Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2.

L Hibrand,J Dunez,O Le Gall,T Candresse

doi:10.1099/0022-1317-73-8-2093

L Hibrand, J Dunez + Show 2 more

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https://doi.org/10.1099/0022-1317-73-8-2093

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Abstract

Fragments of the putative non-structural proteins (44K and 46K) encoded by RNA2 of grapevine chrome mosaic nepovirus (GCMV) were expressed as fusion proteins in Escherichia coli and used to raise specific antisera. All three proteins encoded by GCMV RNA2 (viral coat protein, and the 44K and 46K proteins) were detected by immunoblotting in subcellular fractions prepared from the leaves of infected Chenopodium quinoa plants, confirming a previously proposed model of the GCMV RNA2-encoded polyprotein. In addition to the 44K protein, one of the antisera detected a 90K protein presumably representing a precursor of the 44K and 46K proteins. Whereas the 44K and coat proteins could be detected in both soluble and membrane fractions, the 46K protein was found to be specific to the membrane fraction. Analysis of the kinetics of accumulation of the proteins showed that the 44K and 46K proteins were very transient whereas the coat protein was more stable and could be detected up to 21 days after inoculation. These results provide the first direct in vivo data supporting the maturation map of the GCMV RNA2 polyprotein deduced from in vitro experiments.

Highlights

The genome of Hungarian grapevine chrome mosaic nepovirus (GCMV) (Martelli & Quacquarelli, 1972) consists of two positive-sense ssRNAs of 7212 nucleotides (RNA1 ; Le Gall et al, 1989) and 4441 nucleotides (RNA2; Brault et al, 1989)
Two fragments of the polyprotein encoded by GCMV RNA2 were expressed in E. coli as fusions with the cro-flgalactosidase hybrid protein encoded by vector pEX2
The model derived by Demangeat et al (1991) from in vitro translation/maturation experiments predicts that the polyprotein matures in two steps: (i) cleavage of the C-terminal coat protein leaving a 90K precursor and (ii) cleavage of the 90K precursor to yield two mature products, an N-terminal 44K protein and an internal 46K protein

Summary

Introduction

The genome of Hungarian grapevine chrome mosaic nepovirus (GCMV) (Martelli & Quacquarelli, 1972) consists of two positive-sense ssRNAs of 7212 nucleotides (RNA1 ; Le Gall et al, 1989) and 4441 nucleotides (RNA2; Brault et al, 1989). These RNAs are polyadenylated at their 3' ends and, to those of the closely related tomato black ring nepovirus (TBRV) (Mayo et al, 1982), are probably covalently linked to a small protein (VPg) at their 5' end. The N-terminal sequence of the GCMV coat protein has been determined (Brault et al, 1989), demonstrating that this protein maps to the C terminus of the 146K polyprotein encoded by RNA2

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