Formation of a Novel SARS-CoV-2 Nucleocapsid Dimer: Impacts on Viral and Cellular Processes - While most SARS-CoV-2 research to date has focused on the biological consequences of mutations seen in the Spike (S) protein, the nucleocapsid protein (N) is also under selective pressure and an array of mutations within this protein have been documented in different Variants of Concern (VOCs). In this study, we identified three SARS-CoV-2 variants (Beta, Iota, and Delta) that encode different cysteine mutations, all introduced into the linker region of N. These mutations facilitate a highly stable N-N dimer mediated by the introduction of a cysteine and the formation of a di-sulfide bond. Beta, Iota, and Delta variants isolated and grown at BSL-3 all contained a novel cysteine residue in the linker region of N, which appear to be unique introductions amongst pandemic- causing Betacoronaviruses. Nucleoproteins encoding these cysteine mutations and transiently expressed in HEK-293T cells also form a dimer in the absence of other viral machinery. Removal of these cysteine mutations in the linker abolishes dimer formation. Notably, our biochemical studies also revealed this dimer is highly stable and can be visualized on standard non-reducing SDS-PAGE gels. Our proposal focuses on the G215C mutation, which quickly rose to dominance within the Delta lineages and mutations back to wildtype within transmission chains were quickly followed by a reversion to a cysteine at this position. Using reverse genetics, Drs Johnson and Menachery will construct a SARS-CoV-2 Delta virus that reverts the nucleocapsid cysteine back to the ancestral sequence to specifically evaluate N dimer impact on infection. This proposal aims to study the biological impact of stable N dimer formation during infection by characterizing viral growth kinetics (in vitro and in vivo) as well as the effect on viral fitness and transmission in the hamster model. Notably, a related virus (G215C in the WA1 background) showed substantially increased growth both in vitro and in vivo, suggesting that stable N dimer formation is important for viral replication. The stably dimerized form of N is highly enriched in virions (vs. the cellular environment) and we hypothesize it is increasing the efficiency of encapsidation and thus the stability of the viral RNA during transmission. As the cysteines we observe in the nucleocapsid linker lie near/on the N/NSP3 binding interface we will use proteomics to determine how the cellular and viral binding partners of the nucleoprotein change with/without this disulfide bond. Overall, the observation that mutations introducing a cysteine in the N linker have arisen multiple independent times and been maintained during human transmission, as well as our preliminary viral growth kinetics suggest that stable N dimer formation may drive positive selection and convey a growth advantage during SARS-CoV-2 infection and/or a selective benefit during animal-to-animal transmission.
