Hallmarks of Alpha and Betacoronavirus Non-Structural Protein 7+8 Complexes



Boris Krichel1, Ganesh Bylapudi2, Christina Schmidt3, Clement Blanchet4, Robin Schubert3, Lea Brings3, Martin Koehler5, Renato Zenobi5, Dmitri Svergun4, Kristina Lorenzen3, Ramakanth Madhugiri2, John Ziebuhr2, Charlotte Uetrecht1,3


  1. Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
  2. Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany
  3. European XFEL GmbH, Schenefeld, Germany
  4. EMBL Hamburg c/o DESY, Notkestraße 85, 22607 Hamburg, Germany
  5. ETH Zurich D-CHAB Lab of Organic Chemistry, Zürich, Switzerland


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.

CovalX Technology Used

Crosslinking Mass Spectrometry (XL-MS)


RNA-dependent RNA-polymerase and other enzymes in the replication-transcription complex (RTC) are interacted and regulated by the coronaviral non-structural proteins (nsp) 7 and nsp8. To understand the structural plasticity of nsp7+8 complex various methods including cross-linking mass spectrometry (XL-MS) based on Matrix-Assisted Laser Desorption Ionization (MALDI) were used. Before the MALDI process, pre-purified FIPV nsp7+8 and HCoV-229E nsp7+8 at 20 μM were cross-linked with 0.15 % glutaraldehyde (Sigma-Aldrich) at 4 °C for 25 minutes. Then the prepared solution was diluted to 1 μM in MALDI matrix solution containing sinapinic acid 10 mg/mL in acetonitrile/ water/ TFA, 49.95/49.95/0.1, v/v/v. 1μL of the solution was spotted on a MALDI target plate that was analyzed by the MALDI-TOF MS modified with CovalX high-mass detector (HM2) in linear mode. MALDI mass spectrum of FIPV nsp7+8 showed the most abundant nsp7+8 complexes: (1:1) heterodimer (32.4 kDa) and (2:1) heterotrimer (42.5 kDa), and the MALDI mass spectrum of HCoV-229E nsp7+8 showed the most abundant nsp7+8 complexes: (1:1) heterodimer (32.2 kDa) and (2:2) heterotetramer (64.6 kDa).



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Categories : Publications, High-Mass MS