Tobacco Mosaic Research Articles

High-yield production of a functional bacteriophage lysin with antipneumococcal activity using a plant virus-based expression system

Streptococcus pneumoniae is the causative agent of several serious infectious diseases. It is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are needed. One alternative to antibiotics may be the use of peptidoglycan hydrolases, the bacteriophage lytic enzymes.In this study, we demonstrated high level expression of the S. pneumoniae bacteriophage lysin Pal in Nicotiana benthamiana – TMV (Tobacco Mosaic Virus) transient expression system. The protein was purified to homogeneity and tested for streptococci killing activity in vitro and in vivo. In vitro, Pal was able to lyse three tested S. pneumoniae strains: NCTC12695, NCTC12977 and NCTC11888. The treatment of BALB/c mice with 100μg, 200μg and 400μg of Pal 1h post-challenge with double lethal dose of S. pneumoniae NCTC12695 strain showed a clear dose response and protected from lethal sepsis 30%, 40% and 50% of mice, respectively. The improved mice survival correlated with decreased blood bacterial titers. In conclusion, these results suggest that plant-expressed bacteriophage lysins may have potential use as antimicrobial agents.

PeBL1, a novel protein elicitor from Brevibacillus laterosporus strain A60, activates defense responses and systemic resistance in Nicotiana benthamiana.

We report the identification, characterization, and gene cloning of a novel protein elicitor (PeBL1) secreted from Brevibacillus laterosporus strain A60. Through a purification process consisting of ion-exchange chromatography and high-performance liquid chromatography (HPLC), we isolated a protein that was identified by electrospray ionization quadrupole time of flight tandem mass spectrometry (ESI-Q-TOF-MS-MS). The 351-bp PeBL1 gene produces a 12,833-Da protein with 116 amino acids that contains a 30-residue signal peptide. The PeBL1 protein was expressed in Escherichia coli. The recombinant protein can induce a typical hypersensitive response (HR) and systemic resistance in Nicotiana benthamiana, like the endogenous protein. PeBL1-treated N. benthamiana exhibited strong resistance to the infection of tobacco mosaic virus-green fluorescent protein (TMV-GFP) and Pseudomonas syringae pv. tabaci compared to control N. benthamiana. In addition, PeBL1 triggered a cascade of events that resulted in defense responses in plants, including reactive oxygen species (ROS) production, extracellular-medium alkalization, phenolic-compound deposition, and expression of several defense-related genes. Real-time quantitative-PCR analysis indicated that the known defense-related genes PR-1, PR-5, PDF1.2, NPR1, and PAL were upregulated to varying degrees by PeBL1. This research not only provides insights into the mechanism by which beneficial bacteria activate plant systemic resistance, but also sheds new light on a novel strategy for biocontrol using strain A60.

Site-selective nucleation and controlled growth of gold nanostructures in tobacco mosaic virus nanotubulars.

Site-selective biomineralization of Au nanostructures in the interior channel of Tobacco Mosaic Virus (TMV) is achieved by mutating threonine 103 in TMV to cysteine (T103C-TMV) to introduce the strong coordination interaction between the arrayed sulfhydryl ligands and gold species. By finely tuning the reaction conditions, Au nanoparticle chains and Au nanorods are successfully and exclusively synthesized inside the T103C-TMV nanotubes.

RNA-controlled assembly of tobacco mosaic virus-derived complex structures: from nanoboomerangs to tetrapods.

The in vitro assembly of artificial nanotubular nucleoprotein shapes based on tobacco mosaic virus-(TMV-)-derived building blocks yielded different spatial organizations of viral coat protein subunits on genetically engineered RNA molecules, containing two or multiple TMV origins of assembly (OAs). The growth of kinked nanoboomerangs as well as of branched multipods was determined by the encapsidated RNAs. A largely simultaneous initiation at two origins and subsequent bidirectional tube elongation could be visualized by transmission electron microscopy of intermediates and final products. Collision of the nascent tubes' ends produced angular particles with well-defined arm lengths. RNAs with three to five OAs generated branched multipods with a maximum of four arms. The potential of such an RNA-directed self-assembly of uncommon nanotubular architectures for the fabrication of complex multivalent nanotemplates used in functional hybrid materials is discussed.

