(A) Venn diagram illustrating the degree of overlap between the proteins identified in the three proteomics approaches. The greatest overlap between VPg and the replication complex proteome data was observed between VPg and NS1/2. G3BP1 was enriched in pulldowns of all three bait proteins (VPg, NS1/2 and NS4). (B) The baits NS1/2 and NS4 both enriched VPg during replication complex pulldowns, but NS1/2 also enriched the VPg binding partner eIF4G, while NS4 did not. Immunoblot analysis revealed that NS4 enriched primarily the NS4/5 (VPg) polyprotein precursor, and not mature VPg, while NS1/2 enriched primarily the mature form of VPg (Figure 2C). (C) Plot of all MNV VPg binding partners identified in Figure 1 which overlapped with proteins associated with the replication complex through NS1/2 and NS4 purification. Most VPg interaction partners were better enriched by NS1/2 than NS4, except G3BP1. (D) Table showing Gene Ontology analysis of proteins enriched by VPg and NS1/2 during infection using PANTHER overrepresentation analysis, revealing a subset of the replication complex dedicated to translation and RNA metabolism.