Lisa Selzer

Detection of Late Intermediates in Virus Capsid Assembly by Charge Detection Mass Spectrometry

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain.

Background: Exposure and phosphorylation of the core protein C-terminal domain (CTD) regulate important viral functions. Results: Empty capsids contain at least two populations of CTDs with different rates of exposure that are influenced by phosphorylation. Conclusion: Adding negative charge to CTDs increases capsid stability and decreases CTD exposure. Significance: Phosphorylation is used to tune HBV core protein function. Hepatitis B virus core protein has 183 amino acids divided into an assembly domain and an arginine-rich C-terminal domain (CTD) that regulates essential functions including genome packaging, reverse transcription, and intracellular trafficking. Here, we investigated the CTD in empty hepatitis B virus (HBV) T=4 capsids. We examined wild-type core protein (Cp183-WT) and a mutant core protein (Cp183-EEE), in which three CTD serines are replaced with glutamate to mimic phosphorylated protein. We found that Cp183-WT capsids were less stable than Cp183-EEE capsids. When we tested CTD sensitivity to trypsin, we detected two different populations of CTDs differentiated by their rate of trypsin cleavage. Interestingly, CTDs from Cp183-EEE capsids exhibited a much slower rate of proteolytic cleavage when compared with CTDs of Cp183-WT capsids. Cryo-electron microscopy studies of trypsin-digested capsids show that CTDs at five-fold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at five-fold, increase capsid stability and reduce CTD exposure. Our studies show that quasi-equivalent CTDs exhibit different rates of exposure and thus might perform distinct functions during the hepatitis B virus lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

Single-particle electrophoresis in nanochannels.

Electrophoretic mobilities and particle sizes of individual Hepatitis B Virus (HBV) capsids were measured in nanofluidic channels with two nanopores in series. The channels and pores had three-dimensional topography and were milled directly in glass substrates with a focused ion beam instrument assisted by an electron flood gun. The nanochannel between the two pores was 300 nm wide, 100 nm deep, and 2.5 μm long, and the nanopores at each end had dimensions 45 nm wide, 45 nm deep, and 400 nm long. With resistive-pulse sensing, the nanopores fully resolved pulse amplitude distributions of T = 3 HBV capsids (32 nm outer diameter) and T = 4 HBV capsids (35 nm outer diameter) and had sufficient peak capacity to discriminate intermediate species from the T = 3 and T = 4 capsid distributions in an assembly reaction. Because the T = 3 and T = 4 capsids have a wiffle-ball geometry with a hollow core, the observed change in current due to the capsid transiting the nanopore is proportional to the volume of electrolyte displaced by the volume of capsid protein, not the volume of the entire capsid. Both the signal-to-noise ratio of the pulse amplitude and resolution between the T = 3 and T = 4 distributions of the pulse amplitudes increase as the electric field strength is increased. At low field strengths, transport of the larger T = 4 capsid through the nanopores is hindered relative to the smaller T = 3 capsid due to interaction with the pores, but at sufficiently high field strengths, the T = 3 and T = 4 capsids had the same electrophoretic mobilities (7.4 × 10–5 cm2 V–1 s–1) in the nanopores and in the nanochannel with the larger cross-sectional area.

The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability.

During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer–dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61–C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome.

Monitoring Assembly of Virus Capsids with Nanofluidic Devices.

Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of hepatitis B virus core protein dimers into T = 3 and T = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single-particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudocritical dimer concentration of 0.5 μM, we observe a switch in the ratio of T = 3 to T = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudocritical dimer concentration, kinetically trapped, incomplete T = 4 particles assemble rapidly, then slowly anneal into T = 4 capsids. At all dimer concentrations tested, T = 3 capsids form more rapidly than T = 4 capsids, suggesting distinct pathways for the two forms.

Assembly and Release of Hepatitis B Virus

The hepatitis B virus (HBV) core protein is a dynamic and versatile protein that directs many viral processes. During capsid assembly, core protein allosteric changes ensure efficient formation of a stable capsid that assembles while packaging viral RNA-polymerase complex. Reverse transcription of the RNA genome as well as transport of the capsid to multiple cellular compartments are directed by dynamic phosphorylation and structural changes of core protein. Subsequently, interactions of the capsid with the surface proteins and/or host proteins trigger envelopment and release of the viral capsids or the transport to the nucleus. Held together by many weak protein-protein interactions, the viral capsid is an extraordinary metastable machine that is stable enough to persist in the cellular and extracellular environment but dissociates to allow release of the viral genome at the right time during infection.

One Protein, At Least Three Structures, and Many Functions

Hepatitis B virus core gene products can adopt different conformations to perform their functional roles. In this issue of Structure, DiMattia and colleagues show the crystal structure of immuno-modulating HBeAg and thereby reveal the similarities and differences between it and HBcAg, the variant found in virions.