Browsing by Subject "potyvirus"

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  • Lohmus, Andres; Hafren, Anders; Mäkinen, Kristiina (2017)
    We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication. IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3' end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70.
  • De, Swarnalok; Chavez-Calvillo, Gabriela; Wahlsten, Matti; Mäkinen, Kristiina (2018)
    Infection caused by the synergistic interaction of two plant viruses is typically manifested by severe symptoms and increased accumulation of either virus. In potex-potyviral synergism, the potyviral RNA silencing suppressor helper component proteinase (HCPro) is known to enhance the pathogenicity of the potexvirus counterpart. In line with this, Potato virus X (PVX; genus Potexvirus) genomic RNA (gRNA) accumulation and gene expression from subgenomic RNA (sgRNA) are increased in Nicotiana benthamiana by Potato virus A (PVA; genus Potyvirus) HCPro expression. Recently, we have demonstrated that PVA HCPro interferes with the host cell methionine cycle by interacting with its key enzymes S-adenosyl-l-methionine synthetase (SAMS) and S-adenosyl-l-homocysteine hydrolase (SAHH). To study the involvement of methionine cycle enzymes in PVX infection, we knocked down SAMS and SAHH. Increased PVX sgRNA expression between 3 and 9 days post-infiltration (dpi) and upregulation of (-)-strand gRNA accumulation at 9 dpi were observed in the SAHH-silenced background. We found that SAMS and SAHH silencing also caused a significant reduction in glutathione (GSH) concentration, specifically in PVX-infected plants between 2 and 9 dpi. Interestingly, HCPro expression in PVX-infected plants caused an even stronger reduction in GSH levels than did SAMS+SAHH silencing and a similar level of reduction was also achieved by knocking down GSH synthetase. PVX sgRNA expression was increased in the GSH synthetase-silenced background. GSH is a major antioxidant of plant cells and therefore GSH shortage may explain the strong oxidative stress and severe symptoms observed during potex-potyvirus mixed infection.
  • Saha, Shreya; Hafren, Anders; Makinen, Kristiina (2019)
    One large open reading frame (ORF) encodes 10 potyviral proteins. We compared the accumulation of cylindrical inclusion (CI) protein from the middle, coat protein (CP) from the 3'end, and Renilla luciferase (RLUC) from two distinct locations in potato virus A (PVA) RNA. 5' RLUC was expressed from an rluc gene inserted between the P1 and helper component proteinase (HCPro) cistrons, and 3' RLUC was expressed from the gene inserted between the RNA polymerase and CP cistrons. Viral protein and RNA accumulation were quantitated (i) when expressed from PVA RNA in the presence of ectopically expressed genome-linked viral protein (VPg) and auxiliary proteins and (ii) at different time points during natural infection. The rate and timing of 3' RLUC and CP accumulation were found to be different from those of 5' RLUC and Cl. Ectopic expression of VPg boosted PVA RNA, 3' RLUC, and, together with HCPro, CP accumulation, whereas 5' RLUC and CI accumulation remained unaffected regardless of the increased viral RNA amount. In natural infection, the rate of the noteworthy minute early accumulation of 3' RLUC accelerated toward the end of infection. 5' RLUC accumulation, which was already pronounced at 2 days postinfection, increased moderately and stabilized to a constant level by day 5, whereas PVA RNA and CP levels continued to increase throughout the infection. We propose that these observations connect with the mechanisms by which potyvirus infection limits CP accumulation during early infection and specifically supports its accumulation late in infection, but follow-up studies are required to understand the mechanism of how this occurs. IMPORTANCE The results of this study suggest that the dynamics of potyviral protein accumulation are regulated differentially from the 3' end of viral RNA than from the rest of the genome, the significance of which would be to satisfy the needs of replication early and particle assembly late in infection.
  • 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.
  • Streng, Janne (Helsingfors universitet, 2013)
    RNA silencing is a sequence specific RNA degradation mechanism which is used by plants to regulate gene expression and to combat virus infections. However, viruses have developed so called silencing suppressors, which can prevent and interfere silencing reaction by many ways. For example, virus proteins can bind to maintaining proteins of the silencing reaction or to molecules which are responsible for signaling of the silencing reaction. This thesis focused on the study of protein-protein-interactions between known silencing suppressors of crini- and potyviruses and four maintaining plant proteins of RNA silencing. Protein-protein-interactions were studied using the yeast two-hybrid system (YTHS) and the bimolecular fluorescence complementation assay (BiFC). The latter method enables visualization of the studied protein interactions in plant cells. Protein expression of the cloned genes in yeast vectors were studied by using western blot. BiFC analysis was focused on protein interactions which were found by YTHS. This study detected three previously unknown protein interactions. Two virus proteins were found for the first time to bind directly to silencing maintaining proteins that are known to be targets of other silencing suppressors. Because the functions of these silencing maintaining proteins are known, it is possible that the three interactions described in this study interfere RNA silencing by impeding the functions of the plant proteins.
