Direct Inhibition of Tombusvirus Plus-Strand RNA Synthesis by a Dominant Negative Mutant of a Host Metabolic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, in Yeast and Plants

Abstract

The replication of plus-strand RNA viruses depends on many cellular factors. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant metabolic enzyme that is recruited to the replicase complex of Tomato bushy stunt virus (TBSV) and affects asymmetric viral RNA synthesis. To further our understanding on the role of GAPDH in TBSV replication, we used an in vitro TBSV replication assay based on recombinant p33 and p92 pol viral replication proteins and cell-free yeast extract. We found that the addition of purified recombinant GAPDH to the cell extract prepared from GAPDH-depleted yeast results in increased plus-strand RNA synthesis and asymmetric production of viral RNAs. Our data also demonstrate that GAPDH interacts with p92 pol viral replication protein, which may facilitate the recruitment of GAPDH into the viral replicase complex in the yeast model host. In addition, we have identified a dominant negative mutant of GAPDH, which inhibits RNA synthesis and RNA recruitment in vitro. Moreover, this mutant also exhibits strong suppression of tombusvirus accumulation in yeast and in virus-infected Nicotiana benthamiana. Overall, the obtained data support the model that the co-opted GAPDH plays a direct role in TBSV replication by stimulating plus-strand synthesis by the viral replicase.Plus-strand RNA [(ϩ)RNA] viruses replicate in the cytosol of infected cells using membrane-bound replicase complexes. The viral replicase is a multisubunit complex consisting of viral-coded components, including RNA-dependent RNA polymerase (RdRp), one or more viral auxiliary replication proteins, the viral RNA, and co-opted host proteins and membranes (1,19,22,34,35,52,57). However, the functions of many host components of the viral replicase complex are currently unknown.Among the co-opted host proteins for (ϩ)RNA virus replication are the abundant cellular RNA-binding proteins, such as poly(C)-and poly(A)-binding proteins, nucleolin, elongation factor 1A (eEF1A), and the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (15, 22-24, 29, 50, 63). Additional subverted RNA-binding proteins include ribosomal proteins, translation factors, and RNA-modifying enzymes (1,36,39). Although most of these host RNA-binding proteins have been shown to associate with the viral positive-strand RNAs, there is a small number of host proteins that preferentially bind to the (Ϫ)RNA intermediate template in the replicase complex. For example, the T-cell intracellular antigen-1 (TIA-1) and the TIA-1-related protein (TIAR), which are stress granule proteins, that bind specifically to the 3Ј-stem-loop (SL) of West Nile virus (Ϫ) RNA (20). Mutations in the 3Ј-SL, which reduce TIA1/TIAR binding, can greatly decrease genomic RNA amplification, suggesting that TIA1/TIAR facilitate efficient (ϩ)RNA synthesis (12). Similarly, the cellular hnRNP C, which binds specifically to the 3Ј end of the poliovirus (Ϫ)RNA (6, 51), has also been proposed to affect positive-strand synthesis. The hnRNP C may function to maintain the singlestranded form of poliovirus ...

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response toTomato Spotted Wilt VirusInfection

“…G3PDH has been found to be important in the infection process of other plant-and animalinfecting viruses. For example, G3PDH negatively regulates the replication of bamboo mosaic virus and its associated satellite RNA in vitro (57), and G3PDH mutants directly inhibit the accumulation and plus-strand synthesis of tomato bushy stunt virus in yeast and plants (36). It is also known that G3PDH binds to membranes and regulates endocytosis (71) and exocytosis (30).…”

Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

“…The largest decrease in copurification was observed with the cellular ESCRT proteins (i.e., Vps4p and Bro1p) and Cdc34p E2 ubiquitin-conjugating enzyme, all of which are known to affect the assembly of the tombusvirus VRCs (4,47,51). The extent of inhibition of the additional copurified host proteins was also significant, but somewhat less pronounced for (i) Tef1p (eEF1A) translation elongation factor known to affect many viral functions, including the stability of p33, the recruitment of the viral RNA, and the assembly of the VRCs and (Ϫ)RNA synthesis (46,51,53); (ii) Tdh2p (GAPDH) involved in (ϩ)RNA synthesis (62,63); and (iii) Pex19p cytosolic shuttle protein that targets p33 and p92 pol to the peroxisomes (60). Altogether, the inhibition of recruitment of multiple host factors to the VRCs by WW-domain protein suggests a regulatory function, possibly via competition of the WW-domain protein with these cellular proteins for binding to p33 and p92 pol .…”

“…The list includes eEF1A, the eukaryotic elongation factor 1B␥ (eEF1B␥), the DDX3-, DDX5-, and eIF4AIII-like DEAD box helicases, and Tdh2p (GAPDH [glyceraldehyde-3-phosphate dehydrogenase]), all of which facilitate RNA replication (51,53,59,(61)(62)(63).…”

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

“…Additional subverted host proteins in the VRC include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eEF1A, eEF1B␥, and Ded1 and other DEAD box helicases. These cellular proteins have been shown to affect viral RNA synthesis (35)(36)(37)(38)(39)(40)(41)(42)(43). The auxiliary p33 replication protein is an RNA chaperone involved in recruitment of the TBSV (ϩ)RNA to the site of replication, which is the cytosolic surface of peroxisomal membranes (44)(45)(46)(47).…”