Hallmarks ofAlpha- andBetacoronavirusnon-structural protein 7+8 complexes

Abstract

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.

Top-Down and Bottom-Up Proteomics Methods to Study RNA Virus Biology

“…The recent optimization of robust high-throughput chromatographic methods and fast sample preparation protocols has already shown great promise in different fields, and will likely also enter diagnostic and prognostic pipelines in the clinical setting for infectious diseases. So far, top-down and structural MS approaches have focused on purified assemblies, showing the potential for deducing the determinants of viral protein complex formation [ 25 ]. Moreover, the field is moving towards an analysis of crude extracts in native MS, combinations of labeling and top-down approaches, as well as in situ labelling combined with MS, allowing for the profiling of complete structural networks [ 38 ].…”

“…The order of processing was identified from the intermediate polyprotein products nps7–8 and nsp7–9, which provided much higher precision than e.g., SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) performed for comparison. In a follow-up [ 25 ], nsp7+8 complexes from different alpha- and betacoronaviruses, including SARS-CoV-2, showed variability in stoichiometry and topology, hence pointing to distinct assembly pathways. Notably, few amino acid exchanges in nsp8 and nsp7 determine stoichiometry switching.…”

“…Native MS has also been used to derive the stoichiometry of polymerase assemblies, including nsp7+8 and the helicase, and select ideal conditions for subsequent cryo-EM [ 7 , 26 ]. To further confirm nsp7+8 stoichiometry in conventional buffers, complexes can be stabilized via a chemical labeling method called “crosslinking” followed by MS [ 25 ]. This method is another approach to gain structural information and will be further explained in the next section.…”

Dimeric form of SARS-CoV-2 polymerase

“…The nsp7-nsp7 interaction is however consistent with formation of a (nsp7-nsp8) 2 heterotetramer 16,17 and its dissociation into a stable nsp7-nsp8 heterodimer that was suggested to bind nsp12 18 .…”

In silico screening for natural ligands to non-structural nsp7 conformers of SARS coronaviruses

“…Mutations to Alanine in nsp7 residues K7, H36 and N37 reduced RNA binding by the nsp7+nsp8+nsp12 complexes, polymerase processivity and viral replication 6 . All these results suggested that nsp7 may play a crucial, yet intriguing role(s) in coronavirus replication 8 . On the other hand, for optimal RNA binding and processivity, the nsp12 requires not only the presence of nsp7+nsp8 but also other nsps.…”