  • Lohmus, Andres; Varjosalo, Markku; Mäkinen, Kristiina (2016)
    The definition of the precise molecular composition of membranous replication compartments is a key to understanding the mechanisms of virus multiplication. Here, we set out to investigate the protein composition of the potyviral replication complexes. We purified the potyviral 6K2 protein-induced membranous structures from Potato virus A (PVA)-infected Nicotiana benthamiana plants. For this purpose, the 6K2 protein, which is the main inducer of potyviral membrane rearrangements, was expressed in fusion with an N-terminal Twin-Strep-tag and Cerulean fluorescent protein (SC6K) from the infectious PVA cDNA. A non-tagged Cerulean-6K2 (C6K) virus and the SC6K protein alone in the absence of infection were used as controls. A purification scheme exploiting discontinuous sucrose gradient centrifugation followed by Strep-tag-based affinity chromatography was developed. Both (+)- and (-)-strand PVA RNA and viral protein VPg were co-purified specifically with the affinity tagged PVA-SC6K. The purified samples, which contained individual vesicles and membrane clusters, were subjected to mass spectrometry analysis. Data analysis revealed that many of the detected viral and host proteins were either significantly enriched or fully specifically present in PVA-SC6K samples when compared with the controls. Eight of eleven potyviral proteins were identified with high confidence from the purified membrane structures formed during PVA infection. Ribosomal proteins were identified from the 6K2-induced membranes only in the presence of a replicating virus, reinforcing the tight coupling between replication and translation. A substantial number of proteins associating with chloroplasts and several host proteins previously linked with potyvirus replication complexes were co-purified with PVA-derived SC6K, supporting the conclusion that the host proteins identified in this study may have relevance in PVA replication.
  • Sarmah, Nandita (Helsingfors universitet, 2017)
    The leaves of healthy potato (Solanum tuberosum L.) plants and leaves systemically infected with Potato virus A (PVA; a monopartite positive sense single stranded RNA genome, genus: Potyvirus, family: Potyviridae) have been previously analysed and compared for the nuclear proteome to better understand the role of nucleus in plant virus infection. The analysis indicated that a small GTP-binding protein (ras-related protein; rabE1) is found in the nuclei of PVA-infected leaves but is absent from nuclei of healthy leaves. Therefore, to confirm the differential localization, this study was conducted. Subcellular localization of proteins was studied by tagging the protein with a marker protein, green fluorescent protein (GFP). The mRNA of ras-like protein, rabE1, of potato cv. Pentland Crown was cloned and expressed with GFP fused to the N- or C-terminus of rabE1 (GFP-ras and ras-GFP respectively). Plants of Nicotiana benthamiana were inoculated with PVA by two methods. Firstly, the Agrobacterium strains expressing GFP-ras or ras-GFP were mixed with the Agrobacterium strain expressing red fluorescent protein (RFP) or PVA-RFP and introduced to the healthy leaves of N. benthamiana plants and observed under epifluorescence microscope three days post inoculation. Secondly, PVA-RFP was agroinoculated to two or three basal leaves of young N. benthamiana plants and plants were grown for 12 days to allow systemic infection. Thereafter, PVA infected upper leaves and healthy leaves were infiltrated with GFP-ras or ras-GFP and the leaves were observed under epifluorescence microscope three days after infiltration. In healthy plants, GFP-ras localized to the cytoplasm and ras-GFP localized mostly to the cytoplasm and in few cells it was found both in the nucleus and cytoplasm. Whereas, in PVA infected cells, GFP-ras localized to the cytoplasm and in some cells to both nucleus and cytoplasm suggesting that PVA infection increases GFP-ras localization to the nucleus. However, no difference was observed in localization of ras-GFP in PVA infected and healthy leaves. The hypothesis of the research is thus partially true. Our study shows that rabE1 can be differentially localized in the healthy and PVA-infected plant cells.