Glyceraldehyde 3-phosphate dehydrogenase is co-opted for replication of citrus tristeza virus via interaction with the viral-encoded protein p23.

Citrus tristeza virus (CTV), genus Closterovirus, family Closteroviridae, is an important pathogen that has killed more than 50 million citrus trees in Spain. CTV genome is a positive-sense RNA of approximately 19.3 kb organized in 12 open reading frames (ORFs) potentially coding for at least 17 proteins. One of them, p23, encoded by the 3’-terminal ORF, has no homologues in other closteroviruses and, therefore, is distinctive. CTV-p23 has also peculiar features: i) it is an RNA-binding protein of 209 amino acids with a putative Zn-finger domain and some basic motifs, ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and iii) it mediates many functions including the asymmetric accumulation of CTV RNA strands, the intracellular suppression of RNA silencing, and the induction of some CTV syndromes when expressed from the virus, and of CTV-like symptoms and enhancement of systemic infection when expressed ectopically as a transgene in several Citrus spp. To search for host interactors of p23, an initial yeast two-hybrid (Y2H) screening of an expression library of Nicotiana benthamiana (in which at least one CTV isolate replicates and incites symptoms) led to the identification of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase (GAPDH), further confirmed in 1-by-1 Y2H tests. Bimolecular fluorescence complementation assays in planta corroborated the previous results and provided new insights. Briefly, p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH (in the cytoplasm forming aggregates) and in plasmodesmata. This latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two other deletion mutants affecting the Zn-finger domain and one internal basic motif. Most importantly, qRT-PCR and RNA gel-blot hybridization showed that virus-induced gene silencing of GAPDH mRNA resulted in a significant decrease in CTV titer. Altogether these data suggest that, paralleling the situation observed in a tombusvirus (Wang and Nagy, Cell Host and Microbe 2008), CTV co-opts GAPDH through p23 to convert the host cell into a viral factory. The finding that two very different viruses co-opt for their replication the same host protein (GAPDH) suggests that this protein is endowed with an intrinsic feature, possibly its RNA-binding ability, that facilitates the process.