scholarly journals Application of fluorescent-based technology detecting protein-protein interactions to monitor the binding of hepatitis B virus X protein to DNA-damage-binding protein 1

2021 ◽  
Vol 18 (0) ◽  
pp. 67-77
Author(s):  
Katsumi Omagari ◽  
Kaori Asamitsu ◽  
Yasuhito Tanaka
Keyword(s):  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Interactions ◽  
Binding Protein ◽  
Protein Protein Interactions ◽  
X Protein ◽  
B Virus

scholarly journals RMP, a Novel RNA Polymerase II Subunit 5-Interacting Protein, Counteracts Transactivation by Hepatitis B Virus X Protein

1998 ◽  
Vol 18 (12) ◽  
pp. 7546-7555 ◽  
Author(s):  
Dorjbal Dorjsuren ◽  
Yong Lin ◽  
Wenxiang Wei ◽  
Tatsuya Yamashita ◽  
Takahiro Nomura ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Binding Protein ◽  
Host Protein ◽  
Dependent Manner ◽  
Binding Region ◽  
X Protein ◽  
B Virus

ABSTRACT To modulate transcription, regulatory factors communicate with basal transcription factors and/or RNA polymerases in a variety of ways. Previously, it has been reported that RNA polymerase II subunit 5 (RPB5) is one of the targets of hepatitis B virus X protein (HBx) and that both HBx and RPB5 specifically interact with general transcription factor IIB (TFIIB), implying that RPB5 is one of the communicating subunits of RNA polymerase II involved in transcriptional regulation. In this context, we screened for a host protein(s) that interacts with RPB5. By far-Western blot screening, we cloned a novel gene encoding a 508-amino-acid-residue RPB5-binding protein from a HepG2 cDNA library and designated it RPB5-mediating protein (RMP). Expression of RMP mRNA was detected ubiquitously in various tissues. Bacterially expressed recombinant RMP strongly bound RPB5 but neither HBx nor TATA-binding protein in vitro. Endogenous RMP was immunologically detected interacting with assembled RPB5 in RNA polymerase in mammalian cells. The central part of RMP is responsible for RPB5 binding, and the RMP-binding region covers both the TFIIB- and HBx-binding sites of RPB5. Overexpression of RMP, but not mutant RMP lacking the RPB5-binding region, inhibited HBx transactivation of reporters with different HBx-responsive cis elements in transiently transfected cells. The repression by RMP was counteracted by HBx in a dose-dependent manner. Furthermore, RMP has an inhibitory effect on transcriptional activation by VP16 in the absence of HBx. These results suggest that RMP negatively modulates RNA polymerase II function by binding to RPB5 and that HBx counteracts the negative role of RMP on transcription indirectly by interacting with RPB5.

scholarly journals Phosphorylation of hepatitis B virus Cp at Ser87 facilitates core assembly

2006 ◽  
Vol 398 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Hee Yong Kang ◽  
Seungkeun Lee ◽  
Sung Gyoo Park ◽  
Jaehoon Yu ◽  
Youngsoo Kim ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Protein Interactions ◽  
Present Report ◽  
Protein Protein Interactions ◽  
B Virus ◽  
Dimeric Form ◽  
Genome Encapsidation

Protein–protein interactions can be regulated by protein modifications such as phosphorylation. Some of the phosphorylation sites (Ser155, Ser162 and Ser170) of HBV (hepatitis B virus) Cp have been discovered and these sites are implicated in the regulation of viral genome encapsidation, capsid localization and nucleocapsid maturation. In the present report, the dimeric form of HBV Cp was phosphorylated by PKA (protein kinase A), but not by protein kinase C in vitro, and the phosphorylation of dimeric Cp facilitated HBV core assembly. Matrix-assisted laser-desorption ionization–time-of-flight analysis revealed that the HBV Cp was phosphorylated at Ser87 by PKA. This was further confirmed using a mutant HBV Cp with S87G mutation. The S87G mutation inhibited the phosphorylation and, as a result, the in vitro HBV core assembly was not facilitated by PKA. In addition, when either pCMV/FLAG–Core(WT) or pCMV/FLAG–Core(S87G) was transfected into HepG2 cells, few mutant Cps (S87G) assembled into capsids compared with the wild-type (WT) Cps, although the same level of total Cps was expressed in both cases. In conclusion, PKA facilitates HBV core assembly through phosphorylation of the HBV Cp at Ser87.

