SUMMARYPotyviral 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.

Lactic acid, a biologically derived compound, exists ubiquitously in nature. Its existence ranges from human being to microorganisms. Having paramount industrial significance, lactic acid should be highly pure, devoid of any contaminants. Hence, development of minimum steps of platform technologies to purify it needs urgent attention. The article proposed a novel and simple process for separation of lactic acid from a potential substrate Zizyphus oenophlia, based on ion exchange chromatography. The process herein involves two steps of purification; firstly a weak anion exchange resin was used to separate lactic acid from other anions present in the broth. This was followed by use of strong cation exchanger which washes out the target molecule (lactic acid) while trapped other cations present in the solution. The selected ion exchangers were Amberlite IRA 96 and Amberlite IR 120. Amberlite IRA 96 retained the lactic acid from the broth while washing away other anions. Maximum binding capacity of the resin was found to 210.46 mg lactic acid/g bead. After the simple two-step purification process, the purity of lactic acid improves up to 99.17 % with a recovery yield of 98.9 %. Upon characterization, formation of only levo rotatory form of lactic acid confirms its easy metabolism by the human system, thus triggering its application towards biomaterial sector.

No 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.

In this study, we demonstrate a novel pro-viral role for the Nicotiana benthamiana ARGO-NAUTE 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.