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 HCPro WD. 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 HCPro WD to induce PGs, introduce VCS into PGs, and suppress RNA silencing was impaired. Accordingly, PVA carrying HCPro WD (PVA WD) infected Nicotiana benthamiana less efficiently than wild-type PVA (PVA WT) and HCPro WD 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 HCPro WD. Moreover, VCS and two viral components of the HMW-complexes, viral protein genome-linked and cylindrical inclusion protein were specifically decreased in HCPro WD-containing HMW-complexes. A VPg-mediated boost in translation of replication-deficient PVA (PVA ΔGDD) 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 PVA WD 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.

World Society for Virology first international conference: Tackling global virus epidemics

“…Kristiina Mäkinen, Helsinki University, Helsinki, Finland, presented research on potato virus A-host interactions, specifically by providing structural and functional elucidation of interactions related to potato virus A RNA stability as well as viral protein manipulation to neutralize host antiviral proteins. These interactions are crucial for the accumulation of stable virus particles required for systemic infection of host by potato virus A ( De et al, 2020 ).…”

Cauliflower Mosaic Virus Utilizes Processing Bodies to Escape Translational Repression in Arabidopsis

“…NMD can restrict viral accumulation while at the same time being a target of viral manipulation (May et al ., 2020; Garcia et al ., 2014). Several PB proteins were shown to regulate viral infections; Carbon Catabolite Repression 4 (CCR4) facilitates Barley yellow striate mosaic virus replication in barley (Zhang et al ., 2020), VCS supports Potato virus A infection (De et al ., 2020; Hafrén et al ., 2015) and overexpression of several PB components was shown to restrict Turnip Mosaic Virus (Li & Wang, 2018) . It is becoming increasingly evident that RNA surveillance pathways, including RNA silencing and decapping, play a major role in translational regulation not only through degradation of aberrant RNAs, but by eliciting translational repression on endogenous targets (Hung & Slotkin, 2021; Iwakawa et al ., 2020; Wu et al ., 2020; Jang et al ., 2019; Lanet et al ., 2009; Xu & Chua, 2009; Brodersen et al ., 2008).…”

In planta vs viral expression of HCPro affects its binding of nonplant 21–22 nucleotide small RNAs, but not its preference for 5′‐terminal adenines, or its effects on small RNA methylation

“…This suggests that the suppressor could be interfering specifically with the methylation of sRNAs of exogenous satellite RNA sequence, but not with those of endogenous sequence (Ebhart et al ., 2005). Disruption of HCPro’s interaction with VARICOSE also prevents silencing suppression (De et al ., 2020).…”