scholarly journals Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein

2002 ◽  
Vol 76 (16) ◽  
pp. 8124-8137 ◽  
Author(s):  
Henrik Daub ◽  
Stephanie Blencke ◽  
Peter Habenberger ◽  
Alexander Kurtenbach ◽  
Julia Dennenmoser ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Protein Kinases ◽  
Kinase Activity ◽  
Core Protein ◽  
Cellular Protein ◽  
Total Cell ◽  
B Virus

ABSTRACT Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.

scholarly journals Regulation of HBV Replication by Cyclin Docking Motifs in Core Protein

2021 ◽  
Author(s):  
Haitao Liu ◽  
Ji Xi ◽  
Jianming Hu
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Capsid Protein ◽  
Core Protein ◽  
Hbv Infection ◽  
Capsid Assembly ◽  
Cyclin Dependent Kinase ◽  
Terminal Domain ◽  
B Virus

Hepatitis B virus (HBV) capsid or core protein (HBc) consists of an N-terminal domain (NTD) and C-terminal domain (CTD) connected by a short linker peptide. Dynamic phosphorylation and dephosphorylation of HBc regulate its multiple functions in capsid assembly and viral replication. The cellular cyclin-dependent kinase 2 (CDK2) plays a major role in HBc phosphorylation and furthermore, is incorporated into the viral capsid, accounting for most of the “endogenous kinase” activity associated with the capsid. The packaged CDK2 is thought to play a role in phosphorylating HBc to trigger nucleocapsid disassembly (uncoating), an essential step during viral infection. However, little is currently known on how CDK2 is recruited and packaged into the capsid. We have now identified three RXL motifs, in the HBc NTD, known as cyclin docking motifs (CDMs), which mediates the interactions of various CDK substrates/regulators with CDK/cyclin complexes. Mutations of the CDMs in the HBc NTD reduced CTD phosphorylation and diminished CDK2 packaging into the capsid. Also, the CDM mutations showed little effects on capsid assembly and pregenomic RNA (pgRNA) packaging but impaired the integrity of mature nucleocapsids. Furthermore, the CDM mutations blocked covalently closed circular DNA (CCC DNA) formation during infection while having no effect on or enhancing CCC DNA formation via intracellular amplification. These results indicate that the HBc NTD CDMs play a role in CDK2 recruitment and packaging, which, in turn, is important for productive infection. Author Summary Hepatitis B virus (HBV) is an important global human pathogen and persistently infects hundreds of millions of people, who are at high risk of cirrhosis and liver cancer. HBV capsid packages a host cell protein kinase, the cyclin-dependent kinase 2 (CDK2), which is thought to be required to trigger disassembly of the viral nucleocapsid during infection by phosphorylating the capsid protein, a prerequisite for successful infection. We have identified docking sites on the capsid protein for recruiting CDK2, in complex with its cyclin partner, to facilitate capsid protein phosphorylation and CDK2 packaging. Mutations of these docking sites reduced capsid protein phosphorylation, impaired CDK2 packaging into HBV capsids, and blocked HBV infection. These results provide novel insights regarding CDK2 packaging into HBV capsids and the role of CDK2 in HBV infection and should facilitate the development of antiviral drugs that target the HBV capsid protein.

scholarly journals Phosphorylation of the Core Protein of Hepatitis B Virus by a 46-Kilodalton Serine Kinase

1998 ◽  
Vol 72 (5) ◽  
pp. 3796-3803 ◽  
Author(s):  
Jyh-Hwa Kau ◽  
Ling-Pai Ting
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Kinase Inhibitors ◽  
Core Protein ◽  
Core Particle ◽  
Terminal Part ◽  
Serine Kinase ◽  
The Core ◽  
B Virus

ABSTRACT Core protein is the major component of the core particle (nucleocapsid) of human hepatitis B virus. Core particles and core proteins are involved in a number of important functions in the replication cycle of the virus, including RNA packaging, DNA synthesis, and recognition of viral envelope proteins. Core protein is a phosphoprotein with most, if not all, of the phosphorylation on C-terminal serine residues. In this study, we identified a serine kinase activity from the ribosome-associated protein fraction of cytoplasm that could specifically bind and phosphorylate the C-terminal portion of recombinant core protein. This kinase is referred to as core-associated kinase (CAK). CAK could be inhibited by the kinase inhibitors heparin and manganese ions but not by spermidine, DRB, H89, or H7, indicating that CAK is distinct from protein kinase A and protein kinase C. CAK could be partially purified by heparin-Sepharose CL-6B and phosphocellulose P11 columns. By using a far-Western assay, three specific proteins, of 46, 35, and 13 kDa, were shown to interact with the C-terminal part of the core protein. These three proteins were present only in the eluted fractions that contains the CAK activity. An in-gel kinase assay showed that a 46-kDa kinase in the same fraction could bind and phosphorylate the C-terminal part of the recombinant core protein. These results indicate that this 46-kDa kinase is most probably CAK. A similar 46-kDa kinase, which exhibits the same profile of sensitivity to kinase inhibitors as that of CAK, is present in both purified intracellular core particles and extracellular 42-nm virions, suggesting that CAK is a candidate for the core particle-associated kinase.

scholarly journals Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

2018 ◽  
Vol 14 (12) ◽  
pp. e1007488 ◽  
Author(s):  
Julia Heger-Stevic ◽  
Peter Zimmermann ◽  
Lauriane Lecoq ◽  
Bettina Böttcher ◽  
Michael Nassal
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Protein Phosphorylation ◽  
Rna Binding ◽  
Core Protein ◽  
Virus Core ◽  
B Virus ◽  
Target Sites

Protein Kinase Activity in Hepatitis B Virus

1980 ◽  
Vol 34 (1) ◽  
pp. 297-302 ◽  
Author(s):  
Catherine Albin ◽  
William S. Robinson
Keyword(s):  
Hepatitis B Virus ◽  
Protein Kinase ◽  
Hepatitis B ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
B Virus

scholarly journals Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation

2018 ◽  
Vol 92 (13) ◽  
pp. e02139-17 ◽  
Author(s):  
Qiong Zhao ◽  
Zhanying Hu ◽  
Junjun Cheng ◽  
Shuo Wu ◽  
Yue Luo ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Viral Replication ◽  
Protein Phosphorylation ◽  
Core Protein ◽  
Regulatory Function ◽  
Core Proteins ◽  
B Virus ◽  
Immunoblotting Assay ◽  
Protein Dephosphorylation

ABSTRACTHepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically “normal” capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly.IMPORTANCEDynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids.

Sign in / Sign up

Export Citation Format

Share Document