protein phosphorylation. For example, the activity of a transcription factor, CHOP, could also be influenced by CK II (Ubeda and Habener 2003). As HDV replication depends on cellular transcription machinery, (Lai 1995, also see the chapters by J.M. Taylor, and T.B. Macnaughton and M.M.C. Lai, this volume), it is possible that blocking the phosphorylation of these transcription factors by kinase inhibitors indirectly impaired HDV replication. More experiments will be required to clarify this issue.

Protein kinase C (PKC) inhibitor suppressed HDV replication more significantly. Mutation of S-HDAg at the putative PKC phosphorylation site did not have any effect on HDV replication, implying that the influence of PKC inhibitor on HDV is an indirect consequence of modulating the activity of cellular proteins. The nucelolar protein, B23 probably is the communicator. It is a substrate of PKC and its interaction with S-HDAg enhances HDV replication (Beckmann et al. 1992; Huang et al. 2001). Histone and serine/threonine phosphatase can also be phosphorylated by PKC (Jakes et al. 1988). The effects of PKC on these proteins and their roles in the HDV life cycle require more detailed studies. Furthermore, the phosphatase responsible for HDAg dephosphorylation is still unknown.

Although L-HDAg is more heavily phosphorylated, phosphorylation seems not to affect the biological functions of L-HDAg significantly. The only effect found so far is that L-HDAg with mutation at serine 123 was translocated from nucleolus to nuclear speckle SC35 (Tan et al. 2004). The same phenomenon was observed when the cells expressing wild-type L-HDAg were treated with the CK II inhibitor, dichlororibofuranosyl benzimidazole. Compared with wild type L-HDAg and serine 2-mutated L-HDAg, L-HDAg mutated at serine 123 was transported to the cytoplasm less efficiently and resulted in a lower level of HDV particle secretion.

0 0

Post a comment