Tobacco Mosaic Virus Particles Research Articles

Magnetron sputtering deposition of ultra-thin metal coatings for the visualization of protein-containing objects of nanometer size by electron microscopy

Electron microscopy is widely used in biological and medical research, enabling to obtain images of the investigated objects with nanometer resolution. Exposure of biological objects under electron beam during the visualization can, however, cause their damage. Destructive effect of the electron beam can be significantly decreased by formation of metal coating on the surface of the objects to be studied. In the present study, an approach for the formation of ultra-thin metal coatings on the surface of protein-containing structures is proposed. Magnetron sputtering in argon plasma in DC mode was employed to form 8 Å-thick tungsten films on the surface of model protein-containing objects— tobacco mosaic virus particles — for their subsequent visualization by electron microscopy.

Atmospheric-pressure plasma irradiation can disrupt tobacco mosaic virus particles and RNAs to inactivate their infectivity.

Low-temperature atmospheric-pressure air plasma is a source of charged and neutral gas species. In this study, N-carrying tobacco plants were inoculated with plasma irradiated and non-irradiated tobacco mosaic virus (TMV) solution, resulting in necrotic local lesions on non-irradiated, but not on irradiated, TMV-inoculated leaves. Virus particles were disrupted by plasma irradiation in an exposure-dependent manner, but the viral coat protein subunit was not. TMV RNA was also fragmented in a time-dependent manner. These results indicate that plasma irradiation of TMV can collapse viral particles to the subunit level, degrading TMV RNA and thereby leading to a loss of infectivity.

Field-effect biosensor using virus particles as scaffolds for enzyme immobilization

A field-effect biosensor employing tobacco mosaic virus (TMV) particles as scaffolds for enzyme immobilization is presented. Nanotubular TMV scaffolds allow a dense immobilization of precisely positioned enzymes with retained activity. To demonstrate feasibility of this new strategy, a penicillin sensor has been developed by coupling a penicillinase with virus particles as a model system. The developed field-effect penicillin biosensor consists of an Al-p-Si-SiO2-Ta2O5-TMV structure and has been electrochemically characterized in buffer solutions containing different concentrations of penicillin G. In addition, the morphology of the biosensor surface with virus particles was characterized by scanning electron microscopy and atomic force microscopy methods. The sensors possessed a high penicillin sensitivity of ~ 92 mV/dec in a nearly-linear range from 0.1 mM to 10 mM, and a low detection limit of about 50 µM. The long-term stability of the penicillin biosensor was periodically tested over a time period of about one year without any significant loss of sensitivity. The biosensor has also been successfully applied for penicillin detection in bovine milk samples.

Speciation of Phenanthriplatin and Its Analogs in the Core of Tobacco Mosaic Virus.

Efficient loading of drugs in novel delivery agents has the potential to substantially improve therapy by targeting the diseased tissue while avoiding unwanted side effects. Here we report the first systematic study of the loading mechanism of phenanthriplatin and its analogs into tobacco mosaic virus (TMV), previously used by our group as an efficient carrier for anticancer drug delivery. A detailed investigation of the preferential uptake of phenanthriplatin in its aquated form (∼2000 molecules per TMV particle versus ∼1000 for the chlorido form) is provided. Whereas the net charge of phenanthriplatin analogs and their ionic mobilities have no effect on loading, the reactivity of aqua phenanthriplatin with the glutamates, lining the interior walls of the channel of TMV, has a pronounced effect on its loading. MALDI-MS analysis along with NMR spectroscopic studies of a model reaction of hydroxy-phenanthriplatin with acetate establish the formation of stable covalent adducts. The increased number of heteroaromatic rings on the platinum ligand appears to enhance loading, possibly by stabilizing hydrophobic stacking interactions with TMV core components, specifically Pro102 and Thr103 residues neighboring Glu97 and Glu106 in the channel. Electron transfer dissociation MS/MS fragmentation, a technique that can prevent mass-condition-vulnerable modification of proteins, reveals that Glu97 preferentially participates over Glu106 in covalent bond formation to the platinum center.

Tobacco Mosaic Virus with Peroxidase-Like Activity for Cancer Cell Detection through Colorimetric Assay.

