Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection.
Association of host protein VARICOSE with HCPro within a multiprotein complex is crucial for RNA silencing suppression, translation, encapsidation and systemic spread of potato virus A infection.
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DOI：
10.1371/journal.ppat.1008956
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发表时间：
2020-10
期刊：
影响因子：
6.7
通讯作者：
Mäkinen K
中科院分区：
文献类型：
--
作者：
De S;Pollari M;Varjosalo M;Mäkinen K
在本研究中，我们研究了马铃薯A病毒(PVA；马铃薯Y病毒属)辅助成分-蛋白水解酶(HCPro)C-末端一个保守的5个氨基酸基序‘AELPR’在马铃薯A病毒(PVA)侵染中的意义。这个基序可能是包含WD40结构域的蛋白质的相互作用位点，包括VCS。我们通过用丙氨酸(A)取代谷氨酸(E)和精氨酸(R)来产生HCProWD，从而取消了HCPro中的相互作用位点。这些突变部分消除了HCPro-VCS在细胞中的共定位。我们早些时候已经描述了马铃薯Y病毒诱导的RNA颗粒(PG)，其中HCPro和VCS共同定位，并提出它们在RNA沉默抑制中具有作用。我们现在证明，HCProWD诱导PG，将VCS引入PG，以及抑制RNA沉默的能力受到了损害。因此，携带HCProWD的PVA(PVAWD)对烟草的侵染效率低于野生型PVA(PVAWT)，并且HCProWD仅部分弥补了PVA基因表达的缺失。HCPro是从PVA侵染的叶片中纯化出来的，是高分子量核糖核蛋白(RNP)复合体的一部分。与野生型HCPro结合时，这些复合体比与HCProWD结合时更稳定。此外，在含有HCProWD的HMW-复合体中，VCS和HMW-复合体中的两个病毒组分--病毒蛋白基因组连接蛋白和柱状包涵体蛋白被特异性地降低。只有当病毒表达野生型Δ时，才能观察到VPG介导的对复制缺陷的PVA(PVAHCPro GDD)翻译的促进作用。VCS沉默和过度表达实验的结果进一步支持了VCS-VPG-HCPro协调在PVA翻译中的作用，并在VPG-VCS共表达时显著提高了PVA衍生的Renilla荧光素酶与PVA RNA的比率。最后，我们发现PVAWD不能在受感染的植物中形成病毒颗粒或系统传播。我们强调了含有与PVA RNA相关的多蛋白组件的HCPro-VCS在保护其免受降解、确保有效翻译、形成稳定的病毒粒子和建立全身感染方面的作用。本研究揭示了马铃薯Y病毒辅助成分蛋白水解酶(HCPro)和宿主蛋白VCS在抑制RNA沉默、形成马铃薯Y病毒诱导的RNA颗粒、病毒蛋白的翻译、病毒粒子的稳定性以及系统感染马铃薯A病毒(PVA)的发展中起重要作用。这些结果表明，HCPro和VCS是调控PVA感染的大型RNP复合体的核心成分。我们认为，这些复合体从复制复合体中释放出来后，保护细胞质中的病毒RNA，并将它们引导到翻译和完整的病毒颗粒中。
In this study, we investigated the significance of a conserved five-amino acid motif ‘AELPR’ in the C-terminal region of helper component–proteinase (HCPro) for potato virus A (PVA; genus Potyvirus) infection. This motif is a putative interaction site for WD40 domain-containing proteins, including VARICOSE (VCS). We abolished the interaction site in HCPro by replacing glutamic acid (E) and arginine (R) with alanines (A) to generate HCProWD. These mutations partially eliminated HCPro-VCS co-localization in cells. We have earlier described potyvirus-induced RNA granules (PGs) in which HCPro and VCS co-localize and proposed that they have a role in RNA silencing suppression. We now demonstrate that the ability of HCProWD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCProWD (PVAWD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVAWT) and HCProWD complemented the lack of HCPro in PVA gene expression only partially. HCPro was purified from PVA-infected leaves as part of high molecular weight (HMW) ribonucleoprotein (RNP) complexes. These complexes were more stable when associated with wild-type HCPro than with HCProWD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCProWD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVAΔGDD) was observed only if viral RNA expressed wild-type HCPro. The role of VCS-VPg-HCPro coordination in PVA translation was further supported by results from VCS silencing and overexpression experiments and by significantly elevated PVA-derived Renilla luciferase vs PVA RNA ratio upon VPg-VCS co-expression. Finally, we found that PVAWD was unable to form virus particles or to spread systemically in the infected plant. We highlight the role of HCPro-VCS containing multiprotein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection. This study revealed that the potyviral helper component proteinase (HCPro) and the host protein VARICOSE (VCS) are linked in a manner that is important for suppression of RNA silencing, formation of potyvirus-induced RNA granules, translation of viral proteins, stability of virions, and development of systemic potato virus A (PVA) infection. The results suggest that HCPro and VCS belong to the core components of large RNP complexes regulating PVA infection. We suggest that these complexes protect viral RNAs in the cytoplasm after release from the replication complex and direct them to translation and intact to the viral particles.
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