ERK1/2-Mediated Phosphorylation of Small Hepatitis Delta Antigen at Serine 177 Enhances Hepatitis Delta Virus Antigenomic RNA Replication

Abstract

The small hepatitis delta virus (HDV) antigen (SHDAg) plays an essential role in HDV RNA double-rollingcircle replication. Several posttranslational modifications (PTMs) of HDAgs, including phosphorylation, acetylation, and methylation, have been characterized. Among the PTMs, the serine 177 residue of SHDAg is a phosphorylation site, and its mutation preferentially abolishes HDV RNA replication from antigenomic RNA to genomic RNA. Using coimmunoprecipitation analysis, the cellular kinases extracellular signal-related kinases 1 and 2 (ERK1/2) are found to be associated with the Flag-tagged SHDAg mutant (Ser-177 replaced with Cys-177). In an in vitro kinase assay, serine 177 of SHDAg was phosphorylated directly by either Flag-ERK1 or Flag-ERK2. Activation of endogenous ERK1/2 by a constitutively active MEK1 (hemagglutininAcMEK1) increased phosphorylation of SHDAg at Ser-177; this phosphorylation was confirmed by immunoblotting using an antibody against phosphorylated S177 and mass spectrometric analysis. Interestingly, we found an increase in the HDV replication from antigenomic RNA to genomic RNA but not in that from genomic RNA to antigenomic RNA. The Ser-177 residue was critical for SHDAg interaction with RNA polymerase II (RNAPII), the enzyme proposed to regulate antigenomic RNA replication. These results demonstrate the role of ERK1/2-mediated Ser-177 phosphorylation in modulating HDV antigenomic RNA replication, possibly through RNAPII regulation. The results may shed light on the mechanisms of HDV RNA replication.Hepatitis delta virus (HDV) is a subviral pathogen that can self-replicate but relies on its helper hepatitis B virus to provide envelope proteins for HDV infection and virion assembly (3). HDV is a negative-stranded RNA virus with 1.7-kb singlestranded circular RNA (genomic RNA) that, because of the intramolecular base pairing, is folded into an unbranched rodlike structure (11, 53). The genomic strand of HDV RNA does not encode any protein, but the complementary strand (antigenomic HDV RNA), which replicates from genomic RNA, encodes hepatitis delta antigen (HDAg) (53). There are two forms of HDAg: p24 (small HDAg [SHDAg]) and p27 (large HDAg [LHDAg]) (54). SHDAg is translated from a 0.8-kb subantigenomic message RNA that transcribes from HDV genomic RNA (19). SHDAg is essential for HDV genomic and antigenomic RNA replication (11,35). During HDV replication, the cells accumulate multimeric HDV genomic or antigenomic RNA, which exhibit ribozyme activity for autocatalytic cleavage and self-ligation to form monomeric circular RNAs (1,5,28,44,56). In the late stage of the viral replication cycle, the LHDAg is synthesized after antigenomic RNA editing (45, 51). LHDAg shares the 195-amino-acid sequence with SHDAg, except that the C terminus of LHDAg has an additional 19-amino-acid extension (62). The function of LHDAg is important for packaging with hepatitis B virus surface antigen to form the mature HDV virus particle (8,15,24).Whether cellular RNA polymerase (RNAP) can trigger HDV replication is co...

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK

“…Thr 178 is part of the well conserved P-E-T/S-P motif and only found in the hinge domain of ␤-arrestin-2 (Fig. 5, A and B) (27,28). Replacing Thr 178 for an Ala residue abolished the effect of PD98059 treatment on the receptor/␤-arrestin-2 complex half-life (Fig.…”

Hepatitis D: Thirty years after

“…Phosphorylation [32,33], acetylation [34], methylation [35], prenylation [36] and several other post translational modifications of the HD antigens [37][38][39] have been shown to orchestrate the life cycle of the virus. Different phosphorylation patterns of the small and large HD-Ag account for their distinct biological functions [32] and phosphorilated SHD-Ag increases replication from antigenomic to genomic RNA [33].…”

“…Different phosphorylation patterns of the small and large HD-Ag account for their distinct biological functions [32] and phosphorilated SHD-Ag increases replication from antigenomic to genomic RNA [33]. Acetylation and deacetylation of HD-Ag may provide a molecular switch for the synthesis of the different RNA species [34], and interactions with the multifunctional transcription factor YY1 and its associated acetyltransferase selectively modulates replication of the genomic and antigenomic forms [39].…”

Phosphorylation of Serine 177 of the Small Hepatitis Delta Antigen Regulates Viral Antigenomic RNA Replication by Interacting with the Processive RNA Polymerase II

“…Our previous studies revealed that a Ser 177 mutant of SHDAg lost the ability to interact with RNAP II and to support HDV replication from antigenomic to genomic RNA. Moreover, we found that extracellular signal-regulated kinase 1/2 (ERK1/2) is an SHDAgassociated kinase, and that its activation enhances the phosphorylation levels of Ser 177 of SHDAg and the antigenomic RNA replication of HDV in vivo (11,52). However, the role of SHDAg phosphorylation in regulating HDV antigenomic RNA replication remains unclear.…”

“…Our previous studies revealed that SHDAg is a phosphorylated protein, and that Ser 177 is the residue that is phosphorylated predominantly (52,54). In addition, the S177A SHDAg mutant (replacement of Ser 177 with Ala 177 ) exhibits the disruption of the synthesis of HDV genomic RNA with the preservation of the synthesis of antigenomic RNA (11,52) (pSer 177 -HDAg) specifically. 293T cells were transfected with HDV-expressing plasmids that were either antigenomic RNA replication competent (2AGwt) or replication deficient (2AG 177 ).…”