Cell-based ELISA (CELLISA) has been widely used in disease diagnosis due to its simplicity and low cost. Recently, peroxidase-like nanomaterials have emerged as promising systems for CELLISA applications. In this work, tobacco mosaic virus (TMV) was simultaneously tailored with peroxidase-like inorganic nanoparticles (platinum nanoparticles) and cancer cell target groups (folic acid, FA) to obtain TMV-FA-Pt nanoparticles for cancer cell detection. Induced by the uniformly distributed reactive groups and well-defined structure of the TMV particle, platinum nanoparticles could be grown in situ on the exterior surface of TMV with excellent monodispersity and uniform spatial distribution. Meanwhile, FA with a PEG1000 linker was successfully conjugated to the coat proteins of TMV through the Cu(I)-catalyzed alkyne-azide cycloaddition reaction, an efficient "click" chemistry. Our study demonstrated that the resultant TMV-FA-Pt had specific affinity to cancer cells and was successfully used to detect cancer cells through CELLISA. Less than 1.0 × 104 cells/mL of cancer cells could be readily detected.

Covalent incorporation of tobacco mosaic virus increases the stiffness of poly(ethylene glycol) diacrylate hydrogels.

Hydrogels are versatile materials, finding applications as adsorbers, supports for biosensors and biocatalysts or as scaffolds for tissue engineering. A frequently used building block for chemically cross-linked hydrogels is poly(ethylene glycol) diacrylate (PEG-DA). However, after curing, PEG-DA hydrogels cannot be functionalized easily. In this contribution, the stiff, rod-like tobacco mosaic virus (TMV) is investigated as a functional additive to PEG-DA hydrogels. TMV consists of more than 2000 identical coat proteins and can therefore present more than 2000 functional sites per TMV available for coupling, and thus has been used as a template or building block for nano-scaled hybrid materials for many years. Here, PEG-DA (Mn = 700 g mol−1) hydrogels are combined with a thiol-group presenting TMV mutant (TMVCys). By covalent coupling of TMVCys into the hydrogel matrix via the thiol-Michael reaction, the storage modulus of the hydrogels is increased compared to pure PEG-DA hydrogels and to hydrogels containing wildtype TMV (wt-TMV) which is not coupled covalently into the hydrogel matrix. In contrast, the swelling behaviour of the hydrogels is not altered by TMVCys or wt-TMV. Transmission electron microscopy reveals that the TMV particles are well dispersed in the hydrogels without any large aggregates. These findings give rise to the conclusion that well-defined hydrogels were obtained which offer the possibility to use the incorporated TMV as multivalent carrier templates e.g. for enzymes in future studies.

TMV-Based Adapter Templates for Enhanced Enzyme Loading in Biosensor Applications.

Nanotubular tobacco mosaic virus (TMV) particles and RNA-free lower-order coat protein (CP) aggregates have been employed as enzyme carriers in different diagnostic layouts and compared for their influence on biosensor performance. In the following, we describe a label-free electrochemical biosensor for improved glucose detection by use of TMV adapters and the enzyme glucose oxidase (GOD). A specific and efficient immobilization of streptavidin-conjugated GOD ([SA]-GOD) complexes on biotinylated TMV nanotubes or CP aggregates was achieved via bioaffinity binding. Glucose sensors with adsorptively immobilized [SA]-GOD, and with [SA]-GOD cross-linked with glutardialdehyde, respectively, were tested in parallel on the same sensor chip. Comparison of these sensors revealed that TMV adapters enhanced the amperometric glucose detection remarkably, conveying highest sensitivity, an extended linear detection range and fastest response times. These results underline a great potential of an integration of virus/biomolecule hybrids with electronic transducers for applications in biosensorics and biochips. Here, we describe the fabrication and use of amperometric sensor chips combining an array of circular Pt electrodes, their loading with GOD-modified TMV nanotubes (and other GOD immobilization methods), and the subsequent investigations of the sensor performance.

Penicillin Detection by Tobacco Mosaic Virus-Assisted Colorimetric Biosensors.

