Helper Components Research Articles

The intriguing success of helper components in vector-transmission of plant viruses.

An intriguing aspect of vector-transmission of plant viruses is the frequent involvement of a helper component (HC). HCs are virus-encoded non-structural proteins produced in infected plant cells that are mandatory for the transmission success. Over five decades, all data collected on HCs from unrelated viral species transmitted by distinct vector species were consistent with a unique mode of action designated “the bridge hypothesis”: the HC has two functional domains, one binding the virus particle and the other binding a putative receptor in the vector, creating a reversible molecular bridge between the two. This hypothesis appeared fully satisfactory as HCs were reported solely in viruses transmitted non-circulatively -- i.e. the virus particle binds externally to the mouthpart of its vector, and can later be released therefrom and inoculated. Recently, however, HCs have also been reported in viruses transmitted circulatively, where the virus particles are internalized in gut cells and cycle within the body to reach the salivary glands. In this more complex scheme of virus-vector interaction, a simple mode of action of HC compatible with the bridge hypothesis becomes questionable. In addition, while it had consistently been shown that the sequential acquisition of HC and virus particles could only work when HC was acquired first, a recent report shows that the reverse acquisition sequence can work in some case, again questioning the bridge hypothesis as a universal mode of action. Because of the importance of HC molecules in the vector-transmission of plant viruses, we here propose an exhaustive review of the field, of its historical perspective and most recent development.

Nanodiamond Integration into Niosomes as an Emerging and Efficient Gene Therapy Nanoplatform for Central Nervous System Diseases.

Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy.

Non-viral mediated gene therapy in human cystic fibrosis airway epithelial cells recovers chloride channel functionality.

Gene therapy strategies based on non-viral vectors are currently considered as a promising therapeutic option for the treatment of cystic fibrosis (CF), being liposomes the most commonly used gene carriers. Niosomes offer a powerful alternative to liposomes due to their higher stability and lower cytotoxicity, provided by their non-ionic surfactant and helper components. In this work, a three-formulation screening is performed, in terms of physicochemical and biological behavior, in CF patient derived airway epithelial cells. The most efficient niosome formulation reaches 28% of EGFP expressing live cells and follows caveolae-mediated endocytosis. Transfection with therapeutic cystic fibrosis transmembrane conductance regulator (CFTR) gene results in 5-fold increase of CFTR protein expression in transfected versus non-transfected cells, which leads to 1.5-fold increment of the chloride channel functionality. These findings highlight the relevance of niosome-based systems as an encouraging non-viral gene therapy platform with potential therapeutic benefits for CF.

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltppro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as “altruistic transformation.”

Structural Analyses of AC4 Protein of Sri Lankan Cassava Mosaic Virus

RNA silencing is one of the important phenomenon in plant defense mechanism, it actively protect host plants against viral infections. Existing viruses must have developed counter defense strategies to survive this arms race. Such counter defense strategy is the viral silencing suppressor (VSRs) which have been reported to directly interfere with the various steps leading to the interference of viral RNAs. Most identified VSRs are multifunctional, besides being RNA-silencing suppressors, they often perform essential roles by functioning as coat proteins, helper components for viral transmission, replicases and movement proteins, proteases or transcriptional regulators. One such identified VSR is AC4 of Sri Lankan cassava mosaic virus strain. Trivial knowledge about the structure –function relationship of this VSR leads to this work, where we focus on the structure generation by modelling to identify the mode of interactions with the various effector molecules of the silencing pathways. Structural analyses have been performed to screen interacting residues. Results indicate conserved structural features which signify propensity of functional interactions and further shows that this VSR can be a potent tool for the analysis of RNA silencing mechanisms and the relationships between different silencing pathways and VSRs.

Task allocation in organic computing systems: networks with reconfigurable helper units

In this paper, computing systems that have no central control and consist of many partially autonomous components are studied. Two types of components are distinguished which are called workers and helpers respectively. The components are connected via a network and the helper components perform service tasks for the worker components. It is assumed that the helper components exhibit reconfigurable hardware to be able to execute different service tasks efficiently. The problem addressed in this paper is how to organise such a decentralised system in a way that requests of the workers are executed by suitable helpers and the total reconfiguration costs of the helpers are small. Several decentralised task allocation methods are proposed. One of them uses a combination of a fully decentralised dynamic clustering algorithm and a self-organised task allocation system. The clustering algorithm is used to classify the service requests that are sent through the network in order to give the helpers hints which requests are suitable to be executed by them. Simulations are done for static and dynamic scenarios to investigate the reconfiguration costs and the number of dropped requests, i.e., requests that could not be satisfied. The results show that the clustering-based system has a strong adaptive behaviour and that the decentralised clustering is able to reduce the reconfiguration costs significantly.

