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 w ere modified by addition of sequences that encode hexahistidine (His-tag) a t the amino- (N-) or carboxy- (C-) terminus of the la and 2a proteins. Thes e proteins are essential components of the viral RNA-dependent RNA polymera se (RdRp). In all but one case, addition of the His-tag did not significant ly affect the yields of the corresponding viruses and the His-tag-encoding sequences were maintained after mechanical passages. No differences were ob served among the in vitro activities of the modified vs. wild-type viral Rd Rps. Subcellular fractionation showed that 2a protein was found both membra ne-associated and in the 30,000 x g soluble fraction. Both termini of the n ative His-tag 2a protein could bind to a resin containing nickel-nitrilotri acetic acid (Ni2+-NTA). Detergent-treated RdRp containing C-terminal His-ta gged 1a and 2a proteins was chromatographed on Ni2+-NTA resin. The activity of the eluted RdRp was template-dependent, in contrast to pre-chromatograp hy 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-denatu ring conditions.