title: Molecular insights into the function of the viral RNA silencing suppressor HCPro.
abstract: Potyviral helper component proteinase (HCPro) is a well-characterized suppressor of antiviral RNA silencing, but its mechanism of action is not yet fully understood. In this study, we used affinity purification coupled with mass spectrometry to identify binding partners of HCPro in potyvirus-infected plant cells. This approach led to identification of various HCPro interactors, including two key enzymes of the methionine cycle, S-adenosyl-L-methionine synthase and S-adenosyl-L-homocysteine hydrolase. This finding, together with the results of enzymatic activity and gene knockdown experiments, suggests a mechanism in which HCPro complexes containing viral and host proteins act to suppress antiviral RNA silencing through local disruption of the methionine cycle. Another group of HCPro interactors identified in this study comprised ribosomal proteins. Immunoaffinity purification of ribosomes demonstrated that HCPro is associated with ribosomes in virus-infected cells. Furthermore, we show that HCPro and ARGONAUTE1 (AGO1), the core component of the RNA-induced silencing complex (RISC), interact with each other and are both associated with ribosomes in planta. These results, together with the fact that AGO1 association with ribosomes is a hallmark of RISC-mediated translational repression, suggest a second mechanism of HCPro action, whereby ribosome-associated multiprotein complexes containing HCPro relieve viral RNA translational repression through interaction with AGO1.
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Konstantin I
authors.initials: KI
authors.lastname: Ivanov
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Katri
authors.initials: K
authors.lastname: Eskelin
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Marta
authors.initials: M
authors.lastname: Bašić
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Swarnalok
authors.initials: S
authors.lastname: De
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Andres
authors.initials: A
authors.lastname: Lõhmus
authors.affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Markku
authors.initials: M
authors.lastname: Varjosalo
authors.affiliation: Department of Food and Environmental Sciences, Viikki Plant Science Centre, University of Helsinki, Helsinki, 00014, Finland.
authors.forename: Kristiina
authors.initials: K
authors.lastname: Mäkinen
mesh_terms: D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D013058:Mass Spectrometry; D008715:Methionine / Q000378:metabolism; D010935:Plant Diseases / Q000821:virology*; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000235:genetics* / Q000378:metabolism; D046188:Protein Modification, Translational; D034622:RNA Interference; D012367:RNA, Viral / Q000235:genetics; D034743:RNA-Induced Silencing Complex / Q000235:genetics / Q000378:metabolism*; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D034743:RNA-Induced Silencing Complex; D012269:Ribosomal Proteins; D014764:Viral Proteins; D008715:Methionine; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus
keywords: Argonaute; HCPro; methionine cycle; post-transcriptional gene silencing; potyvirus; silencing suppressor