C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich 167RRRSQSPRR175 Domain Is Critical for HBV Replication

Jaesung Jung,Sun Park,Hee-Young Kim,Ho-Joon Shin,Taeyeung Kim,Kyongmin Kim,Bo-Hye Shin,Gil-Soon Park,Yong-Joon Chwae,Wang-Shick Ryu

doi:10.1371/journal.pone.0041087

Abstract

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221–262 amino acids of DHBV C protein, in place of 146–185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221–241 and 251–262 amino acids of DHBV C, in place of HBV C 146–166 and 176–185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242–250 of DHBV C (242RAGSPLPRS 250) introduced in place of 167–175 of HBV C (167RRRSQSPRR 175) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich 167RRRSQSPRR175 domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.

Highlights

Hepadnaviruses are small, enveloped DNA viruses that replicate preferentially in liver cells and are associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]
By analyzing the A168R, G169R, P171Q, L172S, and S175R mutants in HHDH C chimera, we further demonstrated that the R169 and R175 residues may be important for Hepatitis B virus (HBV) replication and that S172 may be important for core particle formation, but not for pregenomic RNA (pgRNA) encapsidation or DNA synthesis
To investigate residues in the carboxyl-terminal nucleic acid binding domain of HBV C protein required for HBV replication, various chimeric C proteins were constructed by substituting the corresponding regions of duck hepatitis B virus (DHBV) C protein for the carboxyl-terminus of HBV C protein (Figure 1A)

Summary

Introduction

Hepadnaviruses are small, enveloped DNA viruses that replicate preferentially in liver cells and are associated with acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma [1]. The amino-terminus of C protein (amino acids 1–144) participates in core particle assembly through protein-protein interaction and is, by itself, assembly competent [3,4] The carboxyl-terminus of C protein contains a protamine-like nucleic acid binding domain rich in arginine. This region is dispensable for core particle assembly, it is involved in hepadnaviral replication, including pgRNA encapsidation and DNA replication [3,4,5,6,7,8,9,10]. It has been suggested that these residues are important for selective and/or productive viral RNA encapsidation in deletion- and site-directed mutants [9], a direct demonstration of the amino acid residues or motif in the carboxyl-terminus of

Methods

Results

Conclusion