Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23

[EN] Key messageCitrus tristeza virus encodes a unique protein, p23, with multiple functional roles that include co-option of the cytoplasmic glyceraldehyde 3-phosphate dehydrogenase to facilitate the viral infectious cycle.AbstractThe genome of citrus tristeza virus (CTV), genus Closterovirus family Closteroviridae, is a single-stranded (+) RNA potentially encoding at least 17 proteins. One (p23), an RNA-binding protein of 209 amino acids with a putative Zn-finger and some basic motifs, displays singular features: (i) it has no homologues in other closteroviruses, (ii) it accumulates mainly in the nucleolus and Cajal bodies, and in plasmodesmata, and (iii) it mediates asymmetric accumulation of CTV RNA strands, intracellular suppression of RNA silencing, induction of some CTV syndromes and enhancement of systemic infection when expressed as a transgene ectopically or in phloem-associated cells in several Citrus spp. Here, a yeast two-hybrid screening of an expression library of Nicotiana benthamiana (a symptomatic experimental host for CTV), identified a transducin/WD40 domain protein and the cytosolic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as potential host interactors with p23. Bimolecular fluorescence complementation corroborated the p23-GAPDH interaction in planta and showed that p23 interacts with itself in the nucleolus, Cajal bodies and plasmodesmata, and with GAPDH in the cytoplasm (forming aggregates) and in plasmodesmata. The latter interaction was preserved in a p23 deletion mutant affecting the C-terminal domain, but not in two others affecting the Zn-finger and one internal basic motif. Virus-induced gene silencing of GAPDH mRNA resulted in a decrease of CTV titer as revealed by real-time RT-quantitative PCR and RNA gel-blot hybridization. Thus, like other viruses, CTV seems to co-opt GAPDH, via interaction with p23, to facilitate its infectious cycle.This research was supported by a Grant (Prometeo/2008/121) from the Generalitat Valenciana, Spain, and by a Grant (AGL2009-08052) from the Ministerio de Ciencia e Innovación-Fondo Europeo de Desarrollo Regional. S. Ruiz-Ruiz has been additionally supported by a postdoctoral contract from the Generalitat Valenciana (APOSTD/2012/020, Program VALi+d).Ruiz Ruiz, S.; Spano, R.; Navarro, L.; Moreno, P.; Peña, L.; Flores Pedauye, R. (2018). Citrus tristeza virus co-opts glyceraldehyde 3-phosphate dehydrogenase for its infectious cycle by interacting with the viral-encoded protein p23. Plant Molecular Biology. 98(4-5):363-373. https://doi.org/10.1007/s11103-018-0783-0363373984-5Albiach-Martí MR, Mawassi M, Gowda S, Satanarayana T, Hilf ME, Shanker S, Almira EC, Vives MC, López C, Guerri J, Flores R, Moren P, Garnsey SM, Dawson WO (2000) Sequences of citrus tristeza virus separated in time and space are essentially identical. J Virol 74:6856–6865Ambrós S, El-Mohtar C, Ruiz-Ruiz S, Peña L, Guerri J, Dawson WO, Moren P (2011) Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana. Mol Plant Microb Interact 24:1119–1131Bar-Joseph M, Marcus R, Lee RF (1989) The continuous challenge of citrus tristeza virus control. Annu Rev Phytopathol 27:291–316Bartel PL, Chien C-T, Sternglanz R, Fields S (1993) Using the two-hybrid system to detect protein-protein interactions. In: Hartley DA (ed) Cellular interactions in development: a practical approach. Oxford University Press, Oxford, pp 153–179Bewick V, Cheek L, Ball J (2004) Statistics review 9: one-way analysis of variance. Crit Care 8:130–136Chambers TJ, Hahn CS, Galler R, Rice CM (1990) Flavivirus genome organization, expression, and replication. Annu Rev Microbiol 44:649–688Comellas M (2009) Estudio de la interacción entre naranjo amargo y el virus de la tristeza de los cítricos. Tesis doctoral. Universidad Politécnica de