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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.

ABSTRACT: 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 (PVA?GDD) 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.

SUBMITTER: De S

PROVIDER: S-EPMC7581364 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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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.

De Swarnalok S Pollari Maija M Varjosalo Markku M Mäkinen Kristiina K

PLoS pathogens 20201012 10

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-local ...[more]

PMID: 33045020

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Project description:We have investigated short and small RNAs (sRNAs) that were bound to a biologically active hexahistidine-tagged Potato virus Y (PVY) HCPro suppressor of silencing, expressed from a heterologous virus vector in Nicotiana benthamiana plants, and purified under nondenaturing conditions. We found that RNAs in purified preparations were differentially enriched in 21-nucleotide (nt) and, to a much lesser extent, 22-nt sRNAs of viral sequences (viral sRNAs [vsRNAs]) compared to those found in a control plant protein background bound to nickel resin in the absence of HCPro or in a purified HCPro alanine substitution mutant (HCPro mutB) control that lacked suppressor-of-silencing activity. In both controls, sRNAs were composed almost entirely of molecules of plant sequence, indicating that the resin-bound protein background had no affinity for vsRNAs and also that HCPro mutB failed to bind to vsRNAs. Therefore, PVY HCPro suppressor activity correlated with its ability to bind to 21- and 22-nt vsRNAs. HCPro constituted at least 54% of the total protein content in purified preparations, and we were able to calculate its contribution to the 21- and the 22-nt pools of sRNAs present in the purified samples and its binding strength relative to the background. We also found that in the 21-nt vsRNAs of the HCPro preparation, 5'-terminal adenines were overrepresented relative to the controls, but this was not observed in vsRNAs of other sizes or of plant sequences.IMPORTANCE It was previously shown that HCPro can bind to long RNAs and small RNAs (sRNAs) in vitro and, in the case of Turnip mosaic virus HCPro, also in vivo in arabidopsis AGO2-deficient plants. Our data show that PVY HCPro binds in vivo to sRNAs during infection in wild-type Nicotiana benthamiana plants when expressed from a heterologous virus vector. Using a suppression-of-silencing-deficient HCPro mutant that can accumulate in this host when expressed from a virus vector, we also show that sRNA binding correlates with silencing suppression activity. We demonstrate that HCPro binds at least to sRNAs with viral sequences of 21 nucleotides (nt) and, to a much lesser extent, of 22 nt, which were are also differentially enriched in 5'-end adenines relative to the purified controls. Together, our results support the physical binding of HCPro to vsRNAs of 21 and 22 nt as a means to interfere with antiviral silencing.

Dataset Information

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.

Publications

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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