Co-Authors:
Gal-On, A., Department of Virology, Institute of Plant Protection, Volcani Center, Bet Dagan, Israel, Department of Plant Pathology, Cornell University, Ithaca, NY, United States
Canto, T., Department of Plant Pathology, Cornell University, Ithaca, NY, United States, Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom
Palukaitis, P., Department of Plant Pathology, Cornell University, Ithaca, NY, United States, Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee, United Kingdom, Virology Department, Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, United Kingdom
Abstract:
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 (Ni2+-NTA). Detergent-treated RdRp containing C-terminal His-tagged 1a and 2a proteins was chromatographed on Ni2+-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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