title: Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.
abstract: Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405.
authors.forename: Lisa
authors.initials: L
authors.lastname: Selzer
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Ravi
authors.initials: R
authors.lastname: Kant
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405.
authors.forename: Joseph C-Y
authors.initials: JC
authors.lastname: Wang
authors.affiliation: Department of Chemistry and Biochemistry, Montana State University, Bozeman, Montana 59717.
authors.forename: Brian
authors.initials: B
authors.lastname: Bothner
authors.affiliation: Department of Molecular and Cellular Biochemistry, Indiana University, Bloomington, Indiana 47405. Electronic address: azlotnic@indiana.edu.
authors.forename: Adam
authors.initials: A
authors.lastname: Zlotnick
mesh_terms: D000595:Amino Acid Sequence; D002213:Capsid; D020285:Cryoelectron Microscopy; D006512:Hepatitis B Core Antigens / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation
chemical_list: D006512:Hepatitis B Core Antigens
keywords: capsid stability; cryo-electron microscopy; hepatitis B virus (HBV, Hep B); limited proteolysis; mass spectrometry (MS); phosphorylation; protein dynamics