Proteomics analysis of the tombusvirus replicase: Hsp70 molecular chaperone is associated with the replicase and enhances viral RNA replication.
Plus-strand RNA virus replication occurs via the assembly of viral replicase complexes involving multiple viral and host proteins. To identify host proteins present in the cucumber necrosis tombusvirus (CNV) replicase, we affinity purified functional viral replicase complexes from yeast. Mass spectrometry analysis of proteins resolved by two-dimensional gel electrophoresis revealed the presence of CNV p33 and p92 replicase proteins as well as four major host proteins in the CNV replicase. The host proteins included the Ssa1/2p molecular chaperones (yeast homologues of Hsp70 proteins), Tdh2/3p (glyceraldehyde-3-phosphate dehydrogenase, an RNA-binding protein), Pdc1p (pyruvate decarboxylase), and an unknown approximately 35-kDa acidic protein. Copurification experiments demonstrated that Ssa1p bound to p33 replication protein in vivo, and surface plasmon resonance measurements with purified recombinant proteins confirmed this interaction in vitro. The double mutant strain (ssa1 ssa2) showed 75% reduction in viral RNA accumulation, whereas overexpression of either Ssa1p or Ssa2p stimulated viral RNA replication by approximately threefold. The activity of the purified CNV replicase correlated with viral RNA replication in the above-mentioned ssa1 ssa2 mutant and in the Ssa overexpression strains, suggesting that Ssa1/2p likely plays an important role in the assembly of the CNV replicase.
Serva
Saulius
S
Department of Plant Pathology, University of Kentucky, Lexington, 40546-0312, USA.
Nagy
Peter D
PD
D000251:Adenosine Triphosphatases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D005656:Fungal Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D040901:Proteomics; D011767:Pyruvate Decarboxylase / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D020349:Surface Plasmon Resonance; D017864:Tombusvirus / Q000201:enzymology / Q000502:physiology*
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D012367:RNA, Viral; C542288:SSA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); C465225:TDH2 protein, S cerevisiae; C465226:TDH3 protein, S cerevisiae; D012324:RNA-Dependent RNA Polymerase; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae; C101504:PDC1 protein, S cerevisiae; D011767:Pyruvate Decarboxylase