Abstract
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.
Highlights
Positive-strand RNA viruses replicate inside the infected plant or animal cells by utilizing subcellular membranes and co-opting multiple host proteins
Replication of RNA viruses infecting various eukaryotic organisms is the central step in the infection process that leads to generation of progeny viruses
Expression of phosphatidylinositol 3-kinase (PI3K) mutants and the PI(3)P phosphatase revealed that the PI(3)P phosphoinositide produced by Vps34 is crucial for tombusvirus replication
Summary
Positive-strand RNA viruses replicate inside the infected plant or animal cells by utilizing subcellular membranes and co-opting multiple host proteins. These viruses generate membranous viral replication compartments, often harboring numerous vesicle-like membrane invaginations with narrow openings towards the cytosol [1,2,3,4,5]. The viral replication compartment help sequestering viral proteins, viral RNAs and co-opted host factors in confined areas, which facilitate efficient viral replicase complex (VRC) assembly and robust viral RNA replication. The plant-infecting tombusviruses, such as Tomato bushy stunt virus (TBSV), induce complex rearrangements of cellular membranes, alter metabolic processes with the help of a number of co-opted host proteins [7,8,9]. TBSV exploits sterols and phospholipids to induce a membranous replication compartment harboring numerous spherules, which are vesicle-like invaginations in the peroxisomal membranes [7,13,14,15]
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