Structural mass spectrometry goes viral

Dülfer, Jasmin; Kádek, Alan; Kopicki, Janine-Denise; Krichel, Boris; Uetrecht, Charlotte

doi:10.1016/bs.aivir.2019.07.003

Cited by 37 publications

(30 citation statements)

References 207 publications

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“…Detailed studies of polyprotein processing and complex formation require a method that follows these dynamic processes at a molecular level. Native mass spectrometry (MS) probes and detects protein complexes in protein mixtures, while keeping their non-covalent interactions intact through nano-electrospray ionization (nanoESI) [30,31]. Furthermore, collision-induced dissociation (CID) enables the stoichiometry and topology of natively sprayed protein complexes to be deciphered, by dissociating non-covalent interactions in vacuo.…”

Section: Introductionmentioning

confidence: 99%

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

Krichel

Falke

Hilgenfeld

et al. 2020

Biochemical Journal

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108

119

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Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/ transcription complex, essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease M pro and subsequent complex formation of the released nsp's. 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.

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Section: Introductionmentioning

confidence: 99%

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

Krichel

Falke

Hilgenfeld

et al. 2020

Biochemical Journal

Self Cite

108

119

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“…To fill these knowledge gaps, we analyzed nsp7+8 complexes derived from seven viruses of the Alpha-and Betacoronavirus genera, including a range of human coronaviruses, namely SARS-CoV, SARS-CoV-2, MERS-CoV and HCoV-229E (Table S 2). We used native mass spectrometry (MS) to illustrate the landscape of nsp7+8 complexes in vacuo, collision induced dissociation tandem MS (CID-MS/MS) to reconstruct complex topology and complementary methods to verify the results [24,25]. Our findings reveal distinct sets of nsp7+8 complexes for the different CoV species.…”

Section: Introductionmentioning

confidence: 99%

Hallmarks ofAlpha- andBetacoronavirusnon-structural protein 7+8 complexes

Krichel

Bylapudi

Schmidt

et al. 2020

Preprint

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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.

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“…Thus, hNoVLP particle sizes are polymorphic and dynamic. Native mass spectrometry (MS) is a perfect biophysical tool to characterize these structural dynamics [23]. Previously, VLPs of three different norovirus variants have been investigated with native MS [24,25].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Structural mass spectrometry goes viral

Cited by 37 publications

References 207 publications

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

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

Hallmarks ofAlpha- andBetacoronavirusnon-structural protein 7+8 complexes

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

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