Differential Effects of Viral Coat Protein on Induction of Hypersensitive Response and Systemic Movement of Tobacco Mosaic Virus and Tobacco Mild Green Mosaic Virus in Nicotiana megalosiphon

Tobacco mosaic virus (TMV) and Tobacco mild green mosaic virus (TMGMV) are both known to induce hypersensitive response (HR) local lesions in Nicotiana megalosiphon, a hybrid plant from a cross between N. suaveolens and N. fragrans, but only TMV is capable of systemic movement. Therefore, the determinants of induction of hypersensitive response and systemic movement of TMV and TMGMV in N. megalosiphon were further analyzed. HR was shown to be independent of the temperature indicating that the resistance responses are different from N gene responses induced in tobacco (N. tabacum cv. Xanthi-nc.) to TMV. Comparison of lesion growth between wild-type and transgenic tobacco plants encoding salicylate hydroxylase (nahG) indicated that TMV spread similarly in N. megalosiphon and Xanthi-nc./nahG plants. In addition, exogenous application of SA did not prevent systemic movement of TMV. Coat protein-deficient TMV failed to induce HR and move systemically in N. megalosiphon indicating that CP is the inducer of HR and determinant for systemic movement. However, single epidermal cells expressing TMV-CP did not undergo cell death suggesting that formation of HR cell death requires viral movement out of the initially infected epidermal cells or the presence of intact virus particles. Furthermore, comparison of different TMV strains, including virus vector carrying the CP of TMGMV-U5 in place of TMV-CP, showed no differences in the induction of HR and timing of systemic virus movement suggesting that CP is not the determinant for differential invasion of TMV and TMGMV.

Genome-Wide Characterization of miRNAs Involved in N Gene-Mediated Immunity in Response to Tobacco Mosaic Virus in Nicotiana benthamiana.

microRNAs (miRNAs) are a class of endogenous small RNAs (sRNAs) that play pivotal roles in plant development, abiotic stress response, and pathogen response. miRNAs have been extensively studied in plants, but rarely in Nicotiana benthamiana, despite its wide use in plant virology studies, particularly for studying N protein–tobacco mosaic virus (TMV) interactions. We report an efficient method using high-throughput sequencing and bioinformatics to identify genome-wide miRNAs in N. benthamiana. A total of 30 conserved miRNA families and 113 novel miRNAs belonging to 93 families were identified. Some miRNAs were clustered on chromosomes, and some were embedded in host gene introns. The predicted miRNA targets were involved in diverse biological processes, such as metabolism, signaling, and responses to stimuli. miRNA expression profiling revealed that most of them were differentially expressed during N-mediated immunity to TMV. This study provides a framework for further analysis of miRNA functions in plant immunity.

Nanomanufacturing of Tobacco Mosaic Virus-Based Spherical Biomaterials Using a Continuous Flow Method.

Nanomanufacturing of nanoparticles is critical for potential translation and commercialization. Continuous flow devices can alleviate this need through unceasing production of nanoparticles. Here we demonstrate the scaled-up production of spherical nanoparticles functionalized with biomedical cargos from the rod-shaped plant virus tobacco mosaic virus (TMV) using a mesofluidic, continued flow method. Production yields were increased 30-fold comparing the mesofluidic device versus batch methods. Finally, we produced MRI contrast agents of select sizes, with per particle relaxivity reaching 979,218 mM–1 s–1 at 60 MHz. These TMV-based spherical nanoparticle MRI contrast agents are in the top echelon of relaxivity per nanoparticle.

Weed Control in Sweet Bell Pepper Using Sequential Postdirected Applications of Pelargonic Acid

