Biology subjects

Uetrecht, C.

Publications and source records attributed to Uetrecht, C..

24 records · Page 2Linked to original sources

bioRxiv2020

N-terminal VP1 truncations favor T=1 norovirus-like particles

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.

biophysics↗

bioRxiv2020

Glycan-induced protein dynamics in human norovirus P dimers depend on virus strain and deamidation status

Noroviruses are the major cause of gastroenteritis and re-emerge worldwide every year, with GII.4 currently being the most frequent human genotype. The norovirus capsid protein VP1 is essential for host immune response. The P domain mediates cell attachment via histo blood-group antigens (HBGAs) in a strain-dependent manner but how these glycan-interactions actually relate to cell entry remains unclear. Here, hydrogen/deuterium exchange mass spectrometry (HDX-MS) is used to investigate glycan-induced protein dynamics in P dimers of different strains, which exhibit high structural similarity but different prevalence in humans. While the almost identical strains GII.4 Saga and GII.4 MI001 share glycan-induced dynamics, the dynamics differ in the emerging GII.17 Kawasaki 308 and rare GII.10 Vietnam 026 strain. We also further examine structural effects of N373 deamidation upon glycan binding in partially deamidated GII.4 P dimers, which are likely present during infection. Such mixed species exhibit increased exposure to solvent in the P dimer upon glycan binding as opposed to pure wildtype. Furthermore, deamidated P dimers display increased flexibility and a monomeric population. Our results indicate that glycan binding induces strain-dependent structural dynamics, which are further altered by N373 deamidation, and hence hint at a role of deamidation in modulating cell attachment and entry in GII.4 strains.

biophysics↗

bioRxiv2020

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

bioRxiv2019

Processing of the SARS-CoV pp1a/ab nsp7-10 region

1.1.Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsps. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2:2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

biophysics↗

bioRxiv2019

Flagella methylation promotes bacterial adhesion and host cell invasion

The flagellum is the motility device of many bacteria and the long external filament is made of several thousand copies of a single protein, flagellin. While posttranslational modifications of flagellin are common among bacterial pathogens, the role of lysine methylation remained unknown. Here, we show that both flagellins of Salmonella enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A Salmonella mutant deficient in flagellin methylation was outcompeted for gut colonization in a gastroenteritis mouse model. In support, methylation of flagellin promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization and successful infection of the host.

microbiology↗

bioRxiv2019

Molecular organization of soluble type III secretion system sorting platform complexes

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components is unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

microbiology↗