Factors affecting translation at the programmed −1 ribosomal frameshifting site of Cocksfoot mottle virus RNA in vivo

The ratio between proteins P27 and replicase of Cocksfoot mottle virus (CfMV) is regulated via a −1 programmed ribosomal frameshift (−1 PRF). A minimal frameshift signal with a slippery U UUA AAC heptamer and a downstream stem–loop structure was inserted into a dual reporter vector and directed −1 PRF with an efficiency of 14.4 ± 1.9% in yeast and 2.4 ± 0.7% in bacteria. P27-encoding CfMV sequence flanking the minimal frameshift signal caused ∼2-fold increase in the −1 PRF efficiencies both in yeast and in bacteria. In addition to the expected fusion proteins, termination products ending putatively at the frameshift site were found in yeast cells. We propose that the amount of premature translation termination from control mRNAs played a role in determining the calculated −1PRF efficiency. Co-expression of CfMV P27 with the dual reporter vector containing the minimal frameshift signal reduced the production of the downstream reporter, whereas replicase co-expression had no pronounced effect. This finding allows us to propose that CfMV protein P27 may influence translation at the frameshift site but the mechanism needs to be elucidated

Insights into the Functions of eIF4E-Binding Motif of VPg in Potato Virus A Infection

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 (YXXXLΦ) 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 (PVAVPgmut). PVAVPgmut 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. VPgmut 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 VPgmut 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 YXXXLΦ 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

Special Issue: “The Complexity of the Potyviral Interaction Network”

Many potyvirus species are among the most economically-significant plant viruses as they cause substantial yield losses to crop plants globally [...

Special Issue: “The Complexity of the Potyviral Interaction Network”

Many potyvirus species are among the most economically-significant plant viruses as they cause substantial yield losses to crop plants globally [...]Non peer reviewe

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host’s metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth

Puuttuvat laboratoriotutkimuspyynnöt potilaan siirtyessä erikoissairaanhoidosta perusterveydenhuoltoon

Laboratoriotutkimusten virheistä jopa 50–75 % syntyy preanalyyttisessä vaiheessa. Näistä n. 26 % aiheuttaa haittaa tai ylimääräistä vaivaa potilaille. Laboratoriotutkimusta varten potilaalla tulee olla hoitavan tahon tekemä lähete eli laboratoriotutkimuspyyntö. Toisinaan näytteenottoon tulee kuitenkin potilaita, joilla ei syystä tai toisesta ole voimassa olevaa laboratoriotutkimuspyyntöä. Erityisesti erikoissairaanhoidosta perusterveydenhuoltoon siirtyvien potilaiden pyyntöjen puuttuminen kuluttaa usean eri tahon resursseja, kun esimerkiksi laboratoriohoitaja alkaa muiden töidensä ohessa selvittämään asiaa. Terveyskeskuksen vastaanotossa työskentelevät joutuvat etsimään, tekemään ja uusimaan tutkimuspyyntöjä, jotka eivät varsinaisesti välttämättä olisi heidän tehtäviään. Tämä hidastaa näytteenottotoimintaa ja voi pahimmillaan uhata potilaan tietosuojaa. Potilaalle tai terveydenhuollon asiakkaalle puuttuvat laboratoriotutkimuspyynnöt tuovat epävarmuutta ja turhaa ylimääräistä odotusaikaa. Tämän tutkimuksen tavoitteena oli selvittää, miksi laboratoriotutkimuspyyntöjä puuttuu potilaan saapuessa laboratorioon. Tutkimus suoritettiin Wepropol-pohjaisena kyselytutkimuksena yhteistyössä Jesse Liukkosen kanssa. Kyselyn avulla selvitimme asiaan liittyviä ongelmia ja puutteita. Kysely ohjattiin Tyksin osastonhoitajille, Kliinisen kemian laboratoriohoitajille sekä terveysasemien vastaaville ylilääkäreille. Kyselyn aukioloaika oli 12.12.2022-20.1.2023. Tässä tutkimuksessa keskityttiin Tyksin osastonhoitajilta saatujen vastauksien tulkintaan. Kokonaisuudessaan kyselyyn vastasi yhteensä 59 henkilöä. Jesse Liukkosen osa kyselytutkimuksesta kohdistui puolestaan perusterveydenhuollon Kliinisen kemian laboratoriohoitajille sekä terveysasemien vastaaville ylilääkäreille. Kyselystä saamamme tulokset osoittavat, että potilasohjauksessa on puutteita potilaan siirtyessä erikoissairaanhoidosta jatkohoitoon perusterveydenhuollon puolelle. Potilaat menevät hyvin usein laboratorioon ilman asianmukaista laboratoriotutkimuspyyntöä. He eivät ole läheskään aina tietoisia, että heidän tulisi ottaa ensin yhteyttä omaan terveyskeskukseensa laboratoriotutkimuspyynnön saamista varten. On selkeästi olemassa tarve käytäntöjen yhtenäistämiseksi näitä tilanteita varten. Asiasanat: laboratoriotutkimuspyyntö, erikoissairaanhoito, perusterveydenhuolto, näytteenott

Effects of Poty-Potexvirus Synergism on Growth, Photosynthesis and Metabolite Status of Nicotiana benthamiana

Mixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host’s metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth

The potyviral silencing suppressor HCPro recruits and employs host ARGONAUTE1 in pro-viral functions

Author summary In the course of an infection viral proteins come regularly into contact with host factors. Knowledge of these virus-host interactions in plants is essential for understanding the progress of infections and for addressing challenges in the production of healthy plants, which is important for global food security. In this study we report that the potyviral Helper component proteinase (HCPro) recruits host ARGONAUTE1 protein and engages it in pro-viral functions. We found that the interaction of potato virus A HCPro with ARGONAUTE1 benefited the infection by promoting the stability and accumulation of virus particles. When HCPro was engineered to disrupt the interaction, we detected very few virus particles and observed that the mutated virus did not spread efficiently. Thus, we propose that the interaction between HCPro and ARGONAUTE1 has biological relevance as it is important for formation of stable particles and achieving optimal systemic infection. In this study, we demonstrate a novel pro-viral role for theNicotiana benthamianaARGONAUTE 1 (AGO1) in potyvirus infection. AGO1 strongly enhanced potato virus A (PVA) particle production and benefited the infection when supplied in excess. We subsequently identified the potyviral silencing suppressor, helper-component protease (HCPro), as the recruiter of host AGO1. After the identification of a conserved AGO1-binding GW/WG motif in potyviral HCPros, we used site-directed mutagenesis to introduce a tryptophan-to-alanine change into the HCPro (HCPro(AG)) of PVA (PVA(AG)) and turnip mosaic virus (TuMV(AG)). AGO1 co-localization and co-immunoprecipitation with PVA HCPro was significantly reduced by the mutation suggesting the interaction was compromised. Although the mutation did not interfere with HCPro's complementation or silencing suppression capacity, it nevertheless impaired virus particle accumulation and the systemic spread of both PVA and TuMV. Furthermore, we found that the HCPro-AGO1 interaction was important for AGO1's association with the PVA coat protein. The coat protein was also more stable in wild type PVA infection than in PVA(AG)infection. Based on these findings we suggest that potyviral HCPro recruits host AGO1 through its WG motif and engages AGO1 in the production of stable virus particles, which are required for an efficient systemic infection.Peer reviewe

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

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.Peer reviewe