Abstract
Replication of the ∼30 kb-long coronavirus genome is mediated by a complex of non-structural proteins (NSP), in which NSP7 and NSP8 play a critical role in regulating the RNA-dependent RNA polymerase (RdRP) activity of NSP12. The assembly of NSP7, NSP8 and NSP12 proteins is highly dynamic in solution, yet the underlying mechanism remains elusive. We report the crystal structure of the complex between NSP7 and NSP8 of SARS-CoV-2, revealing a 2:2 heterotetrameric form. Formation of the NSP7-NSP8 complex is mediated by two distinct oligomer interfaces, with interface I responsible for heterodimeric NSP7-NSP8 assembly, and interface II mediating the heterotetrameric interaction between the two NSP7-NSP8 dimers. Structure-guided mutagenesis, combined with biochemical and enzymatic assays, further reveals a structural coupling between the two oligomer interfaces, as well as the importance of these interfaces for the RdRP activity of the NSP7-NSP8-NSP12 complex. Finally, we identify an NSP7 mutation that differentially affects the stability of the NSP7-NSP8 and NSP7-NSP8-NSP12 complexes leading to a selective impairment of the RdRP activity. Together, this study provides deep insights into the structure and mechanism for the dynamic assembly of NSP7 and NSP8 in regulating the replication of the SARS-CoV-2 genome, with important implications for antiviral drug development.
Highlights
Coronaviruses are positive-strand RNA viruses that belong to the family of Coronaviridae
The NSP7 protein eluted at a volume close to what is expected for its monomeric form, whereas NSP8 eluted at a volume corresponding to its dimeric form (Figure 1B), in line with previous reports that NSP7 and NSP8 proteins individually exist as a monomer and a dimer in solution, respectively [16,21]
The NSP7NSP8 mixture eluted at a volume corresponding to what is expected for a 2:2 tetrameric form (∼62 kDa) (Figure 1B), far lower than what is expected for a hexadecameric NSP7NSP8 complex (∼240 kDa)
Summary
Coronaviruses are positive-strand RNA viruses that belong to the family of Coronaviridae. Members of its betasubtype have become a grave threat to public health, causing three major outbreaks in the past two decades: the Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) in 2003, the Middle East Respiratory Syndrome-associated Coronavirus (MERS-CoV) in 2012, and currently, the Severe Acute Respiratory Syndromeassociated Coronavirus 2 (SARS-CoV-2) [1,2]. SARS-CoV-2 has caused the current pandemic of coronavirus disease 2019 (COVID-19), with over 140 million confirmed infected cases and over three million deaths globally, leading to social, societal, and economic disruptions not seen in many generations. Despite recent progress in vaccine development, there is no highly effective therapeutic against SARS-CoV, MERS-CoV or SARS-CoV-2. Effective RNA synthesis is essential for the life cycle of RNA viruses, which makes the RNA replication machinery an appealing target for antiviral drug development
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