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.

A novel sweet potato potyvirus open reading frame (ORF) is expressed via polymerase slippage and suppresses RNA silencing

“…Surprisingly, we found that both P1N‐PISPO and P1 are RNA silencing suppressors in SPFMV. Intrinsic silencing suppression has not been described previously for P1 proteins of potyviruses (Rajamäki et al ., ; Tena Fernández et al ., ), nor was it found herein for P1 of SPLV (which clusters as an immediate outgroup to the SPFMV group). Most recent reports assign potyvirus P1 a role for increasing the accumulation of HCpro and consequently enhancing its silencing activity (Tena Fernández et al ., ).…”

“…Intrinsic silencing suppression has not been described previously for P1 proteins of potyviruses (Rajamäki et al ., ; Tena Fernández et al ., ), nor was it found herein for P1 of SPLV (which clusters as an immediate outgroup to the SPFMV group). Most recent reports assign potyvirus P1 a role for increasing the accumulation of HCpro and consequently enhancing its silencing activity (Tena Fernández et al ., ). We also showed that P1 and P1N‐PISPO seem to have different silencing activities in SPFMV, a strategy described for different RSS proteins of other viruses as well (Lu et al ., ; Senshu et al ., ).…”

The P1N-PISPO trans -Frame Gene of Sweet Potato Feathery Mottle Potyvirus Is Produced during Virus Infection and Functions as an RNA Silencing Suppressor

“…In this regard, for instance, the VPg of the potyvirus PVA has been found to counteract RNA silencing (46,61). Additionally, the levels of expression of HCPro in other potyviruses were reported to be regulated by P1 (62). In the case of SPFMV, although our transient agroinfiltration experiments with individual gene products showed no clear antisilencing activity for P1 or HCPro, preliminary results suggest that P1-HCPro might display some activity.…”

The P25 Protein of Potato Virus X (PVX) Is the Main Pathogenicity Determinant Responsible for Systemic Necrosis in PVX-Associated Synergisms

“…The primer pairs used to amplify all these genes are available upon request. Viral proteins were analyzed by Western blotting as described by Tena et al (33). T7-tagged P25 was detected with horseradish peroxidase (HRP)-conjugated anti-T7 antibody (Novagen) (1:5,000 dilution).…”