Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins.
Biological active cDNA clones of cucumber mosaic virus (CMV) RNAs 1 and 2 were modified by addition of sequences that encode hexahistidine (His-tag) at the amino- (N-) or carboxy- (C-) terminus of the 1a and 2a proteins. These proteins are essential components of the viral RNA-dependent RNA polymerase (RdRp). In all but one case, addition of the His-tag did not significantly affect the yields of the corresponding viruses and the His-tag-encoding sequences were maintained after mechanical passages. No differences were observed among the in vitro activities of the modified vs. wild-type viral RdRps. Subcellular fractionation showed that 2a protein was found both membrane-associated and in the 30,000 x g soluble fraction. Both termini of the native His-tag 2a protein could bind to a resin containing nickel-nitrilotriacetic acid (Ni(2+)-NTA). Detergent-treated RdRp containing C-terminal His-tagged 1a and 2a proteins was chromatographed on Ni(2+)-NTA resin. The activity of the eluted RdRp was template- dependent, in contrast to pre-chromatography fractions. However, only a small proportion of the viral RdRp as well as numerous host proteins bound to and eluted from the resin under non-denaturing conditions.
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Department of Virology, Institute of Plant Protection, The Volcani Center, Bet Dagan, Israel.
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Palukaitis
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D002462:Cell Membrane / Q000378:metabolism / Q000821:virology; D002846:Chromatography, Affinity; D017799:Cucumovirus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005818:Genetic Engineering; D006639:Histidine / Q000235:genetics* / Q000378:metabolism; D010641:Phenotype; D010947:Plants, Toxic; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013347:Subcellular Fractions; D013698:Templates, Genetic; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D014764:Viral Proteins; D006639:Histidine; D012324:RNA-Dependent RNA Polymerase