Volume 86, June 2024, 102787Author links open overlay panel, , Highlights•

Viruses are metastable macromolecular assemblies that display intrinsic breathing dynamics and perturbation-induced conformational transitions.

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HDXMS is a powerful probe of allostery in whole viral particles at peptide resolution.

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Applications of HDXMS in epitope-paratope mapping, antibody binding-induced allostery, and virus-host receptor interactions.

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Complementation of HDXMS with structural and computational methods provide a complete molecular landscape of viruses.

Abstract

X-ray crystallography and cryo-electron microscopy have enabled the determination of structures of numerous viruses at high resolution and have greatly advanced the field of structural virology. These structures represent only a subset of snapshot end-state conformations, without describing all conformational transitions that virus particles undergo. Allostery plays a critical role in relaying the effects of varied perturbations both on the surface through environmental changes and protein (receptor/antibody) interactions into the genomic core of the virus. Correspondingly, allostery carries implications for communicating changes in genome packaging to the overall stability of the virus particle. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of whole viruses is a powerful probe for uncovering virus allostery. Here we critically discuss advancements in understanding virus dynamics by HDXMS with single particle cryo-EM and computational approaches.

Section snippetsViral particle dynamics and allostery in virus lifecycles

The inherent dynamic ability of proteins to exist in ensembles of multiple conformations is a prerequisite for function [1,2]. Protein dynamics is therefore crucial to correlate static views of 3-dimensional tertiary folded end-state conformations with function [3]. Protein conformational dynamics are critical to map how perturbations at specific singular loci are allosterically propagated through long-range conformational changes to distal regions [4, 5, 6]. Uncovering allostery in

Limitations of static snapshots of virus particles in uncovering allostery

Advances in structural virology by single particle cryo-EM have provided high-resolution insights into the quaternary structural organization of icosahedral viruses across multiple families [13, 14∗, 15∗∗]. A compilation of icosahedral virus structures that have been determined by crystallography and cryo-EM can be accessed in http://viperdb.scripps.edu. A limitation of these structures is that they describe a static view of virus particles that only represent snapshots of endpoint

Virus breathing by structural mass spectrometry

While symmetry has greatly improved the resolution of virus structures, this has been at the expense of asymmetric detail and inherent viral particle heterogeneity in solution [23]. Furthermore, it has generated an erroneous impression of viruses operating as static rock-like vehicles for moving viral genomes from one host cell to another. In the late 1990s, it was recognized by limited proteolysis mass spectrometry that viruses are highly dynamic macromolecular assemblies which undergo

Whole virus particle dynamics by amide hydrogen/deuterium exchange mass spectrometry (HDXMS)

While limited proteolysis combined with mass spectrometry was the first structural mass spectrometry approach that defined whole virus dynamics, recently HDXMS [28, 29, 30, 31, 32, 33] has become a probe of choice for measuring intrinsic virus dynamics, mapping virus-receptor/antibody complexes and importantly for monitoring allosteric changes in viruses [34] (Figure 3). In a bottom-up HDXMS approach [35], deuterium exchange is initiated by incubating virus particles in buffers reconstituted in

Virus-host protein interactions and allostery

HDXMS has also been valuable for mapping interactions of host and viral proteins with whole viral particles. For instance, in the bacteriophage HK97, a maturation protease was found to dock at an exosite on the capsid that induced long-range allosteric changes at the distal protease cleavage site [43]. HDXMS has also been used to map epitopes and paratopes of neutralizing antibodies 2D22 [44] and C10 with dengue [45]. Both 2D22 and C10 antibodies showed contrasting allosteric effects on DENV2.

Limitations of HDXMS of viruses and improvements in virus HDXMS workflows

HDXMS describes allostery and dynamics of virus particles at peptide resolution, at the seconds-to-minutes time scales. Further, the dynamics measurements are an average of both of the changes within the repeat units of the individual virus particle and across multiple virus particles in solution. Peptide resolution precludes identification of individual amino acid side chains in viruses that are involved in protein interactions, perturbation sensing, and allostery. Less than complete sequence

Conformational dynamics and allostery of virus particles by HDXMS

Dynamics is an intrinsic property of virus particles that underpins the essential allostery in the virus lifecycle (Figure 4). The outer capsid functions as a stable scaffold for shielding and protecting the nucleic acid genome from degradation by nucleases and physical stressors in the native virus particle. Concomitantly, reversible fluctuations or breathing allow the viruses to respond rapidly to environmental changes associated with host entry. Allostery thus plays a major role in

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We acknowledge startup funds from The Pennsylvania State University (IO 120000001317) and the Patricia and Steve Benkovic Foundation for funding this research.

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