Fucose Binding Cancels out Mechanical Differences between Distinct Human Noroviruses

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-dependentbinding 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-likeparticles (noroVLPs) of three distinct human norovirus variants. We found differences in viral mechanical properties even between the norovirus variants from the same genogroup. The noroVLPswere then subjected to fucose treatment. Surprisingly, after fucose treatment, the previously foundconsiderable differences in viral mechanical properties among these variants were diminished. Weattribute a dynamic switch of the norovirus P domain upon fucose binding to the reduced differencesin viral mechanical properties across the tested norovirus variants. These findings shed light on themechanisms used by norovirus capsids to adapt to environmental changes and, possibly, increasecell infection. Hereby, a new step towards connecting viral mechanical properties to viral prevalenceis taken.<br/

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Leibniz AssociationEU Horizon 2020Indiana University Graduate Training Program in Quantitative and Chemical Biolog

N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles

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

Norovirus assembly and stability

Noroviruses are rapidly evolving RNA viruses and are generally known as the main cause of viral gastroenteritis worldwide. Particle stability is of special interest as transmission occurs via the faecal-oral route and virions are able to persist in the environment. Studies on norovirus capsid assembly and disassembly rely mainly on norovirus-like particles. Notably, stability of several human, murine and bovine variants has been investigated revealing distinct patterns of stability and also distinct assembly mechanisms and intermediates. Gathering information on these differences and common features may deepen our understanding of norovirus emergence and can potentially be used to distinguish variants. However, more systematic studies and standardized approaches are required.The Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit (BMG). RP acknowledges funding from EU Horizon 2020 project VIRUSCAN 731868. CU and JD further thank the DFG FOR2327 Virocarb for support.Peer reviewe

Norovirus-like VP1 particles exhibit isolate dependent stability profiles

Noroviruses are the main cause of viral gastroenteritis with new variants emerging frequently. There are three norovirus genogroups infecting humans. These genogroups are divided based on the sequence of their major capsid protein, which is able to form virus-like particles (VLPs) when expressed recombinantly. VLPs of the prototypical GI.1 Norwalk virus are known to disassemble into specific capsid protein oligomers upon alkaline treatment. Here, native mass spectrometry and electron microscopy on variants of GI.1 and of GII.17 were performed, revealing differences in terms of stability between these groups. Beyond that, these experiments indicate differences even between variants within a genotype. The capsid stability was monitored in different ammonium acetate solutions varying both in ionic strength and pH. The investigated GI.1 West Chester isolate showed comparable disassembly profiles to the previously studied GI.1 Norwalk virus isolate. However, differences were observed with the West Chester being more sensitive to alkaline pH. In stark contrast to that, capsids of the variant belonging to the currently prevalent genogroup GII were stable in all tested conditions. Both variants formed smaller capsid particles already at neutral pH. Certain amino acid substitutions in the S domain of West Chester relative to the Norwalk virus potentially result in the formation of these T  =  1 capsids.The Heinrich-Pette-Institute, Leibniz Institute for Experimental Virology is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Gesundheit (BMG). CU acknowledges funding from the Leibniz Association through SAW-2014-HPI-4 grant. RP acknowledges funding from EU Horizon 2020 project VIRUSCAN 731868. CU, RP and GH further thank the DFG FOR2327 Virocarb for support.Peer reviewe