The Innate Antiviral Factors APOBEC3G and APOBEC3H Interact with the Nucleocapsids of Human Coronaviruses in an RNA-Dependent Manner

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) evolution has been marked by the rapid accumulation of mutations, among which cytosine-to-uracil (C-to-U) transitions represent a major proportion of observed genomic changes. These mutations have been proposed to result from the activity of host APOBEC3 cytidine deaminases, innate immune enzymes capable of editing viral RNA. However, the molecular mechanisms underlying APOBEC3 involvement in SARS-CoV-2 biology remain poorly understood. Here, we systematically investigated physical interactions between APOBEC family members and the SARS-CoV-2 proteins using a Gaussia princeps protein complementation assay. Screening of APOBEC family proteins against the viral proteome identified specific interactions between APOBEC3G (A3G) and APOBEC3H (A3H) with the viral nucleocapsid (N) protein. These interactions were validated by co-immunoprecipitation and were found to be conserved across nucleocapsid proteins from all seven human coronaviruses, suggesting conserved structural determinants. Mechanistic analyses revealed that the RNA-binding and oligomerization capacities of A3G and A3H are key for their interaction with the SARS-CoV-2 nucleocapsid. Mapping experiments further showed that the C-terminal domain of N constitutes the minimal interacting region, with stronger binding observed in larger constructs encompassing adjacent regions, indicating cooperative stabilization. Further work will be needed to determine whether A3G and/or A3H can restrict viral replication and whether their interaction with the nucleocapsid allows them to access and mutate the viral genome.