(A–B) Rab7 puncta (green) are abundant in HEK293 cells expressing (A) SARS-CoV-2 (CoV-2) Orf3aHALO (magenta), but not (B) SARS-CoV-1 (CoV-1) Orf3aHALO (magenta; Hoechst 33342, blue). (C) Co-immunoprecipitation (co-IP) evaluating the interaction of VPS39GFP with CoV-1 and CoV-2 Orf3a2x-STREP, detected by western blot with antibodies against GFP and streptavidin, respectively. VPS39GFP elutes with CoV-2 Orf3a2x-STREP in a concentration-dependent manner, but does not elute with purified CoV-1 Orf3a2x-STREP (compare VPS39 in d lanes, orange). Control, co-IP without Orf3a2x-STREP added (bottom left, no protein). VPS39GFP and Orf3a2x-STREP migrate at ~130 and 35 kDa, respectively, by SDS-PAGE. (D–I) An unstructured loop of CoV-2 Orf3a partially mediates its interaction with VPS39. (D) Side view of CoV-2 Orf3a structure with the subunits (dark and light pink) and unstructured loop highlighted (yellow, dotted box). Zoom-in of the loop from the cytosol (solid box) with resolved loop residues. (E) CoV-2 Orf3a (red) and CoV-1 Orf3a (blue green) loop sequences. Orf3a wild-type (WT) and loop chimeras (LC) are color matched or swapped. Created with Biorender.com (F) Co-IP as in Figure 6C with CoV-2 Orf3a constructs showing loss of VPS39GFP elution with CoV-2 Orf3a LC2x-STREP (compare VPS39 in d lanes, orange). The co-IPs presented in this figure represent three to seven independent experiments. (G) Co-IP of VPS39GFP with CoV-1 Orf3a constructs shows an enrichment with CoV-1 Orf3a LC2x-STREP. (H, I) Rab7 puncta (green) are absent in CoV-2 Orf3a LCHALO (H, magenta) or CoV-1 Orf3a LCHALO-expressing HEK293 cells (I, magenta; Hoechst 33342, blue), consistent with Chen et al., 2021. (J–K) Cumming estimation plots of Rab7 puncta from (A, H) (J) and (B, I) (K) (Ho et al., 2019).