N-terminal VP1 truncations favorT=1 norovirus-like particles

Abstract

Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on VP1 sequence, further enhance this public threat. Self-assembling properties of the human norovirus major capsid protein VP1 are crucial for using virus-like particles (VLPs) for vaccine development. However, there is no vaccine available yet. Here, VLPs from different variants produced in insect cells are characterized in detail using a set of biophysical and structural tools. We are using native mass spectrometry, gas-phase electrophoretic mobility molecular analysis and proteomics to get clear insights into particle size, structure, composition as well as stability. Generally, noroviruses have been known to form mainly T=3 particles. Importantly, we identify a major truncation in the capsid proteins as a likely cause for the formation of merely T=1 particles. For vaccine development, particle production needs to be a reproducible, reliable process. Understanding the underlying processes in capsid size variation will help to produce particles of a defined capsid size presenting antigens consistent with intact virions. Next to vaccine production itself, this would be immensely beneficial for bio-/nano-technological approaches using viral particles as carriers or triggers for immunological reactions.

Molecular weight determination of adeno‐associate virus serotype 8 virus‐like particle either carrying or lacking genome via native nES gas‐phase electrophoretic molecular mobility analysis and nESI QRTOF mass spectrometry

“…Gas‐phase electrophoresis of several VLPs—based on bacteriophages, a norovirus serotype, hepatitis B virus, cowpea mosaic virus and a human rhinovirus—yielding surface dry particle EMDs has already been described 22,23,42,47 . In addition, AAV8 VLPs have likewise been measured via gas‐phase electrophoresis as described in a previous work focusing on VLP aggregation (submitted manuscript).…”

“…Gas-phase electrophoresis of several VLPs-based on bacteriophages, a norovirus serotype, hepatitis B virus, cowpea mosaic virus and a human rhinovirus-yielding surface dry particle EMDs has already been described. 22,23,42,47 In addition, AAV8 VLPs have likewise been measured via gas-phase electrophoresis as described in a previous work focusing on VLP aggregation (submitted manuscript). Focusing on the molecular weight of bionanoparticles in the current manuscript, Figure 1A depicts the nES GEMMA spectra of "empty" (blue profile) and "filled" (red profile) AAV8 VLPs in their native state.…”

“…The main challenge for this MS approach is to desorb/ionize and detect the multiple‐charged analytes while preserving their labile non‐covalent interactions and structure. Nonetheless, several VLPs have been successfully analyzed by employing commercially available MS instrumentation, such as nESI orbitrap 37–40 and nESI charge detection mass spectrometry (CDMS) 41,42 . In our case, we employed a Synapt G1 (Waters Manchester, UK) modified by MS Vision (Almere, The Netherlands) to study AAV8 nanoparticles.…”

“…Nonetheless, several VLPs have been successfully analyzed by employing commercially available MS instrumentation, such as nESI orbitrap [37][38][39][40] and nESI charge detection mass spectrometry (CDMS). 41,42 In our case, we employed a Synapt G1 (Waters Manchester, UK) modified by MS Vision (Almere, The Netherlands) to study AAV8 nanoparticles. The instrument is equipped with a nano-electrospray ionization source for the production of multi-charged bionanoparticles, and with several custom modifications to properly fine-tune the necessary settings (e.g., application of collision and cooling gas, vacuum levels, and voltage settings) for successful analysis.…”