Structural Analyses of AC4 Protein of Sri Lankan Cassava Mosaic Virus

RNA silencing is one of the important phenomenon in plant defense mechanism, it actively protect host plants against viral infections. Existing viruses must have developed counter defense strategies to survive this arms race. Such counter defense strategy is the viral silencing suppressor (VSRs) which have been reported to directly interfere with the various steps leading to the interference of viral RNAs. Most identified VSRs are multifunctional, besides being RNA-silencing suppressors, they often perform essential roles by functioning as coat proteins, helper components for viral transmission, replicases and movement proteins, proteases or transcriptional regulators. One such identified VSR is AC4 of Sri Lankan cassava mosaic virus strain. Trivial knowledge about the structure –function relationship of this VSR leads to this work, where we focus on the structure generation by modelling to identify the mode of interactions with the various effector molecules of the silencing pathways. Structural analyses have been performed to screen interacting residues. Results indicate conserved structural features which signify propensity of functional interactions and further shows that this VSR can be a potent tool for the analysis of RNA silencing mechanisms and the relationships between different silencing pathways and VSRs.

An Evaluation of the Peer Helper Component of Go !: A Multimessage, Multi-“step” Obesity Prevention Intervention

Background Obesity prevention efforts typically involve changing eating and exercise behaviors as well as the physical and social environment in which those behaviors occur. Due to existing social networks, worksites are a logical choice for implementing such interventions. Purpose This article describes the development and implementation of a peer helper intervention, which was part of a multicomponent obesity prevention program conducted at a hospital worksite. It also presents qualitative evaluation findings of peer helper perceptions, roles, and institutional outcomes. Methods Fifty-one male and female employees were recruited to promote healthy eating and physical activity amongst their peers. They received health and communication skill-building training. Semistructured interviews were conducted with 20 peer helpers to evaluate this component of the intervention. Results Findings showed that peer helpers took on many helping roles, became effective role models, and contributed to creating health-promoting social and environmental changes at work. Discussion We found evidence for synergies between the peer helper component and other components of the campaign and present recommendations and questions related to best practices for peer helper interventions at worksites. Translation to Health Education Practice Training on the use of peer helpers and the incorporation of peer helper components into interventions is needed.

Stability and performance of ant queue inspired task partitioning methods

In this paper, we consider computing systems that have autonomous helper components which fulfill support functions and that possess reconfigurable hardware so that they can specialize to different types of service tasks. Several self-organized task partitioning methods are proposed that can be used by the helper components to decide how to reconfigure and which service tasks to execute. The proposed task partitioning methods are inspired by the so-called ant queue system that can be found in real ants for partitioning tasks between the individuals. The aim of this study is to investigate basic properties of the task partitioning methods, like stability and efficiency, in order to obtain basic insights into the design of task partitioning methods in self-organized service systems. More precisely, the investigations are threefold: (1) discrete event simulations are used to investigate systems, (2) for a simple version of the task partitioning system analytical stability results are obtained by means of delay differential equation systems and (3) by numerically solving initial value problems.

Self-Organized Task Allocation for Service Tasks in Computing Systems with Reconfigurable Components

A self-organized scheme for the allocation service tasks in adaptive or organic computing systems is proposed. Such computing systems are highly self-organized and the components ideally adapt to the needs of users or the environment. Typically, the components of such systems need some service from time to time in order perform their work efficiently. Since the type of service tasks will often change in this systems it is attractive to use reconfigurable hardware to perform the service tasks. The studied system consists of normal worker components and the helper components which have reconfigurable hardware and can perform different service tasks. The speed with which a service task is executed by a helper depends on its actual configuration. Different strategies for the helpers to decide about service task acceptance and reconfiguration are proposed. These task acceptance strategies are inspired by stimulus-threshold models that are used to explain task allocation in social insects. Analytical results for a system with two reconfigurable helpers are presented together with simulation results for larger systems.

