The influence of cis ‐acting P 1 protein and translational elements on the expression of P otato virus Y helper‐component proteinase ( HCP ro) in heterologous systems and its suppression of silencing activity

• Published in: Molecular plant pathology Vol. 14; no. 5; pp. 530 - 541
• Main Authors: Tena Fernández, Fátima, González, Inmaculada, Doblas, Paula, Rodríguez, César, Sahana, Nandita, Kaur, Harpreet, Tenllado, Francisco, Praveen, Shelly, Canto, Tomas
• Format: Journal Article
• Language: English
• Published: 01-06-2013
• ISSN: 1464-6722; 1364-3703
• DOI: 10.1111/mpp.12025

In the
P
otyvirus
genus, the
P
1 protein is the first
N
‐terminal product processed from the viral polyprotein, followed by the helper‐component proteinase (
HCP
ro). In silencing suppression patch assays, we found that
P
otato virus
Y
(
PVY
)
HCP
ro expressed from a
P
1‐
HCP
ro
sequence increased the accumulation of a reporter gene, whereas protein expressed from an
HCP
ro
sequence did not, even with
P
1 supplied
in trans
. This enhancing effect of
P
1 has been noted in other potyviruses, but has remained unexplained. We analysed the accumulation of
PVY HCP
ro in infiltrated tissues and found that it was higher when expressed from
P
1‐
HCP
ro
than from
HCP
ro
sequences. Co‐expression of heterologous suppressors increased the steady‐state level of
mRNA
expressed from the
HCP
ro
sequence, but not that of protein. This suggests that, in the absence of
P
1 upstream, either
HCP
ro acquires a conformation that affects negatively its activity or stability, or that its translation is reduced. To test these options, we purified
HCP
ro expressed in the presence or absence of upstream
P
1, and found no difference in purification pattern and final soluble state. By contrast, alteration of the
K
ozak context in the
HCP
ro

mRNA
sequence to favour translation increased partially suppressor accumulation and activity. Furthermore, protein activity was not lower than in protein expressed from
P
1‐
HCP
ro
sequences. Thus, a direct role for
P
1 on
HCP
ro suppressor activity or stability, by influencing its conformation during translation, can be excluded. However,
P
1 could still have an indirect effect favouring
HCP
ro accumulation. Our data highlight the relevance of
cis
‐acting translational elements in the heterologous expression of
HCP
ro.