Valencia, Valenciaden Boon JA, Ahlquist P (2010) Organelle-like membrane compartmentalization of positive-strand RNA virus replication factories. Annu Rev Microbiol 64:241–256Dinesh-Kumar SP, Anandalakshmi R, Marathe R, Schiff M, Liu Y (2003) Virus-induced gene silencing. Methods Mol Biol 236:287–294Dollenmaier G, Weitz M (2003) Interaction of glyceraldehyde-3-phosphate dehydrogenase with secondary and tertiary RNA structural elements of the hepatitis A virus 3′ translated and non-translated regions. J Gen Virol 84:403–414Fagoaga C, López C, Moreno P, Navarro L, Flores R, Peña L (2005) Viral-like symptoms induced by the ectopic expression of the p23 of Citrus tristeza virus are citrus specific and do not correlate with the pathogenicity of the virus strain. Mol Plant-Microb Interact 18:435–445Fagoaga C, Pensabene G, Moreno P, Navarro L, Flores R, Peña L (2011) Ectopic expression of the p23 silencing suppressor of Citrus tristeza virus differentially modifies viral accumulation and tropism in two transgenic woody hosts. Mol Plant Pathol 12:898–910Flores R, Ruiz-Ruiz S, Soler N, Sánchez-Navarro J, Fagoaga C, López C, Navarro L, Moreno P, Peña L (2013) Citrus tristeza virus p23: a unique protein mediating key virus–host interactions. Front Microbiol 4:98French R, Ahlquist P (1987) Intercistronic as well as terminal sequences are required for efficient amplification of brome mosaic virus RNA3. J Virol 61:1457–1465Fromont-Racine M, Rain JC, Legrain P (1997) Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens. Nat Genet 16:277–282García JA, Pallás V (2015) Viral factors involved in plant pathogenesis. Curr Opin Virol 11:21–30Ghorbel R, López C, Fagoaga C, Moreno P, Navarro L, Flores R, Peña L (2001) Transgenic citrus plants expressing the Citrus tristeza virus p23 protein exhibit viral-like symptoms. Mol Plant Pathol 2:27–36Gowda S, Satyanarayana T, Davis CL, Navas-Castillo J, Albiach-Martí MR, Mawassi M, Valkov N, Bar-Joseph M, Moreno P, Dawson WO (2000) The p20 gene product of Citrus tristeza virus accumulates in the amorphous inclusion bodies. Virology 274:246–254Harper SJ (2013) Citrus tristeza virus: evolution of complex and varied genotypic groups. Front Microbiol 4:93Heinlein M (2015) Plant virus replication and movement. Virology 479–480:657–671Hilf ME, Karasev AV, Pappu HR, Gumpf DJ, Niblett CL, Garnsey SM (1995) Characterization of citrus tristeza virus subgenomic RNAs in infected tissue. Virology 208:576–582Huang TS, Nagy PD (2011) 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. J Virol 85:9090–9102Jenkins JL, Tanner JJ (2006) High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase. Acta Crystallogr D Biol Crystallogr 62:290–301Kaido M, Abe K, Mine A, Hyodo K, Taniguchi T, Taniguchi H, Mise K, Okuno T (2014) GAPDH-A recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement. PLoS Pathog 10:e1004505Kang SH, Dao TNM, Kim OK, Folimonova SY (2017) Self-interaction of Citrus tristeza virus p33 protein via N-terminal helix. Virus Res 233:29–34Karasev AV, Boyko VP, Gowda S, Nikolaeva O, Hilf ME, Koonin EV, Niblett CL, Cline K, Gumpf DJ, Lee RF, Garnsey SM, Lewandowski DJ, Dawson WO (1995) Complete sequence of the Citrus tristeza virus RNA genome. Virology 208:511–520Lai MMC, Cavanagh D (1997) The molecular biology of coronaviruses. Adv Virus Res 48:1–100López C, Navas-Castillo J, Gowda S, Moreno P, Flores R (2000) The 23 kDa protein coded by the 3′-terminal gene of Citrus tristeza virus is an RNA-binding protein. Virology 269:462–470Lu R, Folimonov A, Shintaku M, Li WX, Falk BW, Dawson WO, Ding SW (2004) Three distinct suppressors of RNA silencing encoded by a 20-kb viral RNA genome. Proc Natl Acad Sci USA 101:15742–15747Marsh LE, Huntley CC, Pogue GP, Connell JP, Hall TC (1991) Regulation of (+):(−) strand asymmetry in replication of brome mosaic virus RNA. Virology 182:76–83Mawassi