In an infected cell, Hepatitis B virus (HBV) cores assemble in the cytoplasm. These cores are comprised of an icosahedral capsid of core protein (Cp) containing a reverse transcriptase-pregenomic RNA complex and associated chaperonins. We show that the dimeric Cp has a strong propensity to self-assemble in vitro and will bind any nucleic acid. Assembly at the wrong time or place, encapsidating the wrong nucleic acid or complement of proteins, would lead to non-productive infections. Preliminary data support our hypothesis that assembly can be allosterically triggered. We will use biophysical and structural approaches to examine assembly in vitro.
In aim 1, we will examine the intra-dimer interface (within a Cp dimer) for evidence of allosteric transitions. Mutations at this interface have dramatic effects on assembly though they are not involved in the inter-dimer contacts that hold a capsid together.
In aim 2, we will dissect that inter-dimer contact. Theoretical studies indicate that tight binding will lead to kinetic traps and thus interfere with capsid assembly. We will examine mutants that enhance and inhibit assembly, quantifying assembly in vitro and observing phenotype in cultured cells.
In aim 3, we will examine how regulation of assembly plays a role in the formation of RNA-filled capsids. When over-expressed in E. coli, HBV Cp packages random bacterial nucleic acid. By examining mechanisms of regulating assembly and assembly on random and viral nucleic, we address whether there is an intrinsic mechanism for specificity in vivo. These experiments will lead to examination of the physical and structural basis of Cp assembly. By considering Cp as a molecular machine, we will be able to identify regulatory elements in the structure that may be targets for interfering with assembly and thus the HBV lifecycle.
Chronic Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis of the liver and hepatocellular carcinoma, contributing to 1 million deaths per year. In a test tube, HBV perfect capsids assemble spontaneously, which indicates that assembly must be tightly regulated. We will determine the structural and physical basis for regulating assembly and thus identify new targets for future anti-viral therapeutics.
|Nair, Smita; Zlotnick, Adam (2018) Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription. J Virol 92:|
|Pierson, Elizabeth E; Keifer, David Z; Kukreja, Alexander A et al. (2016) Charge Detection Mass Spectrometry Identifies Preferred Non-Icosahedral Polymorphs in the Self-Assembly of Woodchuck Hepatitis Virus Capsids. J Mol Biol 428:292-300|
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|Selzer, Lisa; Zlotnick, Adam (2015) Assembly and Release of Hepatitis B Virus. Cold Spring Harb Perspect Med 5:|
|Wang, Joseph Che-Yen; Chen, Chao; Rayaprolu, Vamseedhar et al. (2015) Self-Assembly of an Alphavirus Core-like Particle Is Distinguished by Strong Intersubunit Association Energy and Structural Defects. ACS Nano 9:8898-906|
|Tan, Zhenning; Pionek, Karolyn; Unchwaniwala, Nuruddin et al. (2015) The interface between hepatitis B virus capsid proteins affects self-assembly, pregenomic RNA packaging, and reverse transcription. J Virol 89:3275-84|
|Wang, Joseph Che-Yen; Nickens, David G; Lentz, Thomas B et al. (2014) Encapsidated hepatitis B virus reverse transcriptase is poised on an ordered RNA lattice. Proc Natl Acad Sci U S A 111:11329-34|
|Pierson, Elizabeth E; Keifer, David Z; Selzer, Lisa et al. (2014) Detection of late intermediates in virus capsid assembly by charge detection mass spectrometry. J Am Chem Soc 136:3536-41|
|Selzer, Lisa; Katen, Sarah P; Zlotnick, Adam (2014) The hepatitis B virus core protein intradimer interface modulates capsid assembly and stability. Biochemistry 53:5496-504|
|Wang, Joseph Che-Yen; Zlotnick, Adam; Mecinovi?, Jasmin (2014) Transmission electron microscopy enables the reconstruction of the catenane and ring forms of CS2 hydrolase. Chem Commun (Camb) 50:10281-3|
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