Novel loading protocol combines highly efficient encapsulation of exogenous therapeutic toxin with preservation of extracellular vesicles properties, uptake and cargo activity.

Extracellular vesicles (EVs) have mostly been investigated as carriers of biological therapeutics such as proteins and RNA. Nevertheless, small-molecule drugs of natural or synthetic origin have also been loaded into EVs, resulting in an improvement of their therapeutic properties. A few methods have been employed for EV cargo loading, but poor yield and drastic modifications of vesicles remain unsolved challenges. We tested a different strategy based on temporary pH alteration through incubation of EVs with alkaline sodium carbonate, which resulted in conspicuous exogenous molecule incorporation. In-depth characterization showed that vesicle size, morphology, composition, and uptake were not affected. Our method was more efficient than gold-standard electroporation, particularly for a potential therapeutic toxin: the plant Ribosome Inactivating Protein saporin. The encapsulated saporin resulted protected from degradation, and was efficiently conveyed to receiving cancer cells and triggered cell death. EV-delivered saporin was more cytotoxic compared to the free toxin. This approach allows both the structural preservation of vesicle properties and the transfer of protected cargo in the context of drug delivery.

Interplay of HCPro and CP in the Regulation of Potato Virus A RNA Expression and Encapsidation.

Potyviral coat protein (CP) and helper component-proteinase (HCPro) play key roles in both the regulation of viral gene expression and the formation of viral particles. We investigated the interplay between CP and HCPro during these viral processes. While the endogenous HCPro and a heterologous viral suppressor of gene silencing both complemented HCPro-less potato virus A (PVA) expression, CP stabilization connected to particle formation could be complemented only by the cognate PVA HCPro. We found that HCPro relieves CP-mediated inhibition of PVA RNA expression likely by enabling HCPro-mediated sequestration of CPs to particles. We addressed the question about the role of replication in formation of PVA particles and gained evidence for encapsidation of non-replicating PVA RNA. The extreme instability of these particles substantiates the need for replication in the formation of stable particles. During replication, viral protein genome linked (VPg) becomes covalently attached to PVA RNA and can attract HCPro, cylindrical inclusion protein and host proteins. Based on the results of the current study and our previous findings we propose a model in which a large ribonucleoprotein complex formed around VPg at one end of PVA particles is essential for their integrity.

Infrared and Raman spectroscopy for purity assessment of extracellular vesicles.

Extracellular vesicles (EVs) are a complex and heterogeneous population of nanoparticles involved in cell-to-cell communication. Recently, numerous studies have indicated the potential of EVs as therapeutic agents, drug carriers and diagnostic tools. However, the results of these studies are often difficult to evaluate, since different characterization methods are used to assess the purity, physical and biochemical characteristics of the EV samples. In this study, we compared four methods for the EV sample characterization and purity assessment: i) the particle-to-protein ratio based on particle analyses with nanoparticle tracking and protein concentration by bicinchoninic acid assay, ii) Western Blot analysis for specific EV biomarkers, iii) two spectroscopic lipid-to-protein ratios by either the attenuated total reflection Fourier transform infrared (ATR-FTIR) or Raman spectroscopy. The results confirm the value of Raman and ATR-FTIR spectroscopy as robust, fast and operator independent tools that require only a few microliters of EV sample. We propose that the spectroscopic lipid-to-protein (Li/Pr) ratios are reliable parameters for the purity assessment of EV preparations. Moreover, apart from determining protein concentrations, we show that ATR-FTIR spectroscopy can also be used for indirect measurements of EV concentrations. Nevertheless, the Li/Pr ratios do not represent full characterization of the EV preparations. For a complete characterization of selected EV preparations, we recommend also additional use of particle size distribution and EV biomarker analysis.

Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery.

Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies.

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation.

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.

Protein composition of 6K2-induced membrane structures formed during Potato virus A infection.

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.

Molecular insights into the function of the viral RNA silencing suppressor HCPro.

Potyviral 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.

Formation of Potato Virus A-Induced RNA Granules and Viral Translation Are Interrelated Processes Required for Optimal Virus Accumulation.

RNA granules are cellular structures, which play an important role in mRNA translation, storage, and degradation. Animal (+)RNA viruses often co-opt RNA granule proteins for viral reproduction. However, the role of RNA granules in plant viral infections is poorly understood. Here we use Potato virus A (PVA) as a model potyvirus and demonstrate that the helper component-proteinase (HCpro), the potyviral suppressor of RNA silencing, induces the formation of RNA granules. We used confocal microscopy to demonstrate the presence of host RNA binding proteins including acidic ribosomal protein P0, argonaute 1 (AGO1), oligouridylate-binding protein 1 (UBP1), varicose (VCS) and eukaryotic initiation factor iso4E (eIF(iso)4E) in these potyvirus-induced RNA granules. We show that the number of potyviral RNA granules is down-regulated by the genome-linked viral protein (VPg). We demonstrated previously that VPg is a virus-specific translational regulator that co-operates with potyviral RNA granule components P0 and eIF(iso)4E in PVA translation. In this study we show that HCpro and varicose, components of potyviral RNA granules, stimulate VPg-promoted translation of the PVA, whereas UBP1 inhibits this process. Hence, we propose that PVA translation operates via a pathway that is interrelated with potyviral RNA granules in PVA infection. The importance of these granules is evident from the strong reduction in viral RNA and coat protein amounts that follows knock down of potyviral RNA granule components. HCpro suppresses antiviral RNA silencing during infection, and our results allow us to propose that this is also the functional context of the potyviral RNA granules we describe in this study.

Tetracycline resistance genes persist at aquaculture farms in the absence of selection pressure.

The prophylactic and therapeutic use of tetracyclines in aquaculture has been shown to contribute to the spread of tetracycline resistance in the environment. In this work, the prevalence of four different tetracycline-resistance genes, tetA, tetC, tetH, and tetM, in sediments from four aquaculture farms and their surroundings in the Baltic Sea was monitored by quantitative polymerase chain reaction (qPCR). The presence of three additional tetracycline-resistance genes (tetE, tetG, and tetW) was studied qualitatively by standard PCR, and the amount of bioavailable tetracyclines and total amounts of tetracycline and oxytetracycline in samples were also measured. None of the farms were using tetracycline at the time of the sampling and one of the farms had stopped all antibiotic use six years prior to the first sampling. Two of the farms were sampled over four successive summers and two were sampled once. Our results showed greater copy numbers of tetA, tetC, tetH, and tetM at the farms compared to pristine sites and demonstrated the presence of tetE, tetG, and tetW genes in the sediments under aquaculture farms at most sampling times. However, no resistance genes were found in samples collected 200 m from any of the farms. None of the samples contained therapeutically active concentrations of tetracyclines at any of the sampling times, suggesting that the increase in the prevalence of tetracycline resistance genes is caused by the persistence of these genes in the absence of selection pressure.