Lars Thiede

Probing viral capsid assembly by native MS

Lars Thiede1,2, Ronja Pogan1,2, Jürgen Müller-Guhl1,3, Charlotte Uetrecht1,2,3

1 Centre for Structural Systems Biology (CSSB), Deutsches Elektronen-Synchroton DESY, Leibniz Institute of Virology (LIV), Hamburg, Germany.
2 Faculty V: School of Sciences, University of Siegen, Siegen, Germany.
3 Bernhard Nocht Institute for Tropical Medicine (BNITM), Hamburg, Germany

The assembly of viral capsids is a complex and still not fully deciphered process, that constitutes a vital step in the viral lifecycle. This is due to the morphological diverse nature of viral particles and the dynamics of the formation process, warranting more insight into the mechanisms of capsid assembly. This is even more important when the objective is the application of norovirus capsid proteins to a biotechnological context. Human norovirus (HuNoV) is the largest driver of viral gastroenteritis globally, threatening mostly children, the elderly and immunocompromised people. A vaccine has yet to be developed and the few available cell culture systems are still difficult to manage and unreliable. Hence, virus-like proteins (VLPs) have become the prime method for investigation.

These VLPs are produced by expressing the major capsid protein VP1 in insect cell culture and mimic the original virions in spherical form and icosahedral symmetry. We utilize native mass spectrometry as our main tool for probing the weight and thus state of assembly of these VLPs. Previous work from our lab determined that the capsid assembly is highly strain dependent, which is mirrored in their distinct stability profiles. Further probing by Pogan and colleagues revealed an expression batch specific N-terminal truncation. This truncation, revealed with charge detection mass spectrometry, gas phase electrophoretic mobility analysis and proteomics, seemingly coincided with an exclusive formation of smaller virus particles.This phenomenon was observed across different strains, pointing to a conserved mechanism.

Preliminary experiments have partially confirmed the impact of the N-terminal region on the formation process, though not as homogenous as initially observed. This points towards a more complicated picture and warrant further research. These homogenic small particles are an important step in attaining size-controlled capsids, for both biotechnological purposes, where capsids could act as carriers,  or vaccine development, which benefits from insights into the virion-like proteins.

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