Characteristics of Potato virus Y Isolated from Paprika in Korea

A virus isolate collected from infected paprika (Capsicum annuum var. grossum) was characterized as Potato virus Y (PVY) based on biological, serological, cytopathological, and molecular properties. In host range studies, the paprika isolate produced the mosaic symptom on some tobacco, tomato and pepper (Capsicum annuum). A new paprika isolate also infected potato cultivars which is different biological characteristic compared to the other popular potyvirus infecting paprika, Pepper mottle virus (PepMoV). Previously reported PVY strains, and did not infect pepper and typical PepMoV isolates did not infect potato. Distinctive inclusion patterns of the scroll, pinwheel, long laminated inclusions, and helper components in the cytoplasm of infected cells were also different to those observed by the typical PVY isolate infections. However, the paprika isolate reacted to the monoclonal antibody of strain with high absorbance readings. RT-PCR amplification, cloning, and sequencing of the 3` untranslated region and a part of coat protein gene also added additional evidence of the paprika isolate as the -related isolate. Multiple alignments as well as cluster dendrograms of PVY-paprika isolate revealed close phylogenetic relationship to the subgroup. Altogether, these results suggest that a new PVY isolate infecting paprika contained distinct characteristics compared to the other previously described PVY strains with closer relationship to the strain.

Properties of Sindbis virus vectors produced with a chimeric split helper system

We have evaluated a chimeric, two-component Sindbis virus packaging system. As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis virus vectors required only one of the helper components--the chimera with a deleted Ross River virus capsid and the Sindbis virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split helper Sindbis virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.

845. Properties of Sindbis Virus Vectors Produced with a Chimeric Split Helper System

Top of pageAbstract We have evaluated a chimeric, two-component Sindbis virus packaging system (Frolov et al, 1997, J. Virol. 71, 2819-2829). As expected, use of this combination of two modified helper RNA species prevented formation of infection competent Sindbis viruses as analyzed by serial passaging. We observed, however, that vectors produced using this method were able to spread in BHK cell cultures and formed clusters of transgene positive cells that did not display cytopathic effects for up to 3 days post-transduction. Formation of spreading Sindbis virus vectors required only one of the helper components |[ndash]| the chimera with a deleted Ross River virus capsid and the Sindbis virus envelope glycoproteins. Spreading was also demonstrated in two rat glioma cell lines, 9L and BT4C, showing that this phenomenon was not limited to BHK cells. Our results warrant further characterization of split-helper Sindbis virus vectors and imply their utility in gene therapy approaches where spreading of transgene expression and consequently high gene transfer rate could be beneficial.

Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice.

Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Here we describe the use of naked double-stranded (ds) linear DNA as a way to insert exogenous DNA sequences into chromosomes of mouse hepatocytes in vivo, without helper components such as integrase or transposase. We constructed ds linear DNA vectors with or without adeno-associated virus inverted terminal repeats (AAV-ITRs), introduced them into mouse hepatocytes in vivo using a hydrodynamics-based transfection technique, and analyzed for vector genome integration in various ways. Surprisingly, these linear DNA molecules integrated in mouse hepatocytes in vivo at a level of 0.3-0.5 vector genome, or more, per diploid genomic equivalent irrespective of the AAV-ITR sequences. Our results establish a novel and simple way to engineer chromosomes in vivo and provide further insights into the mechanisms of recombinant AAV vector integration in vivo. In addition, they may provide a clue for developing new nonviral integrating gene delivery vector systems.

Loss of Aphid Transmissibility of Plum Pox Virus Isolates

ABSTRACTThe aphid transmissibility of seven Plum pox virus (PPV) isolates and the amino acid sequences of their coat proteins were analysed. Two aphid transmissible isolates PPV-A and PPV-P contained the DAG amino triplet, while DAL or NAG replaced this triplet in the coat proteins of non-aphid transmissible isolates. In addition, a sequence of 15 or 17 amino acids was deleted in the coat proteins of PPV-P48, PPV-P45 and PPV-II/7. Helper components with Mr. of 52 kDa were successfully purified from N. benthamiana plants inoculated with aphid transmissible and non-aphid transmissible isolates. The helper components of two coat-proteins-defective isolates mediated aphid transmission of non- coat-protein defective isolates, indicating that the helper components were biologically active.

