Background

The hepatitis B virus (HBV) is an important global human pathogen that chronically infects hundreds of millions of people worldwide and represents a major cause of viral hepatitis, liver cirrhosis, and liver cancer (Seeger et al., 2013; Trepo et al., 2014). HBV replicates its genomic DNA, a relaxed circular, partially duplex DNA (RC DNA), via reverse transcription of an RNA intermediate, the so-called pregenomic RNA (pgRNA), within a nucleocapsid (NC) (Summers and Mason, 1982; Hu and Seeger, 2015; Hu, 2016), which packages a copy of pgRNA together with the virally encoded reverse transcriptase (RT) protein (Bartenschlager and Schaller, 1992; Hu and Lin, 2009). It is within NCs that the RT converts pgRNA into RC DNA.

The icosahedral HBV capsid shell consists of multiple copies of a single viral protein, the HBV core (capsid) protein (HBc). HBc is composed of an N-terminal domain (NTD, aa 1-140) and a C-terminal domain (CTD, 150-183 or 185 depending on strains), which are connected by a linker region (141-149). In heterologous overexpression systems including bacteria and insect cells and in vitro assembly reactions using high concentrations of purified HBc and/or salt, NTD alone, without CTD, is sufficient for capsid assembly; thus it is also called the assembly domain (Gallina et al., 1989; Birnbaum and Nassal, 1990; Lanford and Notvall, 1990; Wingfield et al., 1995). Though not required for capsid assembly in these systems, the highly basic and arginine-rich CTD shows non-specific nucleic acid binding activities (Hatton et al., 1992) and plays important roles in viral RNA packaging (Nassal, 1992), DNA synthesis (Nassal, 1992; Yu and Summers, 1994), and nuclear import of capsids (Liao and Ou, 1995; Liu et al., 2015), all of which is further regulated by the dynamic phosphorylation state of CTD controlled by host factors (Kann and Gerlich, 1994; Kann et al., 1999; Daub et al., 2002; Ludgate et al., 2012; Liu et al., 2015). Whether CTD, and its state of phosphorylation, play a role in capsid assembly under physiological conditions remained unclear.

To study HBV capsid assembly under more physiological conditions, we have recently developed a mammalian cell-free assembly system based on the commonly used mammalian cell extract, rabbit reticulocyte lysate (RRL), in which HBc is expressed at physiological (low) concentrations (25-50 nM, monomer) and assembles into capsids under near-physiological conditions (Ludgate et al., 2016). This system allowed us to reveal an unexpected role of CTD in capsid assembly, which is further subjected to regulation by the state of CTD phosphorylation as controlled by endogenous host factors (Ludgate et al., 2016). This protocol is adapted from Ludgate et al. (2016) and more detailed information on this cell-free capsid assembly system is included, and different treatments are applied to address the roles of viral and host factors, such as RNA-binding activities of CTD and host phosphatases, in HBV capsid assembly under near-physiological conditions. This protocol will facilitate detailed studies on capsid assembly and host regulation under physiological conditions and identification of novel antiviral agents targeting HBc.