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. 2023 Jan 25;12:e84477. doi: 10.7554/eLife.84477

Figure 6. SARS-CoV-2 Orf3a, but not SARS-CoV-1 Orf3a, interacts with HOPS protein, VPS39.

(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).

Figure 6—source data 1. Raw unedited western blots and figures with the uncropped blots for Figure 6C.
Figure 6—source data 2. Raw unedited western blots and figures with the uncropped blots for Figure 6F.

Figure 6.

Figure 6—figure supplement 1. Purification of SARS-CoV-1 and SARS CoV-2 Orf3a loop chimeras.

Figure 6—figure supplement 1.

(A, C) Gel filtration traces from (A) SARS-CoV-2 Orf3a loop chimera (CoV-2 Orf3a LC) and (C) SARS-CoV-1 Orf3a loop chimera (CoV-1 Orf3a LC) after elution from Strep-Tactin XT column. Collected peak fraction is highlighted in gray. (B, D) Gel filtration traces of final samples of (B) CoV-2 Orf3a LC and (D) CoV-1 Orf3a LC used for the co-IP experiments (Figure 6F–G).