A molecular breadboard: Removal and replacement of subunits in a hepatitis B virus capsid

Francis

et al. 2018

eLife

Self Cite

148

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection.

Section: Resultsmentioning

confidence: 99%

Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

Schlicksup

Francis

et al. 2018

eLife

Self Cite

148

“…Resection of hybrid capsids to holey capsids. Previously, we attempted to generate holey capsids by removing modi ed subunits that had been incorporated stochastically; we were unable to identify regular, contiguous patches by this method 58 . However, our Janus capsids have a heterodimer hexamer patche in a body of crosslinked Cp150.…”

Section: Resultsmentioning

confidence: 99%

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry

Zhao

Zhang

et al. 2020

Preprint

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Symmetrical protein complexes are ubiquitous in natural biological systems. Many have been reengineered in vitro for chemical and medical applications. Symmetrical viral capsids and their assembly are frequent platforms for these investigations. Lacking a means to create asymmetric capsids may limit broader applications. Here, starting with the homodimeric Hepatitis B Virus capsid protein, we developed a heterodimer, designed a hierarchical assembly pathway, and produced asymmetric capsids. We showed that the heterodimers assemble into hexamers, and such preformed hexamers can nucleate co-assembly, leading to “Janus” capsids with two discrete patches. We removed the hexamer patches specifically and observed asymmetric holey capsids by cryo-EM reconstruction. The resulting holes can be refilled with new engineered dimers. This programmed assembly pathway provides windows for specific engineering and modification inside and outside of the capsid. This strategy can also be generalized to other capsid assembly systems.

“…Because a large fraction of HBV particles formed in vivo are empty [ 52 ], the formation of empty capsids (even in vitro) is biologically relevant. A metastable species was also observed during HBV dissociation, as with assembly, also a ~90-dimer complex; however, no single type of holey capsid was observed in class averages of dissociation reactions, implying that they were an ensemble of structures [ 53 ]. A critical point is that these asymmetric particles, those that are overgrown and those that are holey, would have been obscured during reconstruction that removed non-ideal particles.…”

Section: Symmetry Defects Are Not Rarementioning

confidence: 99%

“…Class 3 has an elliptical morphology not typically seen in HBV. Panels A and B are from Pierson et al [ 20 ], and Panels C and D are from Lee et al [ 53 ]. Figures are used with permission.…”

Section: Figurementioning

confidence: 99%

Geometric Defects and Icosahedral Viruses

Mukhopadhyay

Zlotnick

2018

Viruses

Self Cite

We propose that viruses with geometric defects are not necessarily flawed viruses. A geometric defect may be a reactive site. Defects may facilitate assembly, dissociation, or accessibility of cellular proteins to virion components. In single molecule studies of hepadnavirus assembly, defects and overgrowth are common features. Icosahedral alphaviruses and flaviviruses, among others, have capsids with geometric defects. Similarly, immature retroviruses, which are non-icosahedral, have numerous “errors”. In many viruses, asymmetric exposure of interior features allows for regulated genome release or supports intracellular trafficking. In these viruses, the defects likely serve a biological function. Commonly used approaches for spherical virus structure determination use symmetry averaging, which obscures defects. We suggest that there are three classes of asymmetry: regular asymmetry as might be found in a tailed phage, irregular asymmetry as found, for example, in defects randomly trapped during assembly, and dynamic asymmetry due to Brownian dynamics of virus capsids. Awareness of their presence and recent advances in electron microscopy will allow unprecedented investigation of capsid irregularities to investigate their biological relevance.