Translation of potyvirus RNA in a rabbit reticulocyte lysate: Cell-free translation strategy and a genetic map of the potyviral genome

Abstract

The translations of tobacco etch virus (TEV) RNA and pepper mottle virus (PeMV) in a rabbit reticulocyte lysate were analyzed to determine the effect of RNA quality on template activity and to identify all the products of the potyviral genome. The size distribution of the in vitro translation products, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was dependent on RNA quality. Full-length RNA stimulated the cell-free synthesis primarily of a 87,000 (87K) molecular weight product for TEV and a 78K product for PeMV. RNA with limited fragmentation stimulated a number of discrete products, while highly fragmented RNA did not act as a template. RNA monocistronic for capsid protein was not detected. Four of the six discrete translation products were identified by reactivity with antisera to four virus-specific proteins. A number of other products, which were consistently immunoprecipitated with more than one antiserum, were detected. Products were presumed to be premature terminations and/or gene readthroughs on the basis of serological reactions and of molecular weight estimates. These readthroughs were useful in linking genes and in constructing a genetic map of the potyviral genome. The proposed genetic map for TEV is as follows: 5′ end-87K protein gene-49K nuclear inclusion protein gene-50K protein gene-70K cylindrical inclusion protein gene-54K nuclear inclusion protein gene-30K capsid protein gene-3′ end. The proposed genetic map for PeMV is as follows: 5′ end-78K protein gene-49K protein gene-41K protein gene-68K cylindrical inclusion protein gene-56K protein gene-33K capsid protein gene-3′ end. The proposed genetic map accounts for 95% of the estimated coding capacity of the TEV RNA and 93% of the PeMV RNA.