Activation of Tomato Bushy Stunt Virus RNA-Dependent RNA Polymerase by Cellular Heat Shock Protein 70 Is Enhanced by Phospholipids In Vitro

Abstract

Similar to other positive-strand RNA viruses, tombusviruses are replicated by the membrane-bound viral replicase complex (VRC). The VRC consists of the p92 virus-coded RNA-dependent RNA polymerase (RdRp), the viral p33 RNA chaperone, and several co-opted host proteins. In order to become a functional RdRp after its translation, the p92 replication protein should be incorporated into the VRC, followed by its activation. We have previously shown in a cell-free yeast extract-based assay that the activation of the Tomato bushy stunt virus (TBSV) RdRp requires a soluble host factor(s). In this article, we identify the cellular heat shock protein 70 (Hsp70) as the co-opted host factor required for the activation of an N-terminally truncated recombinant TBSV RdRp. In addition, small-molecule-based blocking of Hsp70 function inhibits RNA synthesis by the tombusvirus RdRp in vitro. Furthermore, we show that neutral phospholipids, namely, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), enhance RdRp activation in vitro. 1-7). Interestingly, the viral RdRp is assumed to be inactive in the cytosol to prevent the formation of a viral double-stranded RNA (dsRNA) intermediate that could trigger efficient gene silencing and RNAinduced innate immunity (8-10). Therefore, the VRC assembly, including the activation of the viral RdRp, could be a critical regulatory step during the infection cycle.The tombusvirus VRCs are housed in membranous spherule structures, which are membrane invaginations into peroxisomes or mitochondria in the cases of Tomato bushy stunt virus (TBSV) and Carnation Italian ringspot virus (CIRV), respectively (11-15). The tombusvirus VRCs contain the virus-coded p92 RdRp and p33 RNA chaperone and the dsRNA intermediate, as well as co-opted cellular proteins, such as the heat shock protein 70 (Hsp70) molecular chaperone (coded for by yeast [Saccharomyces cerevisiae] SSA1/2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH [coded for by TDH2/3 in yeast]), Cdc34p E2 ubiquitinconjugating enzyme, eukaryotic translation elongation factor 1A (eEF1A), eEF1B␥, the Vps4p ESCRT (endosomal sorting complex required for transport) protein, and DDX3-like Ded1p and eIF4AIII-like RH2 DEAD box helicases (16-30). Hsp70, eEF1A, Cdc34p, and ESCRT proteins are involved in the assembly of the tombusviral VRC, while other subverted RNA-binding proteins (eEF1A, eEF1B␥, GAPDH, Ded1p, and RH2) facilitate viral RNA synthesis (1,3,18,31). Tombusvirus replication also depends on lipids, such as sterols and phospholipids, and oxysterol-binding

The role of co-opted ESCRT proteins and lipid factors in protection of tombusviral double-stranded RNA replication intermediate against reconstituted RNAi in yeast

“…Tombusvirus replication greatly depends on phospholipid levels, especially on phosphatidylethanolamine (PE), which is highly enriched within the replication compartment [ 43 ]. PE is required for TBSV replication in an artificial vesicle- (liposome)-based in vitro assay [ 43 ] and it enhances the activation of the p92 RdRp in vitro [ 62 ]. Moreover, the PE level is increased during TBSV replication in yeast and plant cells and high PE level in yeast via modulation of phospholipid biogenesis genes also leads to enhanced TBSV replication [ 43 , 44 ].…”

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

“…The elevated PC has been proposed to serve as a building block for the formations of the viral replication factory [68,69]. Recently, it has been shown that PE and PC stimulate TBSV RdRP activity in vitro [70]. RCNMV p27 showed affinity, not only for PA but also for other phospholipids such as PI4P in vitro.…”

Lipid Tales of Viral Replication and Transmission

“…First, by being on a membrane, viral protein movement becomes restricted to a two dimensional plane, thus potentially saving time for proteins to find each other and form a complex, a significant advantage to getting replication going in the early stages of infection when viral proteins are a minority in the host cell [5]. Secondly, docking on membrane lipids can modulate enzymatic activity [6,7,8]. Lastly the architecture of the membranes can facilitate replication by generating pockets in which to concentrate viral machinery and protect from host innate immune defenses [2,9-11].…”

“…Many plant as well as some insect-vectored animal (+) ssRNA viruses appear to be dependent on phosphatidylethanolamine (PE) enriched membranes for replication and depletion of PE significantly inhibits viral RNA synthesis [6,8, 46,47]. The plant viruses TBSV, Cymbidium ring spot virus and Cucumber Necrosis Tombus virus all generate their PE enriched replication organelles from primarily peroxisome membranes while Carnation Ring Spot virus and the insect-vectored animal viruses Nodamura and flock house exploit the mitochondrial outer membrane for this purpose [6, 47].…”

“…As RNA priming is a pre-requirement for RNA elongation, docking 3CD pol proteins on PI4P lipids may be a mechanism by which these viruses regulate the rate of proteolytic cleavage (in cis or trans ) and thus ensure that there will always be sufficient levels of primed viral RNA for elongation. Similarly, binding to PE lipids has been shown to directly stimulate the enzymatic activity of TBSV RNA polymerase’ and facilitate their association with viral RNA [8] whereas binding to phosphatidylglycerol molecules can inhibit TBSV polymerases enzymatic activities [47]. …”