Inactivation of tobacco mosaic virus using gamma irradiation and its potential modes of action.

Gamma irradiation is a non-thermal processing technique used to disinfect harmful microorganisms in agriculture. This technology has been shown to be an effective method to control bacterial and fungal plant pathogens. However, its effect on viral plant pathogen is less understood. Gamma irradiation was evaluated for the inactivation of tobacco mosaic virus (TMV). TMV infectivity has gradually decreased following irradiation in a dose-dependent manner and virus was completely inactivated at a dose over 40 kGy. Transmission electron microscopy revealed that increased gamma irradiation disrupts the virion structure and degrades viral proteins, which results in TMV inactivation. The mechanisms, through which gamma irradiation inactivates TMV, can be directly associated with the damage to the virus constituents.

Combined small angle X-ray solution scattering with atomic force microscopy for characterizing radiation damage on biological macromolecules.

BACKGROUND: Synchrotron radiation facilities are pillars of modern structural biology. Small-Angle X-ray scattering performed at synchrotron sources is often used to characterize the shape of biological macromolecules. A major challenge with high-energy X-ray beam on such macromolecules is the perturbation of sample due to radiation damage. RESULTS: By employing atomic force microscopy, another common technique to determine the shape of biological macromolecules when deposited on flat substrates, we present a protocol to evaluate and characterize consequences of radiation damage. It requires the acquisition of images of irradiated samples at the single molecule level in a timely manner while using minimal amounts of protein. The protocol has been tested on two different molecular systems: a large globular tetremeric enzyme (ß-Amylase) and a rod-shape plant virus (tobacco mosaic virus). Radiation damage on the globular enzyme leads to an apparent increase in molecular sizes whereas the effect on the long virus is a breakage into smaller pieces resulting in a decrease of the average long-axis radius. CONCLUSIONS: These results show that radiation damage can appear in different forms and strongly support the need to check the effect of radiation damage at synchrotron sources using the presented protocol.

Influence of high hydrostatic pressure on epitope mapping of tobacco mosaic virus coat protein.

In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP.

Photonic approach to the selective inactivation of viruses with a near-infrared subpicosecond fiber laser.

We report a photonic approach for selective inactivation of viruses with a near-infrared subpicosecond laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic viruses such as the M13 bacteriophage and the tobacco mosaic virus to pathogenic viruses such as the human papillomavirus and the human immunodeficiency virus (HIV). At the same time, sensitive materials such as human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed. The laser technology targets the global mechanical properties of the viral protein shell, making it relatively insensitive to the local genetic mutation in the target viruses. As a result, the approach can inactivate both the wild and mutated strains of viruses. This intriguing advantage is particularly important in the treatment of diseases involving rapidly mutating viral species such as HIV. Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the treatment of blood-borne viral diseases in the clinic.

Nonthermal microwave radiations affect the hypersensitive response of tobacco to tobacco mosaic virus.

OBJECTIVES: The aim of the present study was to evaluate the effects of nonthermal extremely high-frequency microwave radiations in a plant-based bioassay, represented by tobacco plants reacting to tobacco mosaic virus with a hypersensitive response leading to the appearance of necrotic lesions at the infection sites. DESIGN: This study was performed blind and different experimental protocols on tobacco plants inoculated with tobacco mosaic virus were used. BIO-OBJECTS: Tobacco plants (Nicotiana tabacum L. cultivar Samsun) carrying the resistance gene N against tobacco mosaic virus. INTERVENTIONS: Tobacco plants or leaf disks were either directly or indirectly (water-mediated) irradiated using a medical device, designed for microwave resonance therapy. It produces nonthermal weak-intensity extremely high-frequency radiations, either modulated at extremely low frequency or in continuous flux of waves, coupled with a nonthermal red/near-infrared radiation. OUTCOME MEASUREMENTS: The working variable was the number of hypersensitive lesions per leaf disk. RESULTS: Both direct and indirect nonthermal microwave radiations led to significant effects on the hypersensitive response of tobacco plants: modulated radiations generally induced a resistance increase, whereas a continuous flux of waves induced a resistance decrease with direct treatments only. CONCLUSIONS: Nonthermal microwave radiations are effective on the hypersensitive response of tobacco to tobacco mosaic virus and their low-frequency modulation seems to be more bioactive than the continuous-flux of waves, particularly in the indirect water-mediated treatments.