The presentation of enzymes on viral scaffolds has beneficial effects such as an increased enzyme loading and a prolonged reusability in comparison to conventional immobilization platforms. Here, we used modified tobacco mosaic virus (TMV) nanorods as enzyme carriers in penicillin G detection for the first time. Penicillinase enzymes were conjugated with streptavidin and coupled to TMV rods by use of a bifunctional biotin-linker. Penicillinase-decorated TMV particles were characterized extensively in halochromic dye-based biosensing. Acidometric analyte detection was performed with bromcresol purple as pH indicator and spectrophotometry. The TMV-assisted sensors exhibited increased enzyme loading and strongly improved reusability, and higher analysis rates compared to layouts without viral adapters. They extended the half-life of the sensors from 4 - 6 days to 5 weeks and thus allowed an at least 8-fold longer use of the sensors. Using a commercial budget-priced penicillinase preparation, a detection limit of 100 µM penicillin was obtained. Initial experiments also indicate that the system may be transferred to label-free detection layouts.

Shear flow induced long-range ordering of rod-like viral nanoparticles within hydrogel

Controlling the alignment of anisotropic nanoparticles within a three-dimensional (3D) environment over large-scale is still a challenge. In this paper, a facile method to align rod-like nanoparticles in hydrogel via shear flow to afford a long-range order is reported. The rod-like tobacco mosaic virus (TMV) was employed as a prototypical anisotropic particles in the study, and the shear force provided by the unidirectional flow of the precursor solution direct the alignment of TMVs, which could be quickly fixed through the fast sol-gel transition in situ. The degree of orientation and the distance between the TMV particles could be regulated by adjusting the concentration of hydrogels and TMVs. While the introduction of TMVs could reduce the degree of swelling of the hydrogel and help maintaining the mechanical strength of resultant hydrogels, both repulsion interaction and shear flow contributed synergistically to the assembly. This method does not require the usage of strong magnetic or electric fields, nor does it require the use of specialized lithography, thus offers a facile way to the fabrication of hydrogel materials with control of anisotropic structural features.

Featured Article: Delivery of chemotherapeutic vcMMAE using tobacco mosaic virus nanoparticles.

The first-line treatment for non-Hodgkin's lymphoma is chemotherapy. While generally well tolerated, off-target effects and chemotherapy-associated complications are still of concern. To overcome the challenges associated with systemic chemotherapy, we developed a biology-inspired, nanoparticle drug delivery system (nanoDDS) making use of the nucleoprotein components of the tobacco mosaic virus (TMV). Virus-based nanoparticles, including the high-aspect ratio soft nanorods formed by TMV, are growing in popularity as nanoDDS due to their simple genetic and chemical engineerability, size and shape tunability, and biocompatibility. In this study, we used bioconjugation to modify TMV as a multivalent carrier for delivery of the antimitotic drug valine-citrulline monomethyl auristatin E (vcMMAE) targeting non-Hodgkin's lymphoma. We demonstrate successful synthesis of the TMV-vcMMAE; data indicate that the TMV-vcMMAE particles remained structurally sound with all of the 2130 identical TMV coat proteins modified to carry the therapeutic payload vcMMAE. Cell uptake using Karpas 299 cells was confirmed with TMV particles trafficking to the endolysosomal compartment, likely allowing for protease-mediated cleavage of the valine-citrulline linker for the release of the active monomethyl auristatin E component. Indeed, effective cell killing of non-Hodgkin's lymphoma invitro was demonstrated; TMV-vcMMAE was shown to exhibit an IC50 of ∼250 nM. This study contributes to the development of viral nanoDDS. Impact statement Due to side effects associated with systemic chemotherapy, there is an urgent need for the development of novel drug delivery systems. We focus on the high-aspect ratio nanotubes formed by tobacco mosaic virus (TMV) to deliver antimitotic drugs targeted to non-Hodgkin's lymphoma. Many synthetic and biologic nanocarriers are in the development pipeline; the majority of systems are spherical in shape. This may not be optimal, because high-aspect ratio filaments exhibit enhanced tumor homing, increased target cell interactions and decreased immune cell uptake, and therefore have favorable properties for drug delivery compared to their spherical counterparts. Nevertheless, the synthesis of high-aspect ratio materials at the nanoscale remains challenging; therefore, we turned toward the nucleoprotein components of TMV as a biologic nanodrug delivery system. This work presents groundwork for the development of plant virus-based vehicles for use in cancer treatment.

Calculation of the cross-sectional shape of a fibril from equatorial scattering.