scholarly journals Proteomic Analysis of Nuclear HBV rcDNA Associated Proteins Identifies UV-DDB as a Host Factor Involved in cccDNA Formation

2021 ◽  
Author(s):  
Alexander L. Marchetti ◽  
Hu Zhang ◽  
Elena S. Kim ◽  
Xiaoyang Yu ◽  
Sunbok Jang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Dna Damage ◽  
Dna Repair ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Binding Protein ◽  
Host Factors ◽  
Circular Dna ◽  
B Virus

Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify said host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the co-precipitated proteins by mass spectrometry. The DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported shRNA screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Trans-complementation of wild type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA-binding, DDB1-binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified a handful of host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNAi screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay we discovered a novel relationship between the UV-DDB complex and cccDNA formation, hence, providing a proof-of-concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.

scholarly journals The Hepatitis B Virus X Protein Sensitizes HepG2 Cells to UV Light-induced DNA Damage

2005 ◽  
Vol 280 (39) ◽  
pp. 33525-33535 ◽  
Author(s):  
Alvin T. C. Lee ◽  
Jianwei Ren ◽  
Ee-Tsin Wong ◽  
Kenneth H. K. Ban ◽  
Linda A. Lee ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Hepg2 Cells ◽  
Uv Light ◽  
X Protein ◽  
B Virus

scholarly journals Correction: The hepatitis B virus X protein functionally interacts with CREB-binding protein/p300 in the regulation of CREB-mediated transcription.

2020 ◽  
Vol 295 (9) ◽  
pp. 2888-2888
Author(s):  
Delphine Cougot ◽  
Yuanfei Wu ◽  
Stefano Cairo ◽  
Julie Caramel ◽  
Claire-Angélique Renard ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Binding Protein ◽  
Creb Binding Protein ◽  
X Protein ◽  
B Virus

Hepatitis B virus X protein promotes DNA damage propagation through disruption of liver polyploidization and enhances hepatocellular carcinoma initiation

Oncogene ◽  
2018 ◽  
Vol 38 (14) ◽  
pp. 2645-2657 ◽  
Author(s):  
James Ahodantin ◽  
Myriam Bou-Nader ◽  
Corinne Cordier ◽  
Jérôme Mégret ◽  
Patrick Soussan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Dna Damage ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
X Protein ◽  
Damage Propagation ◽  
B Virus

scholarly journals The Hepatitis B Virus X Protein Functionally Interacts with CREB-binding Protein/p300 in the Regulation of CREB-mediated Transcription

2006 ◽  
Vol 282 (7) ◽  
pp. 4277-4287 ◽  
Author(s):  
Delphine Cougot ◽  
Yuanfei Wu ◽  
Stefano Cairo ◽  
Julie Caramel ◽  
Claire-Angélique Renard ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Proliferating Cell Nuclear Antigen ◽  
Binding Protein ◽  
Interleukin 8 ◽  
Nuclear Antigen ◽  
Creb Binding Protein ◽  
X Protein ◽  
B Virus ◽  
Proliferating Cell

The hepatitis B virus infects more than 350 million people worldwide and is a leading cause of liver cancer. The virus encodes a multifunctional regulator, the hepatitis B virus X protein (HBx), that is essential for virus replication. HBx is involved in modulating signal transduction pathways and transcription mediated by various factors, notably CREB that requires the recruitment of the co-activators CREB-binding protein (CBP)/p300. Here we investigated the role of HBx and its potential interaction with CBP/p300 in regulating CREB transcriptional activity. We show that HBx and CBP/p300 synergistically enhanced CREB activity and that CREB phosphorylation by protein kinase A was a prerequisite for the cooperative action of HBx and CBP/p300. We further show that HBx interacted directly with CBP/p300 in vitro and in vivo. Using chromatin immunoprecipitation, we provide evidence that HBx physically occupied the CREB-binding domain of CREB-responsive promoters of endogenous cellular genes such as interleukin 8 and proliferating cell nuclear antigen. Moreover expression of HBx increased the recruitment of p300 to the interleukin 8 and proliferating cell nuclear antigen promoters in cells, and this is associated with increased gene expression. As recruitment of CBP/p300 is known to represent the limiting event for activating CREB target genes, HBx may disrupt this cellular regulation, thus predisposing cells to transformation.

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