Core protein: A pleiotropic keystone in the HBV lifecycle

Abstract

Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates almost every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals - while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story."

Keywords: Antiviral therapy; Capsid; Hepatitis B Virus; Self-assembly.

Figure 1. Dimer and capsid structures

(A) A Cp dimer from an icosahedrally averaged capsid structure (PDB ID: 1QGT) is predominantly alpha helical with the dimeric interface comprising of a four helix bundle with two helices from each monomer (Wynne et al., 1999). (B) The HBeAg dimer structure (PDB ID: 3V6Z) has a drastically altered dimeric interface (Dimattia et al., 2013). (C) The structure of the assembly incompetent Y132A mutant (3KXS) is organized as a trimer of dimers with distinct tertiary structure differences from the assembly competent version (Packianathan et al., 2010). (D) The structure of a T=4 wild type capsid has the four helix bundle forming the spikes on the surface. These images are not to scale.

Figure 2

A panel of non-nucleoside antivirals and selected CpAM structures.

Figure 3. Capsid assembly kinetics and thermodynamics

Assembly kinetics (from (Zlotnick and Mukhopadhyay, 2011)) comparing NaCl-induce assembly of Cp 149 at seven concentration 4-10μM to a computation based on a model with thirty subunits. The model subunits, like those of HBV, are tetravalent and associate weakly.

Figure 4. An HBV Cp149 dimer

The two halves of the dimer (from PDB:1QGT) are in green and blue. Residues that affect capsid assembly are colored red. Those that affect secretion are colored magenta. (A) Cp149 dimer side view. (B) Cp149 dimer top view.

Figure 5. Reverse transcription is a multi-step process

(A,B) First, the pregenomic RNA (pgRNA) is encapsidated (encapsidation) when the stem loop structure epsilon (ε) binds the P protein to form the encapsidation signal (Bartenschlager et al., 1990; Bartenschlager and Schaller, 1992; Chiang et al., 1992; Nassal et al., 1990; Pollack and Ganem, 1993). Minusstrand DNA is then primed from tyrosine 63 of the P protein (Gerlich and Robinson, 1980; Nassal and Rieger, 1996; Rieger and Nassal, 1996; Tavis et al., 1994; Wang and Seeger, 1992; Zoulim and Seeger, 1994). (B) The nascent 3- or 4-mer DNA then switches template to a complementary sequence near the 3’ end of the pgRNA (minus-strand template switch) ((Nassal and Schaller, 1996; Rieger and Nassal, 1996; Tavis et al., 1994; Wang and Seeger, 1993)) in a process that is targeted by base pairing between cis-acting sequences ϕ and ω that flank the acceptor site (panel H) (Abraham and Loeb, 2006, 2007). (C) Minus-strand DNA is then elongated on the pgRNA template (minus-strand DNA elongation). RNase H activity of the P protein digests the pgRNA but leaves the 5’-most ~17 nucleotides undigested (Chen and Marion, 1996; Haines and Loeb, 2007; Radziwill et al., 1990; Will et al., 1987). (D, E) The 5’ RNA fragment switches from its location annealed to the 11-nucleotide Direct Repeat 1 (DR1) to the Direct Repeat 2 (DR2) sequence near the 5’ end of the minus-strand DNA (primer translocation). This template switch requires cis-acting sequences h3E, hM and h5E (panel I) (Lewellyn and Loeb, 2007; Seeger et al., 2007; Will et al., 1987). (F) A final template switch between the 5’ and 3’ terminal redundancies (5’r and 3’r) circularizes the DNA molecule (circularization) (Will et al., 1987). (G) Plus-strand DNA synthesis then resumes (plus-strand DNA elongation) to produce a mature, relaxed-circular DNA genome.