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
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany
- Institute of Medical Virology, Justus Liebig University Giessen, Giessen, Germany
- European XFEL GmbH, Schenefeld, Germany
- EMBL Hamburg c/o DESY, Notkestraße 85, 22607 Hamburg, Germany
- 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
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).