Environmental Biology Department, Centro de Investigaciones Biológicas, CIB-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.
Mol Plant Pathol. 2013 Jun;14(5):530-41. doi: 10.1111/mpp.12025. Epub 2013 Mar 3.
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.
在马铃薯 Y 病毒属中，P1 蛋白是病毒多聚蛋白从 N 端切割产生的第一个蛋白，随后是辅助蛋白酶（HCPro）。在沉默抑制补丁分析中，我们发现马铃薯 Y 病毒（PVY）HCPro 由 P1-HCPro 序列表达会增加报告基因的积累，而由 HCPro 序列表达则不会，即使提供了 P1。这种 P1 的增强作用在其他几种 Potyvirus 中也有发现，但尚未得到解释。我们分析了 PVY HCPro 在渗透组织中的积累，发现当它由 P1-HCPro 表达时比由 HCPro 序列表达时更高。异源抑制剂的共表达增加了 HCPro 序列表达的 mRNA 的稳态水平，但不增加蛋白的水平。这表明，在没有上游 P1 的情况下，HCPro 获得了一种影响其活性或稳定性的构象，或者其翻译减少。为了验证这些选择，我们纯化了在存在或不存在上游 P1 的情况下表达的 HCPro，并且在纯化模式和最终可溶性状态上没有发现差异。相比之下，改变 HCPro mRNA 序列中的 Kozak 序列以有利于翻译，部分增加了抑制剂的积累和活性。此外，蛋白活性并不低于由 P1-HCPro 序列表达的蛋白。因此，可以排除 P1 通过影响翻译过程中其构象来直接影响 HCPro 抑制剂活性或稳定性的可能性。然而，P1 仍然可以通过有利于 HCPro 积累的间接作用发挥作用。我们的数据突出了顺式作用翻译元件在 HCPro 异源表达中的重要性。