Specific epitope-induced conversion of CD8+ memory cells into effector cytotoxic T lymphocytes in vitro: presentation of peptide antigen by CD8+ T cells.

The requirements for the conversion of CD8+ memory T cells into effector class I major histocompatibility complex (MHC) Kd-restricted cytotoxic T (Tc) cells in vitro have been studied. Purified CD8+ splenocytes from influenza A/WSN-primed BALB/c (H-2d) mice stimulated with a synthetic nucleoprotein peptide 147-158 R156- (NPP) alone generated Tc cells specific for influenza virus-infected target cells. No additional requirements for accessory cells or their lymphokine products were necessary indicating that peptide antigen (Ag) in association with Kd was presented on CD8+ T cells. The evidence for presentation of NPP by CD8+ T cells was supported by the use of CD8+ memory T cells from semiallogeneic bone marrow radiation chimeras of P1----F1 type (H-2b----[H-2d x H-2b]F1). Memory CD8+ splenocytes from A/WSN-immune chimeras did not develop into secondary effector Tc cells as a result of a 4-day culture with NPP alone, however, were able to do so if NPP was presented by Kd-bearing Ag-presenting cells. In addition, these results exclude the possibility of direct recognition of free NPP molecules by the specific T cell receptor of CD8+ memory T cells. CD8+ memory splenocytes (H-2b) from chimeras were also able to develop into functionally active Tc cells as a result of presentation of Db-restricted synthetic peptide (NP 366-374) with a sequence derived from influenza virus nucleoprotein with high affinity for Db MHC class I molecules. Blockade of endogenously produced interleukin 2 (IL-2) activity by anti-IL-2 or anti-IL-2 receptor monoclonal antibody in the culture of CD8+ memory T cells during a 4-day NPP stimulation completely abolished Tc cell generation, indicating that the utilization of this lymphokine is absolutely required for the secondary Tc cell development. These findings demonstrate that CD8+ memory T cells per se are able to recognize the restimulating epitope as a result of its presentation by CD8+ T cells and develop into cytolytically active and highly specific Tc cells with no requirements for other cellular helper components or their lymphokine products.

Biological and Serological Characterization of Zucchini Yellow Mosaic and Watermelon Mosaic Virus-2 Isolates in Israel

Cucurbit potyviruses were collected in the field in Israel and subcultured in indicator plants in a greenhouse. Partial characterization of the Israeli cucurbit potyviruses was done on the basis of host reaction using cucurbits, peas andChenopodium spp. as hosts. Further classification of potyviruses was done by enzyme-linked immunosorbent assay (ELISA) and serological specific electron microscopy (SSEM). By these methods it was possible to identify three of the four isolates as strains of the zucchini yellow mosaic virus, while the fourth was identified as watermelon mosaic virus-2. Two of the ZYMV isolates were nonaphid-transmissible following prolonged mechanical transmission in a greenhouse. Both of these isolates were found to produce helper components capable of assisting the transmission of virions from a transmissible isolate but not those of their own.

Properties and in vitro Translation of Maize Dwarf Mosaic Virus RNA

SUMMARY The RNA of maize dwarf mosaic virus strain A (MDMV-A) was characterized and compared with that of strain B (MDMV-B). Glyoxal-treated MDMV-A RNA has an M r of 3·32 × 106 measured in agarose gels, compared with that of MDMV-B which is 3·41 × 106 under the same conditions. MDMV-A RNA has a Tm of 50·7 °C and a hyperchromicity of 23·3%, which are higher than those reported for MDMV-B RNA. Translation products of both RNAs in a rabbit reticulocyte cell-free system ranged from 23K to 121K. Four major polypeptide products of 57K, 91K, 100K and 117K were produced by MDMV-A RNA-directed translation, and four major products of 39K, 55K, 86K and 121K by MDMV-B RNA. Protein profiles from the two RNAs were distinct including translation products that comigrated with authentic coat protein at 37K (MDMV-A) or 39K (MDMV-B) for each strain. Anti-virion sera immunoprecipitated polypeptide products from each strain. Distinct subsets of translation products were immunoprecipitated with antisera to tobacco etch virus 49K and 54K nuclear inclusion proteins. No cell-free translation products were recognized by antisera to potato virus Y (PVY) or tobacco vein mottling virus (TVMV) helper components or antisera to the cylindrical inclusion proteins of PVY or TVMV.

