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  • Swarnalok, De; Pollari, Maija; Varjosalo, Markku; Mäkinen, Kristiina (2020)
    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; genusPotyvirus) 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)) infectedNicotiana benthamianaless 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 boost in translation of replication-deficient PVA (PVA(Delta 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-derivedRenillaluciferase 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 multi-protein assemblies associated with PVA RNA in protecting it from degradation, ensuring efficient translation, formation of stable virions and establishment of systemic infection. Author summary 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.
  • Saha, Shreya; Makinen, Kristiina (2020)
    The interaction between the viral protein genome-linked (VPg) and eukaryotic initiation factor 4E (eIF4E) or eIF(iso)4E of the host plays a crucial role in potyvirus infection. The VPg of potato virus A (PVA) contains the Tyr-X-X-X-X-Leu-phi (YXXXL phi) binding motif for eIF(iso)4E. In order to investigate its role in PVA infection, we substituted the conserved tyrosine and leucine residues of the motif with alanine residues in the infectious cDNA of PVA (PVA(VPgmut)). PVA(VPgmut) RNA replicated in infiltrated leaves, but RNA accumulation remained low. Systemic infection occurred only if a reversion to wild type PVA occurred. VPg was able to stabilize PVA RNA and enhance the expression of Renilla luciferase (3'RLUC) from the 3' end of the PVA genome. VPg(mut) could not support either PVA RNA stabilization or enhanced 3'RLUC expression. The RNA silencing suppressor helper-component proteinase (HCPro) is responsible for the formation of PVA-induced RNA granules (PGs) during infection. While VPg(mut) increased the number of PG-like foci, the percentage of PVA RNA co-localization with PGs was reduced from 86% to 20%. A testable hypothesis for future studies based on these results is that the binding of eIF(iso)4E to PVA VPg via the YXXXL phi motif is required for PVA RNA stabilization, as well as the transfer to the RNA silencing suppression pathway and, further, to polysomes for viral protein synthesis.
  • Weinheimer, Isabel; Haikonen, Tuuli; Ala-Poikela, Marjo; Moser, Mirko; Streng, Janne; Rajamaki, Minna-Liisa; Valkonen, Jari P. T. (2016)
    Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA-dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.