An alternate formulation of helical diffraction theory is used to generate cross-sectional shapes of fibrous structures from equatorial scattering. We demonstrate this approach with computationally generated scattering intensities and then apply it to scattering data from Tobacco Mosaic Virus (TMV) and in vitro assembled fibrils of Aβ40 peptides. Refining the cross-sectional shape of TMV from SAXS data collected on a 26mg/ml solution resulted in a circular shape with outer diameter of ∼180Å and inner diameter of ∼40Å consistent with the known structure of TMV. We also utilized this method to analyze the equatorial scattering from TMV collected by Don Caspar from a concentrated (24% ∼295mg/ml) gel of TMV as reported in his Ph.D. thesis in 1955. This data differs from the SAXS data in having a sharp interference peak at ∼250Å spacing, indicative of strong interparticle interactions in the gel. Analysis of this data required consideration of interatomic vectors as long as 2000Å and resulted in generation of images that were interpreted as representative of local organization of TMV particles in the sample. Peaks in the images were separated, on average by about 250Å with a density consistent with Caspar's original measurements. Analysis of SAXS data from Aβ fibrils resulted in a cross-sectional shape that could be interpreted in terms of structural models that have been constructed from ssNMR and cryoEM. These results demonstrate an unexpected use of the small-angle region of fiber diffraction patterns to derive fundamental structural properties of scattering objects.

Key factors of scanning a plant virus with AFM in air and aqueous solution.

For tobacco mosaic virus (TMV) as a model virus, this article shows typical issues of scanning soft biological matter by atomic force microscopy (AFM). TMV adsorbed on chemically different flat surfaces, gold, mica, and APDMES-functionalized silicon, is studied in air and aqueous environment. In air, the TMV particles arrangement shows some variety, depending on the substrate. The height of TMV is reduced to 13.7, 15.8, and 15.6 nm, for gold, APDMES, and mica, respectively while the width is about ∼30 nm due to the influence of the tip radius. In aqueous solution, the surface charges of the virus and the solid support play an important role in the virus adsorption process. While deposition on negatively charged mica is favored only at low pH values, it is shown that positively charged APDMES functionalized silicon can be a suitable substrate to work with at neutral pHs. The effects of cantilever oscillation's free amplitude (A0 ) and the amplitude set-point (A) are also assessed here. While high A0 prompt reversible deformation of TMV in measurements performed in air, irreversible damage of the virus in liquid conditions (water) is observed using stiff cantilevers (0.35 Nm-1 ) and high A0 (81 nm), leading to a 6 nm reduction in the height of TMV after the first scan. Finally, low values of the amplitude set-point (A/A0 = 0.3), which means applying higher forces to the sample, also brings the damage of TMV virus assemblies, reducing its monolayer roughness to 0.3 nm. Microsc. Res. Tech. 80:18-29, 2017. © 2016 Wiley Periodicals, Inc.

Antiviral activity of glycoprotein GP-1 isolated from Streptomyces kanasensis ZX01

Plant virus diseases have seriously damaged global food security. However, current antiviral agents are not efficient enough for the requirement of agriculture production. So, developing new efficient and nontoxic antiviral agents is imperative. GP-1, from Streptomyces kanasensis ZX01, is a new antiviral glycoprotein, of which the antiviral activity and the mode of action against Tobacco mosaic virus (TMV) were investigated in this study. The results showed that GP-1 could fracture TMV particles, and the infection and accumulation of TMV in host plants were inhibited. Moreover, GP-1 could induce systematic resistance against TMV in the host, according to the results of activities of defensive enzymes increasing, MDA decreasing and overexpression of pathogenesis-related proteins. Furthermore, GP-1 could promote growth of the host plant. In conclusion, GP-1 showed the ability to be developed as an efficient antiviral agent and a fertilizer for agriculture.

Chimeric particles of tobacco mosaic virus as a platform for the development of next-generation nanovaccines

The production of vaccines and other proteins in plants for medical purposes offers a number of advantages over other expression systems. The tobacco mosaic virus (TMV) is an appropriate model for the development of a variety of vectors, including those used for the assembly of chimeric particles carrying heterologous peptides on the surface and inducing an effective immune response. To overcome the problems arising during the assembly of such particles from recombinant subunits of the coat protein, peptide linkers, suppressed stop codons, and proteolytic sites are used. To date, it has been shown that TMV-based nanovaccines provide protection against the viruses of influenza A, foot-and-mouth disease, papilloma, and they are also able to overcome B-cell tolerance for cancer-cell suppression. Genetically modified TMV-based virions can accommodate the ions of various metals and act as affinity agents for protein purification.

