Fig. 5: ORF3a interacted with the HOPS component VPS39 and prevented RAB7–VPS39 bundling.

a Outline of co-immunoprecipitation and mass spectrometry experiments performed to identify ORF3a-interacting proteins in HeLa cells. b HOPS component VPS39 was the candidate among the ORF3a-interacting proteins identified by mass spectrometry at the highest confident level. c Scheme showing the function of HOPS–RAB7 in autophagosome–lysosome fusion. d Two experimental studies on ORF3a-interacting proteins and our study simultaneously identified VPS39. e ORF3a directly interacted with VPS39. GST, GST-ORF3a, or His-VPS39 were expressed in bacteria. The purified proteins were used for GST pull-down assays and subsequent western blot analysis. f Schematic representation of ORF3a and the indicated Y160A mutant used to verify the VPS39 interaction and function in autophagy. g The Y160A mutation in ORF3a dramatically reduced the interaction between ORF3a and VPS39. GST, GST-ORF3a, the GST-ORF3a-Y160A mutant or His-VPS39 were expressed in bacteria. Purified proteins were used for GST pull-down assays and subsequent western blot analysis. h The Y160A mutation in ORF3a abolished its function in blocking autophagy. i ORF3a prevented the RAB7 interaction with VPS39. The RAB7–VPS39 interaction was detected by co-immunoprecipitation assays with or without ORF3a expression. j ORF3a disrupted the assembly of SNARE. The interaction between SNARE components SNAP29 and VAMP8 was detected by co-immunoprecipitation assays with or without expression of ORF3a.