Title: CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design. Introduction Upon entry into human cells SARS-CoV-2, the causative agent of COVID-19, produces two large polyproteins, pp1a and pp1ab. These polyproteins are further processed by two viral proteases into 16 individual non-structural proteins (nsp1-nsp16). These non-structural proteins fulfill a number of essential viral functions including RNA replication, replication proofreading, double-membrane vesicle formation, and others1. Many of these also interact with host factors to effectively subvert the host cell to meet the virus’s needs2. Such subversion, for example, includes suppressing host innate immune responses, host translation, nuclear import, and other effects3–5. Despite their central importance to viral pathogenesis, many non-structural proteins remain structurally and functionally uncharacterized. Furthermore, the interactions between