Noroviruses cause immense sporadic gastroenteritis outbreaks worldwide. Emerging genotypes, which are divided based on the sequence of the major capsid protein VP1, 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 were characterized in detail using a set of biophysical and structural tools. We used native mass spectrometry, gas-phase electrophoretic mobility molecular analysis, and proteomics to get clear insights into particle size, structure, and composition, as well as stability. Generally, noroviruses have been known to form mainly T = 3 particles. Importantly, we identified a major truncation in the capsid proteins as a likely cause for the formation of 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.

The majority of nonbacterial gastroenteritis in humans and livestock is caused by noroviruses. Like most RNA viruses, frequent mutations result in various norovirus variants. The strain-dependent binding profiles of noroviruses to fucose are supposed to facilitate norovirus infection. It remains unclear, however, what the molecular mechanism behind strain-dependent functioning is. In this study, by applying atomic force microscopy (AFM) nanoindentation technology, we studied norovirus-like particles (noroVLPs) of three distinct human norovirus variants. We found differences in viral mechanical properties even between the norovirus variants from the same genogroup. The noroVLPs were then subjected to fucose treatment. Surprisingly, after fucose treatment, the previously found considerable differences in viral mechanical properties among these variants were diminished. We attribute a dynamic switch of the norovirus P domain upon fucose binding to the reduced differences in viral mechanical properties across the tested norovirus variants. These findings shed light on the mechanisms used by norovirus capsids to adapt to environmental changes and, possibly, increase cell infection. Hereby, a new step towards connecting viral mechanical properties to viral prevalence is taken.

Electrospray ionization (ESI)-based native mass spectrometry (MS) has been extensively used to study non-covalent protein complexes and their structural aspects in the gas phase. As such it has a stable place in the portfolio of modern structural biology and biopharmaceutical analysis techniques. It is also promising as an approach to deliver pro-teins and their assemblies into the gas phase for single particle experiments at advanced light sources such as free electron lasers and synchrotrons. Here we report on a novel radiofrequency ion transfer interface enabling gas flow laminarization at low pressure for efficient capture, focusing, thermalization, trapping and gating of ions into a mass analyser, boosting up to 19-fold the observable signal intensities especially for notoriously challenging multi-megadalton assemblies such as in-tact viral capsids. The new ion transfer interface offers unique opportunities for interdisciplinary approaches intersecting native MS-guided structural biology, molecular and cluster physics as well as (bio)analytical applications. Additionally, when combined with sensitive detection of individual particles, this technology could eventually also open new avenues in clinical diagnostics, enabling for example the analysis of viruses at very low concentrations directly from dilute patient-derived samples as well as viral therapeutic vectors along the biopharmaceutical production chain, thereby helping to un-lock the full potential of native MS.