Chemical degradation of tobacco mosaic virus followed by infectivity assay, reverse transcription-polymerase chain reaction and gel electrophoresis.

In order to determine the detection limit for chemically treated virions by gel electrophoresis, reverse transcription-polymerase chain reaction (RT-PCR) and infectivity assay, tobacco mosaic virus (TMV) exposed to various concentrations of chemicals was studied. When virions were exposed to 0.2 N HCl for 30 mins, partially degraded TMV particles were observed by gel electrophoresis. Under the same exposure, a major RT-PCR amplified DNA product corresponding to the target size of 806 bp, which decreased as a function of time, could be detected for up to 60 mins of exposure. When virions were treated with NaOH (0.02 N or higher normality) for 5 mins, partially degraded virions were detected by gel electrophoresis, exhibiting multiple band patterns. Exposure of the virions to 0.1 N NaOH for 5 mins revealed severely degraded viral RNA, but disappearance of the amplified RT-PCR products was apparent during 30-60 mins of exposure. Therefore, these data showed clearly the difference in the detection limit of gel electrophoresis and that of RT-PCR for the degraded viral RNA. In addition, the infectivity assay showed that the number of local lesions in Nicotiana rustica were significantly reduced by more than 95% when the virus was exposed to 0.2 N HCl for 15 mins or 0.1 N NaOH for 10 mins. From these results we conclude that loss of infectivity was not related to that of PCR product. Other chemical disinfectants such as phenol or formalin were also found to be effective to reduce the virus infectivity, but a corresponding degradation of viral RNA was detected by neither gel electrophoresis nor RT-PCR.

Visualizing "greengold" clusters in the STEM.

"Greengold" is a large metallic cluster thought to contain 75 gold atoms in a compact 1.4-nm-diameter core surrounded by an organic shell. Scanning transmission electron microscope imaging shows uniform mass and size distributions with an apparent mass of 24 kDa, unaffected by radiation damage. The signal-to-noise ratio is adequate for visualization at low dose and in the presence of a relatively thick biological matrix. Under some conditions these clusters have a slight tendency to form linear chains and 2-D hexagonal arrays with a spacing of 2.6 nm. The parameters presented permit estimation of the feasibility of proposed labeling experiments.

Spectral quality affects disease development of three pathogens on hydroponically grown plants.

Plants were grown under light-emitting diode (LED) arrays with various spectra to determine the effects of light quality on the development of diseases caused by tomato mosaic virus (ToMV) on pepper (Capsicum annuum L.), powdery mildew [Sphaerotheca fuliginea (Schlectend:Fr.) Pollaci] on cucumber (Cucumis sativus L.), and bacterial wilt (Pseudomonas solanacearum Smith) on tomato (Lycopersicon esculentum Mill.). One LED (660) array supplied 99% red light at 660 nm (25 nm bandwidth at half-peak height) and 1% far-red light between 700 to 800 nm. A second LED (660/735) array supplied 83% red light at 660 nm and 17% far-red light at 735 nm (25 nm bandwidth at half-peak height). A third LED (660/BF) array supplied 98% red light at 660 nm, 1% blue light (BF) between 350 to 550 nm, and 1% far-red light between 700 to 800 nm. Control plants were grown under broad-spectrum metal halide (MH) lamps. Plants were grown at a mean photon flux (300 to 800 nm) of 330 micromoles m-2 s-1 under a 12-h day/night photoperiod. Spectral quality affected each pathosystem differently. In the ToMV/pepper pathosystem, disease symptoms developed slower and were less severe in plants grown under light sources that contained blue and UV-A wavelengths (MH and 660/BF treatments) compared to plants grown under light sources that lacked blue and UV-A wavelengths (660 and 660/735 LED arrays). In contrast, the number of colonies per leaf was highest and the mean colony diameters of S. fuliginea on cucumber plants were largest on leaves grown under the MH lamp (highest amount of blue and UV-A light) and least on leaves grown under the 660 LED array (no blue or UV-A light). The addition of far-red irradiation to the primary light source in the 660/735 LED array increased the colony counts per leaf in the S. fuliginea/cucumber pathosystem compared to the red-only (660) LED array. In the P. solanacearum/tomato pathosystem, disease symptoms were less severe in plants grown under the 660 LED array, but the effects of spectral quality on disease development when other wavelengths were included in the light source (MH-, 660/BF-, and 660/735-grown plants) were equivocal. These results demonstrate that spectral quality may be useful as a component of an integrated pest management program for future space-based controlled ecological life support systems.

