Ling-Pai Ting

Human Hepatitis B Viral e Antigen Interacts with Cellular Interleukin-1 Receptor Accessory Protein and Triggers Interleukin-1 Response

Human hepatitis B virus (HBV) can cause acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV e antigen (HBeAg), a secreted protein and not required for viral replication, is thought to play an immunoregulatory role during viral infection. However, the functional involvement of HBeAg in host immune response has not been fully elucidated. We report in this study that HBeAg can bind to interleukin-1 receptor accessory protein (IL-1RAcP). Interleukin-1 (IL-1) plays an important role in inflammation and regulation of immune response, and membrane form of IL-1RAcP (mIL-1RAcP) is an essential component of trimeric IL-1/IL-1 receptor/mIL-1RAcP complex. We show that glutathione S-transferase- or polyhistidine-tagged recombinant HBeAg can interact with endogenous mIL-1RAcP in vitro. Purified (His)6-HBeAg added in the culture medium can interact with overexpressed FLAG-tagged mIL-1RAcP in vivo. Indirect immunofluorescence and confocal microscopy show that HBeAg colocalizes with mIL-1RAcP on the cell surface. Furthermore, HBeAg is able to induce the interaction of IL-1 receptor I (IL-1RI) with mIL-1RAcP and trigger the recruitment of adaptor protein myeloid differentiation factor 88 (MyD88) to the IL-1RI/mIL-1RAcP complex. Assembly and activation of IL-1RI/mIL-1RAcP signaling complex by HBeAg can activate downstream NF-κB pathway through IκB degradation, induce NF-κB-dependent luciferase expression, and induce the expression of IL-1-responsive genes. Silencing of IL-1RAcP by small interfering RNA dramatically abolishes HBeAg-mediated NF-κB activation. These results demonstrate that HBeAg can trigger host IL-1 response by binding to mIL-1RAcP. The interaction of HBeAg with mIL-1RAcP may play an important role in modulating host immune response in acute and chronic HBV infection.

Transcription Repression of Human Hepatitis B Virus Genes by Negative Regulatory Element-binding Protein/SON

A negative regulatory element (NRE) is located immediately upstream of the upstream regulatory sequence of core promoter and second enhancer of human hepatitis B virus (HBV). NRE represses the transcription activation function of the upstream regulatory sequence of core promoter and the second enhancer. In this study, we described the cloning and characterization of an NRE-binding protein (NREBP) through expression cloning. NREBP cDNA is 8266 nucleotides in size and encodes a protein of 2386 amino acids with a predicted molecular mass of 262 kDa. Three previously described cDNAs, DBP-5, SONB, and SONA, are partial sequence and/or alternatively spliced forms of NREBP. The genomic locus of theNREBP/SON gene is composed of 13 exons and 12 introns. The endogenous NREBP protein is localized in the nucleus of human hepatoma HuH-7 cells. Antibody against NREBP protein can specifically block the NRE binding activity present in fractionated nuclear extracts in gel shifting assays, indicating that NREBP is the endogenous nuclear protein that binds to NRE sequence. By polymerase chain reaction-assisted binding site selection assay, we determined that the consensus sequence for NREBP binding is GA(G/T)AN(C/G)(A/G)CC. Overexpression of NREBP enhances the repression of the HBV core promoter activity via NRE. Overexpression of NREBP can also repress the transcription of HBV genes and the production of HBV virions in a transient transfection system that mimics the viral infection in vivo.

Hepatitis B virus core protein interacts with the C-terminal region of actin-binding protein

Hepatitis B viral core protein is present in the nucleus and cytoplasm of infected hepatocytes. There is a strong correlation between the intrahepatic distribution of core protein and the viral replication state and disease activity in patients with chronic hepatitis. To understand the role of core protein in the pathogenesis of HBV, we used a yeast two-hybrid system to search for cellular proteins interacting with the carboxyl terminus of core protein, as this region is involved in a number of important functions in the viral replication cycle including RNA packaging and DNA synthesis. A cDNA encoding the extreme C-terminal region of human actin-binding protein, ABP-276/278, was identified. This interaction was further confirmed both in vitro and in vivo. In addition, the extreme C-terminal region of ABP-276/278 interacted with the nearly full-length HBV core protein. Since this region is present in both the core and the precore proteins, it is likely that both core and precore proteins of HBV can interact with the C-terminal region of ABP-276/278. The minimal region of ABP-276/278 which interacted with the HBV core protein was the C-terminal 199 amino acid residues which correspond to part of the 23rd repeat, the entire 24th repeat and the intervening hinge II region in ABPs. The potential functional outcome of ABP interaction in HBV replication and its contribution to the pathological changes seen in patients with chronic HBV infection are discussed.