Pepper (Capsicum annuum) producers would benefit from additional herbicide options that are safe to the crop and provide effective weed control. Research was conducted in southeastern Oklahoma (Atoka County, Lane, OK) during 2010 and 2011 to determine the impact of pelargonic acid on weed control efficacy, crop injury, and pepper yields. The experiment included pelargonic acid applied unshielded postdirected at 5, 10, and 15 lb/acre, plus an untreated weedy control and an untreated weed-free control. ‘Jupiter’ sweet bell pepper, a tobacco mosaic virus-resistant sweet pepper with a 70-day maturity, was transplanted into single rows on 3-ft centered raised beds with 18 inches between plants (9680 plants/acre) on 28 May 2010 and 27 May 2011, respectively. Weeds included smooth crabgrass (Digitaria ischaemum), cutleaf groundcherry (Physalis angulata), spiny amaranth (Amaranthus spinosus), and yellow nutsedge (Cyperus esculentus). Pelargonic acid was applied postdirected each year in mid-June and then reapplied 8 days later. The 15-lb/acre pelargonic acid treatment resulted in the maximum smooth crabgrass control (56%) and broadleaf weed control (66%) at 1 day after the initial spray treatment (DAIT), and 33% yellow nutsedge control at 3 DAIT. Pelargonic acid at 15 lb/acre provided equal or slightly greater smooth crabgrass and broadleaf (cutleaf groundcherry and spiny amaranth) control compared with the 10-lb/acre application, and consistently greater control than the 5-lb/acre rate and the weedy control. Pelargonic acid was less effective at controlling yellow nutsedge than smooth crabgrass and broadleaf weeds. As the rate of pelargonic acid increased from 5 to 15 lb/acre, yellow nutsedge control also increased significantly for all observation dates. Increasing the pelargonic acid application rate increased the crop injury rating. The maximum crop injury occurred for each application rate at 1 DAIT with 7%, 8.0%, and 13.8% injury for pelargonic acid rates 5, 10, and 15 lb/acre, respectively. There was little or no new crop injury after the second postdirected application of pelargonic acid and crop injury following 3 DAIT for application rates was 2% or less. Only the 15-lb/acre pelargonic acid application produced greater fruit per hectare (4784 fruit/ha) and yields (58.65 kg·ha−1) than the weedy control (1196 fruit/ha and 19.59 kg·ha−1). The weed-free yields (7176 fruit/ha, 178.11 kg·ha−1, and 24.82 g/fruit) were significantly greater than all pelargonic acid treatments and the weedy control. Pelargonic acid provided unsatisfactory weed control for all rates and did not significantly benefit from the sequential applications. The authors suggest the pelargonic acid be applied to smaller weeds to increase the weed control to acceptable levels (>80%).

Trans-Encapsidation of Flock House virus with Tobacco Mosaic virus Structural Proteins

Flock House virus (FHV) encodes a viral polymerase supporting autonomous replication of the FHV genome which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. As FHV viral genome size is strictly limited by native FHV capsid, the goal of this study was to determine if this restriction could be eliminated by inserting a non-native viral packaging signal from Tobacco Mosaic virus (TMV) to allow for trans-encapsidation by TMV coat. FHV was adapted to express enhanced green fluorescent protein (GFP) and several FHV-GFP expressing clones were generated from which full length RNA transcripts were generated. Fluorescent microscopy was used to confirm GFP expression after FHV-GFP RNA transfection into baby hamster kidney (BHK-21) cells. The TMV origin of assembly (OA) was introduced into the most robust GFP expressing FHV construct and GFP expression was used to ensure functional FHV RNA 1 activity. RNA from the best clone, FHV-C2-GFP-OA, was then mixed with TMV coat protein and monitored for encapsidation. The production of TMV-like rod shaped nanoparticles indicated that FHV-GFP-OA RNA can be encapsidated in vitro by purified TMV coat protein. This is the first demonstration of replication-independent but specific encapsidation/packaging of the FHV genome by heterologous plant viral structural proteins while maintaining FHV genome replication capacity.

Probing Viral Genomic Structure: Alternative Viewpoints and Alternative Structures for Satellite Tobacco Mosaic Virus RNA

Viral RNA structure prediction is a valuable tool for development of drugs against viral disease. This work discusses different approaches to predicting encapsidated viral RNA and highlights satellite tobacco mosaic virus (STMV) RNA as a model system with excellent crystallography data. Fundamentally important issues for debate include thermodynamic versus kinetic control of virus assembly and the possible consequences of quasi-species in the primary structure on RNA secondary structure prediction of a single structure or an ensemble of structures. Multiple computational tools and chemical reagents are now available for improved viral RNA structure prediction. Two different predicted structures for encapsidated STMV RNA result from differences in three main areas: a different approach and philosophy to studying encapsidated viral RNA, an emphasis on different RNA motifs, and technical differences in computational methods and chemical reagents. The experiments with traditional chemical probing and SHAPE reagents are compared in terms of chemistry, results, and interpretation for STMV RNA as well as other RNA protein assemblies, such as the 5'UTR of HIV and the ribosome. This discussion of the challenges of viral RNA structure prediction will lead to new experiments and improved future predictions for viral RNA.

