Lisa Selzer

All Publications

• Hepatitis B Virus Core Protein Phosphorylation Sites Affect Capsid Stability and Transient Exposure of the C-terminal Domain JOURNAL OF BIOLOGICAL CHEMISTRY Selzer, L., Kant, R., Wang, J. C., Bothner, B., Zlotnick, A. 2015; 290 (47): 28584-?

  Abstract

  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 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 compared to CTDs of Cp183-WT capsids. Cryo-EM studies of trypsin-digested capsids show that CTDs at fivefold symmetry vertices are most protected. We hypothesize that electrostatic interactions between glutamates and arginines in Cp183-EEE, particularly at fivefolds, 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 HBV lifecycle. Our results demonstrate a structural role for CTD phosphorylation and indicate crosstalk between CTDs within a capsid particle.

  View details for DOI 10.1074/jbc.M115.678441

  View details for Web of Science ID 000365761900049

  View details for PubMedID 26405031

• Monitoring Assembly of Virus Capsids with Nanofluidic Devices. ACS nano Harms, Z. D., Selzer, L., Zlotnick, A., Jacobson, S. C. 2015; 9 (9): 9087-9096

  Abstract

  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.

  View details for DOI 10.1021/acsnano.5b03231

  View details for PubMedID 26266555

• Single-Particle Electrophoresis in Nanochannels ANALYTICAL CHEMISTRY Harms, Z. D., Haywood, D. G., Kneller, A. R., Selzer, L., Zlotnick, A., Jacobson, S. C. 2015; 87 (1): 699-705

  Abstract

  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) cm(2) V(-1) s(-1)) in the nanopores and in the nanochannel with the larger cross-sectional area.

  View details for DOI 10.1021/ac503527d

  View details for Web of Science ID 000347590400074

  View details for PubMedID 25489919

• Assembly and Release of Hepatitis B Virus Capsid Particles Hepatitis B and delta viruses / edited by Christoph Seeger and Stephen Locarnini Selzer, L., Zlotnick, A. Cold Spring Harbor, New York : Cold Spring Harbor Laboratory Press,. 2015
• Structurally Similar Woodchuck and Human Hepadnavirus Core Proteins Have Distinctly Different Temperature Dependences of Assembly JOURNAL OF VIROLOGY Kukreja, A. A., Wang, J. C., Pierson, E., Keifer, D. Z., Selzer, L., Tan, Z., Dragnea, B., Jarrold, M. F., Zlotnick, A. 2014; 88 (24): 14105-14115

  Abstract

  Woodchuck hepatitis virus (WHV), a close relative of human hepatitis B virus (HBV), has been a key model for disease progression and clinical studies. Sequences of the assembly domain of WHV and HBV core proteins (wCp149 and hCp149, respectively) have 65% identity, suggesting similar assembly behaviors. We report a cryo-electron microscopy (cryo-EM) structure of the WHV capsid at nanometer resolution and characterization of wCp149 assembly. At this resolution, the T=4 capsid structures of WHV and HBV are practically identical. In contrast to their structural similarity, wCp149 demonstrates enhanced assembly kinetics and stronger dimer-dimer interactions than hCp149: at 23 °C and at 100 mM ionic strength, the pseudocritical concentrations of assembly of wCp149 and hCp149 are 1.8 μM and 43.3 μM, respectively. Transmission electron microscopy reveals that wCp149 assembles into predominantly T=4 capsids with a sizeable population of larger, nonicosahedral structures. Charge detection mass spectrometry indicates that T=3 particles are extremely rare compared to the ∼ 5% observed in hCp149 reactions. Unlike hCp149, wCp149 capsid assembly is favorable over a temperature range of 4 °C to 37 °C; van't Hoff analyses relate the differences in temperature dependence to the high positive values for heat capacity, enthalpy, and entropy of wCp149 assembly. Because the final capsids are so similar, these findings suggest that free wCp149 and hCp149 undergo different structural transitions leading to assembly. The difference in the temperature dependence of wCp149 assembly may be related to the temperature range of its hibernating host.In this paper, we present a cryo-EM structure of a WHV capsid showing its similarity to HBV. We then observe that the assembly properties of the two homologous proteins are very different. Unlike human HBV, the capsid protein of WHV has evolved to function in a nonhomeostatic environment. These studies yield insight into the interplay between core protein self-assembly and the host environment, which may be particularly relevant to plant viruses and viruses with zoonotic cycles involving insect vectors.

  View details for DOI 10.1128/JVI.01840-14

  View details for Web of Science ID 000345292100017

  View details for PubMedID 25253350

• The Hepatitis B Virus Core Protein Intradimer Interface Modulates Capsid Assembly and Stability BIOCHEMISTRY Selzer, L., Katen, S. P., Zlotnick, A. 2014; 53 (34): 5496-5504

  Abstract

  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.

  View details for DOI 10.1021/bi500732b

  View details for Web of Science ID 000341229800004

  View details for PubMedID 25102363

• Detection of Late Intermediates in Virus Capsid Assembly by Charge Detection Mass Spectrometry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Pierson, E. E., Keifer, D. Z., Selzer, L., Lee, L. S., Contino, N. C., Wang, J. C., Zlotnick, A., Jarrold, M. F. 2014; 136 (9): 3536-3541

  Abstract

  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.

  View details for DOI 10.1021/ja411460w

  View details for Web of Science ID 000332684700033

  View details for PubMedID 24548133

• One Protein, At Least Three Structures, and Many Functions STRUCTURE Zlotnick, A., Tan, Z., Selzer, L. 2013; 21 (1): 6-8

  Abstract

  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.

  View details for DOI 10.1016/j.str.2012.12.003

  View details for Web of Science ID 000313383400004

  View details for PubMedID 23312031