Benefits and Limitations of Low-kV Macromolecular Imaging of Frozen-Hydrated Biological Samples

Object contrast is one of the most important parameters of macromolecular imaging. Low-voltage transmission electron microscopy has shown an increased atom contrast for carbon materials, indicating that amplitude contrast contributions increase at a higher rate than phase contrast and inelastic scattering. Here, we studied image contrast using ice-embedded tobacco mosaic virus particles as test samples at 20–80 keV electron energy. The particles showed the expected increase in contrast for lower energies, but at the same time the 2.3-nm-resolution measure decayed more rapidly. We found a pronounced signal loss below 60 keV, and therefore we conclude that increased inelastic scattering counteracts increased amplitude contrast. This model also implies that as long as the amplitude contrast does not increase with resolution, beam damage and multiple scattering will always win over increased contrast at the lowest energies. Therefore, we cannot expect that low-energy imaging of conventionally prepared samples would provide better data than state-of-the-art 200–300 keV imaging.

Modified TMV Particles as Beneficial Scaffolds to Present Sensor Enzymes.

Tobacco mosaic virus (TMV) is a robust nanotubular nucleoprotein scaffold increasingly employed for the high density presentation of functional molecules such as peptides, fluorescent dyes, and antibodies. We report on its use as advantageous carrier for sensor enzymes. A TMV mutant with a cysteine residue exposed on every coat protein (CP) subunit (TMVCys) enabled the coupling of bifunctional maleimide-polyethylene glycol (PEG)-biotin linkers (TMVCys/Bio). Its surface was equipped with two streptavidin [SA]-conjugated enzymes: glucose oxidase ([SA]-GOx) and horseradish peroxidase ([SA]-HRP). At least 50% of the CPs were decorated with a linker molecule, and all thereof with active enzymes. Upon use as adapter scaffolds in conventional “high-binding” microtiter plates, TMV sticks allowed the immobilization of up to 45-fold higher catalytic activities than control samples with the same input of enzymes. Moreover, they increased storage stability and reusability in relation to enzymes applied directly to microtiter plate wells. The functionalized TMV adsorbed to solid supports showed a homogeneous distribution of the conjugated enzymes and structural integrity of the nanorods upon transmission electron and atomic force microscopy. The high surface-increase and steric accessibility of the viral scaffolds in combination with the biochemical environment provided by the plant viral coat may explain the beneficial effects. TMV can, thus, serve as a favorable multivalent nanoscale platform for the ordered presentation of bioactive proteins.

Atomic force microscope, molecular imaging, and analysis.

Image visibility is a central issue in analyzing all kinds of microscopic images. An increase of intensity contrast helps to raise the image visibility, thereby to reveal fine image features. Accordingly, a proper evaluation of results with current imaging parameters can be used for feedback on future imaging experiments. In this work, we have applied the Laplacian function of image intensity as either an additive component (Laplacian mask) or a multiplying factor (Laplacian weight) for enhancing image contrast of high-resolution AFM images of two molecular systems, an unknown protein imaged in air, provided by AFM COST Action TD1002 (http://www.afm4nanomedbio.eu/), and tobacco mosaic virus (TMV) particles imaged in liquid. Based on both visual inspection and quantitative representation of contrast measurements, we found that the Laplacian weight is more effective than the Laplacian mask for the unknown protein, whereas for the TMV system the strengthened Laplacian mask is superior to the Laplacian weight. The present results indicate that a mathematical function, as exemplified by the Laplacian function, may yield varied processing effects with different operations. To interpret the diversity of molecular structure and topology in images, an explicit expression for processing procedures should be included in scientific reports alongside instrumental setups.

Development of large-scale size-controlled adult pancreatic progenitor cell clusters by an inkjet-printing technique.