Myosins VIII and XI play distinct roles in reproduction and transport of tobacco mosaic virus.

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.

Nanoparticle encapsidation of Flock house virus by auto assembly of Tobacco mosaic virus coat protein.

Tobacco Mosaic virus (TMV) coat protein is well known for its ability to self-assemble into supramolecular nanoparticles, either as protein discs or as rods originating from the ~300 bp genomic RNA origin-of-assembly (OA). We have utilized TMV self-assembly characteristics to create a novel Flock House virus (FHV) RNA nanoparticle. FHV encodes a viral polymerase supporting autonomous replication of the FHV genome, which makes it an attractive candidate for viral transgene expression studies and targeted RNA delivery into host cells. However, FHV viral genome size is strictly limited by native FHV capsid. To determine if this packaging restriction could be eliminated, FHV was adapted to express enhanced green fluorescent protein (GFP), to allow for monitoring of functional FHV RNA activity. Then TMV OA was introduced in six 3' insertion sites, with only site one supporting functional FHV GFP expression. To create nanoparticles, FHV GFP-OA modified genomic RNA was mixed in vitro with TMV coat protein and monitored for encapsidation by agarose electrophoresis and electron microscopy. The production of TMV-like rod shaped nanoparticles indicated that modified FHV RNA can be encapsidated by purified TMV coat protein by self-assembly. This is the first demonstration of replication-independent packaging of the FHV genome by protein self-assembly.

A Novel Method to Determine the Resistance of Biotemplated Nanowires

Genetically engineered Tobacco Mosaic Virus (TMV) was coated with uniform layers of palladium metal particles in aqueous solution without the aid of an external reducing agent while producing nanowires of 300 nm in length and 30–60 nm in diameter. By combining a gold nanogap approach and electron beam assisted Pt deposition approach, an innovative electrical characterization technique has been developed in order to determine the resistance of a single Pallidium-Tobacco Mosaic Virus (Pd-TMV) nanorod. The preliminary results showed that these nanoparticles are indeed electrically conductive and that this technique can provide resistance measurements of these types of biotemplated nanoparticles.

An electrochemical sensor for selective TNT sensing based on Tobacco mosaic virus-like particle binding agents.

This paper presents a selective differential sensing method based on diffusion modulation of the target molecules through suspended Tobacco mosaic virus-like particles modified with binding peptides for TNT sensing in solution.

Effects of Different Treatments on the Inactivation of Various Seedborne Viruses in Some Vegetables

In this study, we considered the efficacy of seed treatments for the inactivation of some seedborne viruses in tomato, pepper, melon, squash, bean and lettuce seeds, which are essential for human nutrition and seed production in our country. A total of 325 seed samples obtained from various farmers and foundations were tested by DAS-ELISA and RT-PCR procedures. Eight seed lots infected with Tomato mosaic tobamovirus (ToMV), Tobacco mosaic tobamovirus (TMV), Cucumber mosaic cucumovirus (CMV), Soybean mosaic potyvirus (SMV) and Lettuce mosaic potyvirus (LMV) were selected as research materials. Virus inactivation treatments were made by using acetic acid (CH3COOH), hydrogen peroxide (H2O2), hydrochloric acid (HCl), sodium hypochlorite (NaClO), Triton X 100, dry heat, heated water, ozone (O3), and UV (305 nm wavelength). The most effective treatments for reducing virus concentration were HCl, heated water (65 °C) and ozone (10 g m−3). These treatments reduced concentrations of seed-borne viruses in ranges of 51%, 42%, and 32%, respectively. Other treatments were less effective and reduced virus concentrations in the range of 27%–12%. HCl and ozone treatments were the most effective and applicable methods because they did not have negative effect on seed germination.

Transient viscoelasticity study of tobacco mosaic virus/Ba2+ superlattice

Recently, we reported a new method to synthesize the rod-like tobacco mosaic virus (TMV) superlattice. To explore its potentials in nanolattice templating and tissue scaffolding, this work focused the viscoelasticity of the superlattice with a novel transient method via atomic force microscopy (AFM). For measuring viscoelasticity, in contrast to previous methods that assessed the oscillating response, the method proposed in this work enabled us to determine the transient response (creep or relaxation) of micro/nanobiomaterials. The mathematical model and numerical process were elaborated to extract the viscoelastic properties from the indentation data. The adhesion between the AFM tip and the sample was included in the indentation model. Through the functional equation method, the elastic solution for the indentation model was extended to the viscoelastic solution so that the time dependent force vs. displacement relation could be attained. To simplify the solving of the differential equation, a standard solid model was modified to obtain the elastic and viscoelastic components of the sample. The viscoelastic responses with different mechanical stimuli and the dynamic properties were also investigated.

Examination of the leaf proteome during flooding stress and the induction of programmed cell death in maize.

BackgroundMaize is a major economic crop worldwide, with substantial crop loss attributed to flooding. During a stress response, programmed cell death (PCD) can be an effective way for plants better adapt. To identify flooding stress related PCD proteins in maize leaves, proteomic analysis was performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) and mass spectrometry.ResultsComparative proteomics was combined with physiological and biochemical analysis of maize leaves under flooding stress. Fv/Fm, qP, qN and relative water content (RWC) were found to be altered in response to flooding stress, with an increase in H2O2 content noted in vivo. Furthermore, DNA ladder detection indicated that PCD had occurred under flooding treatment. The maize leaf proteome was analyzed via 2D-DIGE gel, with a total of 32 differentially expressed spots isolated, 31 spots were successfully identified via MALDI-TOF/TOF MS which represent 28 proteins. The identified proteins were related to energy metabolism and photosynthesis, PCD, phytohormones and polyamines. To better characterize the role of translationally controlled tumor protein (TCTP) in PCD during a stress response, mRNA expression was examined in different plants by stress-induced PCD. These included heat stress induced rice protoplasts, Tobacco Mosaic Virus infected tobacco leaves and dark induced rice and Arabidopsis thaliana leaves, all of which showed active PCD, and TCTP expression was increased in different degrees. Moreover, S-adenosylmethionine synthase 2 (SAMS2) and S-adenosylmethionine decarboxylase (SAMDC) mRNA expression were also increased, but ACC synthase (ACS) and ACC oxidase (ACO) mRNA expression were not found in maize leaves following flooding. Lastly, ethylene and polyamine concentrations were increased in response to flooding treatment in maize leaves.ConclusionsFollowing flooding stress, the photosynthetic systems were damaged, resulting in a disruption in energy metabolism, with the noted photosynthetic decline also possibly attributed to ROS production. The observed PCD could be regulated by TCTP with a possible role for H2O2 in TCTP induction under flooding stress. Additionally, increased SAMS2 expression was closely associated with an increased polyamine synthesis during flooding treatment.

Tobacco mosaic virus efficiently targets DC uptake, activation and antigen-specific T cell responses in vivo

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8+ T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.

English

Tomato crop is primarily infected by various common diseases like Bacterial Canker, bird's-eye fruit spots, Bacterial Spot, Chlorosis, Curly Top, Early Blight, Fusarium Wilt, Gray Leaf, Gray Mold Rot, Leaf Mold, Leaf Roll and Leaf Curl, Powdery mildew, Septoria Leaf Spot, Tobacco Mosaic Virus, Verticillium Wilt. The presented work describes a algorithm for different disease detection based on the infected images of various tomato plants. Images of the infected tomato plants are captured by closed circuit CCD cameras to cover approximately 5 sq. meter area that could acquire good quality images of tomato crop. The acquired images are in jpeg format and are converted to gray scale image. The gray scale image are the enhanced and made noise free. The Otsu algorithm is applied in order to get the thresholded image. The segmentation techniques based in pixel neighborhood are applied to get the segmented leaf and infected part of the leaf. The methods evolved in this system are both image processing and soft computing technique applied on number of diseased tomato plant images. The tomato images are acquired by using a CCD camera of approx. 3 M-Pixel resolution in 24-bits color resolution. The images are then transferred to PC and represented in MATLAB software. The RGB image is then segmented using K-means algorithm for segmentation of fungus in the tomato crop. The segmented fungus part is now analyzed for its percentage presence.

Optimization and Utilization of <em>Agrobacterium</em>-mediated Transient Protein Production in <em>Nicotiana</em>

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana × N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).