Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus
Fig 5
Recruitment of Vps34 by the TBSV p33 replication protein into the viral replication compartment in N. benthamiana.
(A-B) Confocal microscopy images show co-localization of TBSV p33-BFP replication protein and the AtVps34-GFP, the ortholog of the yeast Vps34 protein, in planta. The large replication compartment was visualized via expression of RFP-SKL peroxisomal matrix marker protein. Expression of the above proteins from the 35S promoter was done after co-agroinfiltration into N. benthamiana leaves. Scale bars represent 5 μm. Note that the top panel in panel B is enlargement of the replication compartment portion of the bottom images. (C) Top images: interaction between TBSV p33-cYFP replication protein and the nYFP-AtVps34 protein was detected by BiFC. Co-localization of RFP-SKL with the BiFC signal (see merged image) demonstrates that the interaction between p33 replication protein and Vps34 occurs in the large viral replication compartments in planta. Control BiFC experiments included p33-cYFP protein in combination with nYFP-MBP protein expressed in N. benthamiana infected with TBSV (bottom images). Scale bars represent 10 μm. (D) Top images: interaction between CIRV p36-cYFP replication protein and the nYFP-AtVps34 protein was detected by BiFC. The CIRV replication compartment was visualized with CoxIV-RFP mitochondria marker. Scale bars represent 10 μm. See further details in panel C.