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 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.”

SUMMARY Hepatitis B Virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes, but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized HAP compounds, but favors a unique quasi-equivalent location on the capsid surface. Thus, this pocket is a promiscuous drug binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a new paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses.

We report characterization of Hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ~40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T=3 (90 Cp149 dimer) and T=4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T=3 to T=4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T=3 capsids. These results combined with longer transit times for the T=4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.Nanopores and nanochannels exhibit unique transport properties1 and have a number of potential applications.2 Of particular interest is developing label-free, nondestructive techniques for rapid sensing, characterization, and sorting of particles with nanometer dimensions. The resistive-pulse technique3 measures changes in ion current resulting from transit of particles through an electrically biased nanopore filled with electrolyte. As sensing elements, protein pores,4 e.g., α-hemolysin, exhibit highly reproducible pore composition and dimensions, but lack robustness when suspended in lipid bilayers. Alternatively, microand nanofabrication techniques are used to fabricate solid-state and synthetic nanopores5 , 6 with a wide range of well-defined geometries and dimensions. Forming these pores parallel or perpendicular to the substrate surface permits straightforward integration with other device features. Solid-state and synthetic nanopores exhibit ion depletion/concentration,7 -9 ion permittivity,10 enhanced channel conductance,11 ion current rectification,12 , 13 and pressure-induced salt flux rectification.14 , 15 The ability to control pore dimensions over a range of length scales permits analysis of a variety of samples, including DNA,16 -18 proteins,19 viruses,20 immune complexes,21 nanoparticles,22 and small molecules,23 and similarly designed pores may be used to sequence DNA.24 In some cases, the molecule of interest, e.g., DNA, must overcome an entropic barrier to enter nanoscale slits25 and pores. 26 Related to this work is the characterization of viruses with track-etched pores20 and immune complexes with femtosecond laser-machined pores.21 In both examples, the studied protein complexes are ~100-150 nm in diameter. The reassembly process is inherently of interest, and this system offers a unique opportunity to characterize capsid transport, capsid properties, and nanopore properties. The T=3 and T=4 capsids are similar in diameter, 31 and 36 nm, respectively, and have identic...

We report fabrication and characterization of nanochannel devices with two nanopores in series for resistive-pulse sensing of hepatitis B virus (HBV) capsids. The nanochannel and two pores are patterned by electron beam lithography between two microchannels and etched by reactive ion etching. The two nanopores are 50-nm wide, 50-nm deep, and 40-nm long and are spaced 2.0-μm apart. The nanochannel that brackets the two pores is 20x wider (1 μm) to reduce the electrical resistance adjacent to the two pores and to ensure the current returns to its baseline value between resistive-pulse events. Average pulse amplitudes differ by <2% between the two pores and demonstrate the fabrication technique is able to produce pores with nearly identical geometries. Because the two nanopores in series sense single particles at two discrete locations, particle properties, e.g., electrophoretic mobility, are determined from the pore-to-pore transit time.

Hepatitis B virus (HBV) capsid proteins (Cps) assemble around the pregenomic RNA (pgRNA) and viral reverse transcriptase (P). pgRNA is then reverse transcribed to double-stranded DNA (dsDNA) within the capsid. The Cp assembly domain, which forms the shell of the capsid, regulates assembly kinetics and capsid stability. The Cp, via its nucleic acid-binding C-terminal domain, also affects nucleic acid organization. We hypothesize that the structure of the capsid may also have a direct effect on nucleic acid processing. Using structure-guided design, we made a series of mutations at the interface between Cp subunits that change capsid assembly kinetics and thermodynamics in a predictable manner. Assembly in cell culture mirrored in vitro activity. However, all of these mutations led to defects in pgRNA packaging. The amount of first-strand DNA synthesized was roughly proportional to the amount of RNA packaged. However, the synthesis of second-strand DNA, which requires two template switches, was not supported by any of the substitutions. These data demonstrate that the HBV capsid is far more than an inert container, as mutations in the assembly domain, distant from packaged nucleic acid, affect reverse transcription. We suggest that capsid molecular motion plays a role in regulating genome replication. IMPORTANCE The hepatitis B virus (HBV) capsid plays a central role in the virus life cycle and has been studied as a potential antiviral target.The capsid protein (Cp) packages the viral pregenomic RNA (pgRNA) and polymerase to form the HBV core. The role of the capsid in subsequent nucleic acid metabolism is unknown. Here, guided by the structure of the capsid with bound antiviral molecules, we designed Cp mutants that enhanced or attenuated the assembly of purified Cp in vitro. In cell culture, assembly of mutants was consistent with their in vitro biophysical properties. However, all of these mutations inhibited HBV replication. Specifically, changing the biophysical chemistry of Cp caused defects in pgRNA packaging and synthesis of the second strand of DNA. These results suggest that the HBV Cp assembly domain potentially regulates reverse transcription, extending the activities of the capsid protein beyond its presumed role as an inert compartment. Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide and contributes to 780,000 deaths per year (1). Currently there is no reliable cure for chronic hepatitis (2, 3). HBV is an enveloped double-stranded DNA (dsDNA) virus with an icosahedral core. HBV replicates its genome through an RNA intermediate. During replication, the capsid protein (Cp; also called the core protein) assembles around pregenomic RNA (pgRNA) and reverse transcriptase (the P protein) to form RNAfilled cores (4-8). Unlike HIV, reverse transcription of the HBV genome is a prerequisite for the progeny viruses to leave the host cell (5, 9).HBV reverse transcription is a complex reaction involving three template switches. P protein is incorporated into capsids durin...