title: The influence of cis-acting P1 protein and translational elements on the expression of Potato virus Y helper-component proteinase (HCPro) in heterologous systems and its suppression of silencing activity.
abstract: In the Potyvirus genus, the P1 protein is the first N-terminal product processed from the viral polyprotein, followed by the helper-component proteinase (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 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of PVY HCPro in infiltrated tissues and found that it was 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 suggests that, in the absence of P1 upstream, either HCPro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified HCPro expressed in the presence or absence of upstream P1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the Kozak context in the HCPro mRNA sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in 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 favouring HCPro accumulation. Our data highlight the relevance of cis-acting translational elements in the heterologous expression of HCPro.
authors: Environmental Biology Department, Centro de Investigaciones Biológicas, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors: Fátima
authors: F
authors: Tena Fernández
authors: Inmaculada
authors: I
authors: González
authors: Paula
authors: P
authors: Doblas
authors: César
authors: C
authors: Rodríguez
authors: Nandita
authors: N
authors: Sahana
authors: Harpreet
authors: H
authors: Kaur
authors: Francisco
authors: F
authors: Tenllado
authors: Shelly
authors: S
authors: Praveen
authors: Tomas
authors: T
authors: Canto
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020868:Gene Silencing; D016153:Genes, Suppressor; D008969:Molecular Sequence Data; D017800:Potyvirus / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D012995:Solubility; D013489:Suppression, Genetic; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus