caa a22 4500 475765486 CHVBK 20180406123604.0 cr unu---uuuuu 170329e20000101xx s 000 0 eng 10.1007/s007050050003 doi (NATIONALLICENCE)springer-10.1007/s007050050003 Characterisation of genetically modified cucumber mosaic virus expressing histidine-tagged 1a and 2a proteins [Elektronische Daten] [A. Gal-On, T. Canto, P. Palukaitis] Summary.:  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 × 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. 2000 Springer-Verlag/, Wien Gal-On A. Department of Virology, Institute of Plant Protection, The Volcani Center, Bet Dagan, Israel, IL aut Canto T. Department of Plant Pathology, Cornell University, Ithaca, New York, U.S.A., US aut Palukaitis P. Department of Plant Pathology, Cornell University, Ithaca, New York, U.S.A., US aut https://doi.org/10.1007/s007050050003 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s007050050003 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Gal-On A. Department of Virology, Institute of Plant Protection, The Volcani Center, Bet Dagan, Israel, IL aut NATIONALLICENCE 700 1- Canto T. Department of Plant Pathology, Cornell University, Ithaca, New York, U.S.A., US aut NATIONALLICENCE 700 1- Palukaitis P. Department of Plant Pathology, Cornell University, Ithaca, New York, U.S.A., US aut Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer