Co-opted Cellular Sac1 Lipid Phosphatase and PI(4)P Phosphoinositide Are Key Host Factors during the Biogenesis of the Tombusvirus Replication Compartment

Abstract

Positive-strand RNA [(+)RNA] viruses assemble numerous membrane-bound viral replicase complexes (VRCs) with the help of viral replication proteins and co-opted host proteins within large viral replication compartments in the cytosol of infected cells. In this study, we found that deletion or depletion of Sac1 phosphatidylinositol 4-phosphate [PI(4)P] phosphatase reduced tomato bushy stunt virus (TBSV) replication in yeast (Saccharomyces cerevisiae) and plants. We demonstrate a critical role for Sac1 in TBSV replicase assembly in a cell-free replicase reconstitution assay. The effect of Sac1 seems to be direct, based on its interaction with the TBSV p33 replication protein, its copurification with the tombusvirus replicase, and its presence in the virus-induced membrane contact sites and within the TBSV replication compartment. The proviral functions of Sac1 include manipulation of lipid composition, sterol enrichment within the VRCs, and recruitment of additional host factors into VRCs. Depletion of Sac1 inhibited the recruitment of Rab5 GTPase-positive endosomes and enrichment of phosphatidylethanolamine in the viral replication compartment. We propose that Sac1 might be a component of the assembly hub for VRCs, likely in collaboration with the co-opted the syntaxin18-like Ufe1 SNARE protein within the TBSV replication compartments. This work also led to demonstration of the enrichment of PI(4)P phosphoinositide within the replication compartment. Reduction in the PI(4)P level due to chemical inhibition in plant protoplasts; depletion of two PI(4)P kinases, Stt4p and Pik1p; or sequestration of free PI(4)P via expression of a PI(4)P-binding protein in yeast strongly inhibited TBSV replication. Altogether, Sac1 and PI(4)P play important proviral roles during TBSV replication. IMPORTANCE Replication of positive-strand RNA viruses depends on recruitment of host components into viral replication compartments or organelles. Using TBSV, we uncovered the critical roles of Sac1 PI(4)P phosphatase and its substrate, PI(4)P phosphoinositide, in promoting viral replication. Both Sac1 and PI(4)P are recruited to the site of viral replication to facilitate the assembly of the viral replicase complexes, which perform viral RNA replication. We found that Sac1 affects the recruitment of other host factors and enrichment of phosphatidylethanolamine and sterol lipids within the subverted host membranes to promote optimal viral replication. In summary, this work demonstrates the novel functions of Sac1 and PI(4)P in TBSV replication in the model host yeast and in plants.

Dynamic interplay between the co-opted Fis1 mitochondrial fission protein and membrane contact site proteins in supporting tombusvirus replication

“…Another critical host protein in vMCS formation/function is Sac1p PI4P phosphatase, which allows the directional transfer of sterols from ER to the acceptor membranes through converting PI(4)P phosphoinositide to PI phosphatidylinositol [35]. PI(4)P is used by oxysterol localization of RFP-AtTim21 with the BiFC signal, indicating that the interactions between p33 replication protein and AtFis1A/B occur in the large viral replication compartments consisting of aggregated mitochondria.…”

“…Another proposed important function of vMCS is to provide PI(4)P phosphoinositide for the OSBP-like oxysterol transfer proteins to drive the directional sterol exchange between the opposing membranes [75][76][77][78][79]. We have previously shown that TBSV co-opt the Stt4p PI4K kinase to VROs to produce PI(4)P, which is critical for VRO biogenesis [35]. To test if Fis1p affects the subcellular distribution of Stt4p PI4K during TBSV replication, we used fis1Δ yeast expressing the viral replication proteins.…”

“…In addition, a major role for global phospholipid and sterol biosynthesis has been revealed [26][27][28]. Also critical roles of phospatidylethanolamine (PE), phospatidylserine, phosphoinositides and sterols in formation of VRCs and activation of the viralcoded p92 RdRp has been shown [29][30][31][32][33][34][35][36][37][38]. Sterols and PE bind directly to the p33 replication protein, which is the master regulator of VRC assembly and viral (+)RNA recruitment into VRCs [29,39].…”

Key interplay between the co-opted sorting nexin-BAR proteins and PI3P phosphoinositide in the formation of the tombusvirus replicase

“…Tomato bushy stunt virus (TBSV) and the closely related cucumber necrosis virus (CNV) use peroxisomal membranes, whereas carnation Italian ringspot virus (CIRV) exploits the outer membranes of mitochondria. The ER also contributes to VRO formation [ 16 – 18 ] and ER membranes even support TBSV replication efficiently in the absence of peroxisomes [ 19 ]. TBSV, similar to other (+)RNA viruses, induces major metabolic and structural changes in the infected cells, including aggregation of peroxisomal and ER membranes, membrane deformations by forming hundreds of 40–70 nm spherules that harbor the VRCs [ 6 , 20 , 21 ].…”

Characterization of a DCL2-Insensitive Tomato Bushy Stunt Virus Isolate Infecting Arabidopsis thaliana

“…Thus, over 100 yeast proteins were shown to interact with P33 or P92 and approximately 400 host proteins that could negatively or positively affect TBSV replication or recombination were identified [ 11 ]. These vast number of co-opted proteins comprise cellular translation factors, heat shock proteins, DEAD-box helicases, lipid biosynthesis and transfer enzymes, cytoskeleton components and even cellular energy-metabolism enzymes that altogether are reprogrammed to provide the optimal intracellular environment for TBSV replication [ 11 , 12 , 23 , 24 , 25 , 26 ].…”