Fluorescent protein tagged hepatitis B virus capsid protein with long glycine-serine linker that supports nucleocapsid formation

Abstract

Fusion core proteins of Hepatitis B virus can be used to study core protein functions or capsid trafficking. A problem in constructing fusion core proteins is functional impairment of the individual domains in these fusion proteins, might due to structural interference. We reported a method to construct fusion proteins of Hepatitis B virus core protein (HBc) in which the functions of fused domains were partially kept. This method follows two principles: (1) fuse heterogeneous proteins at the N terminus of HBc; (2) use long Glycine-serine linkers between the two domains. Using EGFP and RFP as examples, we showed that long flexible GS linkers can effectively separate the two domains in function. Among these fusion proteins constructed, GFP-GS186-HBc and RFP-G4S47-HBc showed the best efficiency in rescuing the replication of an HBV replicon deficient in the core protein expression, though both of the two fusion proteins failed to support the formation of the relaxed circular DNA. These fluorescent protein-tagged HBcs might help study related to HBc or capsids tracking in cells.

Identification of Compounds Targeting Hepatitis B Virus Core Protein Dimerization through a Split Luciferase Complementation Assay

“…Each split luciferase fragment was fused to the N terminus of the HBc through a glycine-serine linker (G 4 S) with a length of 92 amino acids (G 4 S92). This long linker was used because our previous results (31) showed that it helped maintain the functions of HBc fusion proteins. The split site of each luciferase, according to previous reports, is shown in Fig.…”

“…To further assess to what extent these split-luc-HBcs keep their functions, we tested these HBc fusion proteins for their capability to support HBV DNA replication. The 6 split-luc-HBcs were cotransfected with HBV1.1c Ϫ , a plasmid that expresses all the elements needed for HBV DNA replication except HBc (31). Southern blotting was used to detect the intracellular core particleassociated HBV DNA.…”

“…Plasmid pCH-9/3091, which expresses an HBV 1.1ϫ genome governed by CMV-IE promoter (52), was kindly provided by Lin Nan (Southwest Hospital, Chongqing, China). pHBV1.1C Ϫ was constructed previously (31) in which the 40th amino acid of the core protein was mutated to a stop codon (GAA to TAA). wtHBc was a plasmid expressing wild-type HBc of genotype D from PCH9/3091.…”

Hepatitis B Virus Core Protein Domains Essential for Viral Capsid Assembly in a Cellular Context

“…In order to study HBc oligomerisation by quantitative fluorescent microscopy, we designed HBc proteins fused to eGFP and mCherry. To this end, several strategies have already been described [37,38], and the insertion of antigens in the c/e1 epitope was shown to poorly impact the capsid formation [39,40]. Indeed, an eGFP protein (or a monomeric mCherry) flanked by glycine-rich linkers was inserted between residues 78 and 80 of HBc and electron microscopy revealed the formation of capsid-like particles of HBc149-eGFP, with surfaceexposed eGFP domains [34,36,41,42].…”