Mapping and quantification of potato virus A RNA genomes within viral particles and polysomes in infected plant cells

Abstract

Potato virus A belongs to the genus Potyvirus, a group of single-stranded positive sense RNA viruses infecting crops worldwide. To initiate infection in a host, its genome takes part in different activities, viz., translation, replication, encapsidation during the infection cycle. Extensive research has been carried out to scrutinize the stages of potyviral infection cycle and decipher the strategies it employs to cause disease. Nonetheless, the amount of viral RNA taking part in translation and virion formation, at a given time point, is missing. In this study, we quantified the percentage of viral RNA that exists as virions and those that associates with host polysome, relative to total viral RNA in infected plant tissue. We employed a revised version of immuno-capture reverse transcription PCR and polysome profiling to address our queries. We tested three different coating antibody concentrations and further optimized the immuno-capture reverse transcription PCR protocol to address its limitation of binding and retaining viral particles. Our results indicate that most of the viral RNA (69 %) exists as encapsidated genomes, while 3 % of total viral RNA associates with host polysomes. These findings are crucial for correct interpretation of quantitative translational studies in which correlation must be made between the number of polysome-associated transcripts and the amount of protein synthesized.