The generation of transplantable β-cells from pancreatic progenitor cells (PPCs) could serve as an ideal cell-based therapy for diabetes. Because the transplant efficiency depends on the size of islet-like clusters, it becomes one of the key research topics to produce PPCs with controlled cluster sizes in a scalable manner. In this study, we used inkjet printing to pattern biogenic nanoparticles, i.e., mutant tobacco mosaic virus (TMV), with different spot sizes to support the formation of multicellular clusters by PPCs. We successfully achieved TMV particle patterns with variable features and sizes by adjusting the surface wettability and printing speed. The spot sizes of cell-adhesive TMV mutant arrays were in the range of 50-150 μm diameter. Mouse PPCs were seeded on the TMV-RGD (arginine-glycine-aspartate)-patterned polystyrene (PS) substrate, which consists of areas that either favor (TMV-RGD) or prohibit (bare PS) cell adhesion. The PPCs stably attached, proliferated on top of the TMV-RGD support, thus resulting in the formation of uniform and confluent PPC clusters. Furthermore, the aggregated PPCs also maintained their multipotency and were positive for E-cadherin, indicating that the formation of cell-cell junctions is critical for enhanced cell-cell contact.

Silica-coated Gd(DOTA)-loaded protein nanoparticles enable magnetic resonance imaging of macrophages.

The molecular imaging of in vivo targets allows non-invasive disease diagnosis. Nanoparticles offer a promising platform for molecular imaging because they can deliver large payloads of imaging reagents to the site of disease. Magnetic resonance imaging (MRI) is often preferred for clinical diagnosis because it uses non-ionizing radiation and offers both high spatial resolution and excellent penetration. We have explored the use of plant viruses as the basis of for MRI contrast reagents, specifically Tobacco mosaic virus (TMV), which can assemble to form either stiff rods or spheres. We loaded TMV particles with paramagnetic Gd ions, increasing the ionic relaxivity compared to free Gd ions. The loaded TMV particles were then coated with silica maintaining high relaxivities. Interestingly, we found that when Gd(DOTA) was loaded into the interior channel of TMV and the exterior was coated with silica, the T1 relaxivities increased by three-fold from 10.9 mM-1 s-1 to 29.7 mM-1s-1 at 60 MHz compared to uncoated Gd-loaded TMV. To test the performance of the contrast agents in a biological setting, we focused on interactions with macrophages because the active or passive targeting of immune cells is a popular strategy to investigate the cellular components involved in disease progression associated with inflammation. In vitro assays and phantom MRI experiments indicate efficient targeting and imaging of macrophages, enhanced contrast-to-noise ratio was observed by shape-engineering (SNP > TMV) and silica-coating (Si-TMV/SNP > TMV/SNP). Because plant viruses are in the food chain, antibodies may be prevalent in the population. Therefore we investigated whether the silica-coating could prevent antibody recognition; indeed our data indicate that mineralization can be used as a stealth coating option to reduce clearance. Therefore, we conclude that the silica-coated protein-based contrast agent may provide an interesting candidate material for further investigation for in vivo delineation of disease through macrophage imaging.

Effect of chitosan on tobacco mosaic virus (TMV) accumulation, hydrolase activity, and morphological abnormalities of the viral particles in leaves of N. tabacum L. cv. Samsun.

The effect of chitosan on the development of infection caused by Tobacco mosaic virus (TMV) in leaves of Nicotiana tabacum L. cv. Samsun has been studied. It was shown that the infectivity and viral coat protein content in leaves inoculated with a mixture of TMV (2 μg/mL) and chitosan (1 mg/mL) were lower in the early period of infection (3 days after inoculation), by 63% and 66% respectively, than in leaves inoculated with TMV only. Treatment of leaves with chitosan 24 h before inoculation with TMV also caused the antiviral effects, but these were less apparent than when the virus and polysaccharide were applied simultaneously. The inhibitory effects of the agent decreased as the infection progressed. Inoculation of leaves with TMV together with chitosan considerably enhanced the activity of hydrolases (proteases, RNases) in the leaves, in comparison with leaves inoculated with TMV alone. Electron microscope assays of phosphotungstic acid (PTA)-stained suspensions from infected tobacco leaves showed that, in addition to the normal TMV particles (18 nm in diameter, 300 nm long), these suspensions contained abnormal (swollen, "thin" and "short") virions. The highest number of abnormal virions was found in suspensions from leaves inoculated with a mixture of TMV and chitosan. Immuno-electron microscopy showed that "thin" virus particles, in contrast to the particles of normal diameter, lost the ability to bind to specific antiserum. It seems that the chitosan-induced activation of hydrolases stimulates the intracellular degradation of TMV particles and hence hydrolase activation may be considered to be one of the polysaccharide-mediated cellular defense mechanisms that limit virus accumulation in cells.