Recruitment of Vps34 PI3K and enrichment of PI3P phosphoinositide in the viral replication compartment is crucial for replication of a positive-strand RNA virus

Tombusviruses depend on subversions of multiple host factors and retarget cellular pathways to support viral replication. In this work, we demonstrate that tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus (CIRV) recruit the cellular Vps34 phosphatidylinositol 3-kinase (PI3K) into the large viral replication compartment. The kinase function of Vps34 is critical for TBSV replication, suggesting that PI(3)P phosphoinositide is utilized by TBSV for building of the replication compartment. We also observed increased expression of Vps34 and the higher abundance of PI(3)P in the presence of the tombusviral replication proteins, which likely leads to more efficient tombusvirus replication. Accordingly, overexpression of PI(3)P phosphatase in yeast or plants inhibited TBSV replication on the peroxisomal membranes and CIRV replication on the mitochondrial membranes. Moreover, the purified PI(3)P phosphatase reduced TBSV replicase assembly in a cell-free system. Detection of PI(3)P with antibody or a bioprobe revealed the enrichment of PI(3)P in the replication compartment. Vps34 is directly recruited into the replication compartment through interaction with p33 replication protein. Gene deletion analysis in surrogate yeast host unraveled that TBSV replication requires the vesicle transport function of Vps34. In the absence of Vps34, TBSV cannot efficiently recruit the Rab5-positive early endosomes, which provide PE-rich membranes for membrane biogenesis of the TBSV replication compartment. We found that Vps34 and PI(3)P needed for the stability of the p33 replication protein, which is degraded by the 26S proteasome when PI(3)P abundance was decreased by an inhibitor of Vps34. In summary, Vps34 and PI(3)P are needed for providing the optimal microenvironment for the replication of the peroxisomal TBSV and the mitochondrial CIRV.
Author summary Replication of RNA viruses infecting various eukaryotic organisms is the central step in the infection process that leads to generation of progeny viruses. The replication process requires the assembly of numerous viral replicase complexes within the large replication compartment, whose formation is not well understood. Using tombusviruses and the model host yeast, the authors discovered that a highly conserved cellular lipid kinase, Vps34 phosphatidylinositol 3-kinase (PI3K), is critical for the formation of the viral replication compartment. Expression of PI3K mutants and the PI(3)P phosphatase revealed that the PI(3)P phosphoinositide produced by Vps34 is crucial for tombusvirus replication. Tombusviruses co-opt Vps34 through interaction with the viral replication protein into the replication compartment. In vitro reconstitution of the tombusvirus replicase revealed the need for Vps34 and PI(3)P for the full-activity of the viral replicase. Chemical inhibition of Vps34 in yeast or plants showed that PI(3)P is important for the replication of several plant viruses within the Tombusviridae family and the insect-infecting Nodamuravirus. These results open up the possibility that the cellular Vps34 PI3K could be a target for new antiviral strategies.