M, Mietkiewska E, Gofman R, Yang G, Bar-Joseph M (1996) Unusual sequence relationships between two isolates of Citrus tristeza virus. J Gen Virol 77:2359–2364Moreno P, Ambrós S, Albiach-Martí MR, Guerri J, Peña L (2008) Citrus tristeza virus: a pathogen that changed the course of the citrus industry. Mol Plant Pathol 9:251–268Nassuth A, Bol JF (1983) Altered balance of the synthesis of plus- and minus-strand RNAs induced by RNAs 1 and 2 of alfalfa mosaic virus in the absence of RNA3. Virology 124:75–85Petrik J, Parker H, Alexander G (1999) Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA. J Gen Virol 80:3109–3113Pogany J, Nagy PD (2008) Authentic replication and recombination of tomato bushy stunt virus RNA in a cell-free extract from yeast. J Virol 82:5967–5980Prasanth KR, Huang YW, Liou MR, Wang RY, Hu CC, Tsai CH, Meng M, Lin NS, Hsu YH (2011) Glyceraldehyde 3-phosphate dehydrogenase negatively regulates the replication of bamboo mosaic virus and its associated satellite RNA. J Virol 85:8829–8840Ruiz-Ruiz S, Moreno P, Guerri J, Ambrós S (2007) A real-time RT-PCR assay for detection and absolute quantitation of citrus tristeza virus in different plant tissues. J Virol Methods 145:96–105Ruiz-Ruiz S, Soler N, Sánchez-Navarro J, Fagoaga C, López C, Navarro L, Moreno P, Peña L, Flores R (2013) Citrus tristeza virus p23: determinants for nucleolar localization and their influence on suppression of RNA silencing and pathogenesis. Mol Plant Microb Interact 26:306–318Sambade A, López C, Rubio L, Flores R, Guerri J, Moreno P (2003) Polymorphism of a specific region in gene p23 of Citrus tristeza virus allows discrimination between mild and severe isolates. Arch Virol 148:2325–2340Satyanarayana T, Gowda S, Boyko VP, Albiach-Martí MR, Mawassi M, Navas-Castillo J, Karasev AV, Dolja V, Hilf ME, Lewandowski DJ, Moreno P, Bar-Joseph M, Garnsey SM, Dawson WO (1999) An engineered closterovirus RNA replicon and analysis of heterologous terminal sequences for replication. Proc Natl Acad Sci USA 96:7433–7438Satyanarayana T, Gowda S, Ayllón MA, Albiach-Martí MR, Rabindram R, Dawson WO (2002) The p23 protein of Citrus tristeza virus controls asymmetrical RNA accumulation. J Virol 76:473–483Soler N, Fagoaga C, López C, Moreno P, Navarro L, Flores R, Peña L (2014) Symptoms induced by transgenic expression of p23 from Citrus tristeza virus in phloem-associated cells of Mexican lime mimic virus infection without the aberrations accompanying constitutive expression. Mol Plant Pathol 16:388–399Vives MC, Rubio L, López C, Navas-Castillo J, Albiach-Martí MR, Dawson WO, Guerri J, Flores R, Moreno P (1999) The complete genome sequence of the major component of a mild Citrus tristeza virus isolate. J Gen Virol 80:811–816Vojtek AB, Hollenberg SM (1995) Ras-Raf interaction: two-hybrid analysis. Methods Enzymol 255:331–342Wang RY, Nagy PD (2008) Tomato bushy stunt virus coopts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis. Cell Host Microb 3:178–187White MR, Garcin ED (2016) The sweet side of RNA regulation: glyceraldehyde-3-phosphate dehydrogenase as a noncanonical RNA-binding protein. Wiley Interdisc Rev RNA 7:53–70Yang ZN, Mathews DH, Dodds JA, Mirkov TE (1999) Molecular characterization of an isolate of Citrus tristeza virus that causes severe symptoms in sweet orange. Virus Genes 19:131–142Yang SH, Liu M, Tien CF, Chou SJ, Chang RY (2009) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) interaction with 3′ ends of Japanese encephalitis virus RNA and colocalization with the viral NS5 protein. J Biomed Sci 16:1–10Yi M, Schultz DE, Lemon SM (2000) Functional significance of the interaction of hepatitis A virus RNA with glyceraldehyde 3-phosphate dehydrogenase (GAPDH): opposing effects of GAPDH and polypyrimidine tract binding protein on internal ribosome entry site function. J Virol 74:6459–646