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The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y HCPro in heterologous systems and its suppression of silencing activity

AuthorsTena, Fátima ; González, Inmaculada; Doblas, Paula; Rodríguez, César; Sahana, Nandita; Kaur, Harpreet; Tenllado, Francisco ; Praveen, Shelly; Canto, Tomás
Issue DateMay-2013
PublisherJohn Wiley & Sons
CitationMolecular Plant Pathology (2013) 14(5), 530–541
AbstractIn the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by HCPro. In silencing suppression patch assays we found that Potato virus Y (PVY) HCPro expressed from a P1-HCPro sequence increased the accumulation of a reporter gene, whereas protein expressed from an HCPro sequence did not, even with P1 supplied in trans. This enhancing effect of P1 had been noticed in other potyviruses, but remained unexplained. We analyzed the accumulation of PVY HCPro in infiltrated tissue and found it higher when expressed from P1-HCPro than from HCPro sequences. Co-expression of heterologous suppressors increased the steady-state level of mRNA expressed from the HCPro sequence, but not that of protein. This suggested that in the absence of P1 upstream, either HCPro acquired a conformation that affected negatively its activity or stability, or that its translation was reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no differences in purification patterns and final soluble states. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favor translation increased partially both suppressor accumulation and activity. Furthermore, it was as active as protein expressed from P1-HCPro sequences. Thus, a direct role for P1 on HCPro suppressor activity or stability by influencing its conformation during translation can be excluded. However, P1 could still have an indirect effect favoring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro.
Description34 p.
Publisher version (URL)http://dx.doi.org/10.1111/mpp.12025
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