Repression of hepatitis B viral gene expression by transcription factor nuclear factor‐kappaB

Infection of human hepatitis B virus (HBV) causes acute hepatitis. Its persistent infection leads to a high risk of developing chronic hepatitis, cirrhosis and hepatocellular carcinoma. The levels of HBV 3.5 kb and 2.4/2.1 kb RNAs transcribed from a replicating HBV expression plasmid in human hepatoma HuH‐7 cells are repressed by tumour necrosis factor alpha treatment or overexpressed p65 in a dose‐dependent manner. The diminished expression of endogenous p65 by a p65‐specific siRNA or IκB‐α overexpression enhances the HBV gene expression. The protein bound to the Specificity protein 1 (Sp1) binding sites (nt 1733–1753) of HBV core promoter is reduced by either tumour necrosis factor alpha treatment or overexpressed p65. The N‐terminal 43‐amino‐acid region of p65, which is required to interact with Sp1, is essential to repress the Sp1‐mediated transactivation. The binding of Sp1 to Sp1 site and the Sp1‐dependent reporter expression are inhibited by p65 in a dose‐dependent manner. Furthermore, nuclear factor‐kappa B‐mediated repression of HBV gene expression is abolished by deletion of Sp1 sites of HBV gene promoter. Together, these results demonstrate that nuclear factor‐kappa B represses the HBV gene expression through its interaction with Sp1 and repression of Sp1‐mediated transcriptional activation.

Suppression of hepatitis B viral gene expression by phosphoinositide 5‐phosphatase SKIP

Human hepatitis B virus (HBV) causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Here we report that HBV core protein interacts with a cellular SKIP (skeletal muscle and kidney enriched inositol phosphatase) protein, an endoplasmic reticulum‐located phosphoinositide 5‐phosphatase, both in vivo and in vitro. The minimal sequence required for interaction is the amino acid region from 116 to 149 for the core protein and the SKIP carboxyl homology (SKICH) domain for SKIP. When HBV replicates in HuH‐7 cells, overexpressed SKIP localizes to nucleus in addition to ER and suppresses HBV gene expression and replication. SKIP loses its nuclear localization and suppressive effect during replication of a core‐negative HBV mutant. HBV gene expression is enhanced significantly when endogenous SKIP expression is knocked down by a SKIP‐specific siRNA. SKIP mutation analysis shows that its 5‐phosphatase activity is not required for the suppressive effect and that the suppression domain maps to amino acids 199–226. These results demonstrate that SKIP is translocated from endoplasmic reticulum into nucleus through its interaction with core protein and suppresses HBV gene expression via a novel suppression domain.

A serine-kinase-containing protein complex interacts with the terminal protein domain of polymerase of hepatitis B virus

Polymerase of human hepatitis B virus is required for viral replication and pregenomic RNA encapsidation. Using recombinant GST fusion proteins, we show that the terminal protein domain of polymerase can interact specifically with a protein complex containing kinase activity and a tightly associated 35-kD protein (p35). This kinase is termed terminal-protein-associated kinase (TPAK). The phosphoamino acid analysis of phosphorylated p35 demonstrates that TPAK is a serine kinase. Analysis of deletion mutants shows that amino acids 1-95 of the terminal protein domain are required for the interaction with TPAK/p35 and phosphorylation of p35. TPAK/p35 are found predominantly in the cytoplasm. Furthermore, TPAK can be inhibited by heparin and manganese ions, but is resistant to spermidine, DRB, H89 or H7. These results indicate that TPAK is not protein kinase A or protein kinase C. Copyright 1997 S. Karger AG, Basel