Kyu-Jin Park

Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase

ABSTRACT Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-α)-induced NF-κB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-α-induced NF-κB and JNK activation. In this study, we have demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-κB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKα. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKα, and then TNF-α-mediated IKKα kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-α-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-α signaling pathways and may contribute to HCV pathogenesis.

IKKα Regulates Estrogen-induced Cell Cycle Progression by Modulating E2F1 Expression

The IκB kinase (IKK) complex consists of the catalytic subunits IKKα and IKKβ and a regulatory subunit, IKKγ/NEMO. Even though IKKα and IKKβ share significant sequence similarity, they have distinct biological roles. It has been demonstrated that IKKs are involved in regulating the proliferation of both normal and tumor cells, although the mechanisms by which they function in this process remain to be better defined. In this study, we demonstrate that IKKα, but not IKKβ, is important for estrogen-induced cell cycle progression by regulating the transcription of the E2F1 gene as well as other E2F1-responsive genes, including thymidine kinase 1, proliferating cell nuclear antigen, cyclin E, and cdc25A. The role of IKKα in regulating E2F1 was not the result of reduced levels of cyclin D1, as overexpression of this gene could not overcome the effects of IKKα knock-down. Furthermore, estrogen treatment increased the association of endogenous IKKα and E2F1, and this interaction occurred on promoters bound by E2F1. IKKα also potentiated the ability of p300/CBP-associated factor to acetylate E2F1. Taken together, these data suggest a novel mechanism by which IKKα can influence estrogen-mediated cell cycle progression through its regulation of E2F1.

Cell cycle arrest mediated by hepatitis delta antigen

Hepatitis delta antigen (HDAg) is the only viral‐encoded protein of the hepatitis delta virus (HDV). This protein has been extensively characterized with respect to its biochemical and functional properties. However, the molecular mechanism responsible for persistent HDV infection is not yet clear. Previously, we reported that overexpression of HDAg protects insect cells from baculovirus‐induced cytolysis [Hwang, S.B. Park, K.‐J. and Kim, Y.S. (1998) Biochem. Biophys. Res. Commun. 244, 652–658]. Here we report that HDAg mediates cell cycle arrest when overexpressed in recombinant baculovirus‐infected insect cells. Flow cytometry analysis has shown that HDAg expression in Spodoptera frugiperda cells causes an accumulation of substantial amounts of polyploid DNA in the absence of cell division. This phenomenon may be partly responsible for the persistent infection of chronic HDV patients.

Large Hepatitis Delta Antigen Is Phosphorylated at Multiple Sites and Phosphorylation Is Associated with Protein Conformational Change

Hepatitis delta antigen (HDAg) consists of two species, small HDAg (SHDAg) and large HDAg (LHDAg), which are identical in sequence with the exception that the large form contains an additional 19 amino acids at the C-terminus. Both HDAgs are nuclear phosphoproteins. However, LHDAg is hyperphosphorylated, i.e. it is at least 10 times more phosphorylated than SHDAg. To determine the phosphorylation site(s) of the LHDAg, we mutated all the conserved serine residues and expressed these mutant proteins using a recombinant baculovirus expression system. By labeling insect cells in vivo with 32P-orthophosphate and immunoprecipitation, we showed that LHDAg is phosphorylated at multiple serine residues. Although LHDAg contains two additional serines at its 19-amino acid extension, mutations of these two amino acids did not affect the overall phosphorylation level. Most importantly, the phosphorylation level of middle domain-deleted LHDAg (M75del) was significantly higher than that of wild-type LHDAg. We conclude that phosphorylation of the LHDAg occurs at multiple sites and that hyperphosphorylation is associated with alteration of protein conformation.