In vitro analysis of allogeneic lymphocyte interaction. VIII. Characterization of helper components of allogeneic effect factor (AEF) that activate Lyb5+ and Lyb5- B cells to respond to thymus-dependent and thymus-independent antigens.

We analyzed the ability of an allogeneic effect factor (AEF), produced using alloactivated A.SW (H-2s) responder T cells and irradiated A/WySN (H-2a) T cell-depleted stimulator spleen cells, to enhance the responsiveness of Lyb5+ and Lyb5- B cells to thymus-dependent (TD) and thymus-independent type 1 (TI-1) and type 2 (TI-2) antigens. We found that normal adult male CBA/CaHN (Lyb5+ + Lyb5-) B cells are activated by unfractionated AEF in primary in vitro IgM antibody responses to the SRBC (TD), TNP-LPS (TI-1), and TNP-Ficoll (TI-2) antigens. Adult male xid CBA/N (Lyb5-) B cells are activated by unfractionated AEF to respond to TNP-LPS but to neither SRBC nor TNP-Ficoll. Gel filtration of AEF on ACA 54 resolves several helper components. One component, which is I-Ak restricted in its activity and may represent a secreted form of an Ia antigen T cell receptor, interacts with histocompatible and functionally competent CBA/CaHN or CBA/N antigen-presenting cells to activate a primary anti-SRBC response of CBA/CaHN B cells but not of CBA/N B cells. A second antigen-nonspecific and H-2-nonrestricted AEF component, which consists of interleukin 2 and possibly also interleukin 1, activates anti-SRBC and anti-TNP-Ficoll responses of both CBA/CaHN B cells and CBA/N B cells. Such CBA/N B cell responses were potentiated by this AEF component only after its biochemical fractionation and only in the presence of a limiting number of T cells. These data indicate that both Lyb5+ and Lyb5- B cells are responsive to TD, TI-1, and TI-2 antigens in the presence of ancillary antigen-nonspecific help. They are also consistent with the notion that T helper cell activation of Lyb5+ B cells is H-2 nonrestricted and that T helper cell activation of Lyb5- B cells is H-2 restricted.

In vitro analysis of allogeneic lymphocyte interaction. VII. I-A-restricted self-reactive and alloreactive helper components of allogeneic effect factor are distinct donor T cell-derived Ia-molecules that recognize Ia determinants on antigen-presenting cells.

An allogeneic effect factor (AEF) derived from mixed lymphocyte reaction (MLR) cultures of alloactivated A.SW (H-2s) responder T cells and irradiated T cell-depleted A/WySn (H-2a) stimulator spleen cells was fractionated on the basis of molecular size and charge into two I-A-restricted helper components. The cellular origin of these components is believed to be an Lyt-1+2- -activated responder T helper (TH) cell. One alloreactive component, TIAH-1, recognizes allo-I-A determinants on an A/WySn antigen-presenting cell (APC). The other self-reactive component, TIAH-2, recognizes self-I-A determinants on an A.SW APC. The interaction of each of these components with the appropriate APC subsequently activates an in vitro primary anti-SRBC PFC response of either stimulator haplotype- or responder haplotype-derived B cells. These data demonstrate that the activity of TIAH-1 and TIAH-2 is dependent on the genotype of the APC and not the B cell, and that the target cell of action of these AEF TH components is an APC. TIAH-1 and TIAH-2 are 68,000 mol wt single polypeptide chains that have an isoelectric point (pI) of 5.8 and 5.5, respectively. Their charge difference is not attributable to altered amounts of sialylation or phosphorylation, but probably is due to other forms of altered glycosylation and/or to changes in their amino acid sequence. They share approximately 80% of their tryptic peptides and likely constitute homologous but nonidentical molecules. Papain cleaves TIAH-1 and TIAH-2 into a 40,000 mol wt fragment. TIAH-1 and TIAH-2 may represent structurally very related but nonidentical secreted forms of activated responder TH cell-derived receptors for allo-I-A and self-I-A determinants, respectively.