Space microbiology--lethality, mutagenicity and cytological effects of terrestrial microorganisms by irradiation of cosmic proton under simulated space condition.

We have been discussing in connection with a space quarantine. The subject is not merely an academic problem, but it contains a fundamental problem which avoid the contamination of other planets by terrestrial microflora. The space environments in the solar system were simulated by using an apparatus of cryostat (low temperature of 110-310K, high vacuum of 1 x 10(-8) torr) and proton irradiation from the Van de Graaff generator. After exposure to a barrage of protons corresponding to about 250 years in solar space, Tobacco mosaic virus, Bacillus subtilis spore, Staphylococcus aureus. Micrococcusflavus, Clostridium mangenoti spore and Aspergillus niger spore showed considerably high survival rates. Furthermore, it was found firstly that an irradiation of proton induced considerable mutation frequency compared to that of spontaneous and caused also the cytological effects based on a damage of chromosome.

Image contrast in high-resolution electron microscopy of biological macromolecules: TMV in ice.

It is shown that the contrast in high-resolution electron micrographs of biological macromolecules, illustrated by a study of TMV in ice, falls considerably below the level which should theoretically be attained. The factors which contribute to the low contrast include radiation damage, inelastic scattering, specimen movement and charging. Future progress depends on improved understanding of their contributions and relative importance. Contrast is defined as the amplitude of a particular Fourier component extracted from an image in comparison to that expected by extrapolation from separate electron or X-ray diffraction measurements. The fall in contrast gets worse with increased resolution and is particularly serious at 10 A and beyond for specimens embedded in vitreous ice, a method of specimen preparation which is otherwise particularly desirable because of the expectation that the embedded molecules should be well preserved in a near-native environment. This low contrast at high resolution is the principal limitation to atomic-resolution structure determination by electron microscopy. In spite of good progress in the direction of better images, it remains a major problem which prevents electron microscopy from becoming a simple and rapid method for biological atomic structure determination.

[Identification of a peptide of the membrane protein of tobacco mosaic virus, cross-linked with intraviral RNA under the effect of UV-radiation]. / Identifikatsiia peptida belka obolochki virusa tabanoi mozaiki, sshivaiushchegosiia s vnutrivirusnoi RNK pod deistviem UF- izlucheniia.

As reported previously, UV-irradiation induces crosslinking between tobacco mosaic virus (TMV) coat protein molecules and intraviral RNA nucleotides. We have irradiated [3H]-uridine labeled TMV and isolated TMV coat protein subunits with the attached nucleotide label. These TMV protein subunits were hydrolyzed with trypsin. The tryptic peptides were separated by high-performance liquid chromatography and [3H]-labeled peptides were identified. The UV-irradiation of TMV was found to result in crosslinking to intraviral RNA of the T8 tryptic peptide (residues 93-112) of TMV coat protein.

UV laser induced RNA-protein crosslinks and RNA chain breaks in tobacco mosaic virus RNA in situ.

The efficiency of RNA-protein crosslink and RNA chain break formation under nanosecond or picosecond UV-laser pulse irradiation of tobacco mosaic virus was determined. It was found that on high-intensity UV-laser irradiation the quantum yields of both reactions increase considerably as compared to the usual (low-intensity) UV-irradiation. The RNA-protein crosslink quantum yield was found to be 1.8 x 10(-5) and 1.2 x 10(-4) and that of RNA chain breaks 1.7 x 10(-4) and 8.9 x 10(-4) for nanosecond and picosecond irradiation, respectively.

[Difference in the mechanism of self-assembly in vitro in tobacco mosaic virus and potato virus X]. / Razlichiia v mekhanizmakh samosborki in vitro virusa tabachnoi mozaiki i X-virusa kartofelia.

The effects of 254 nm UV-irradiation of tobacco mosaic virus (TMV) and potato virus X (PVX) RNA preparations on the RNA ability to self-assembly in vitro with the viral coat proteins were studied. It was found that while TMV RNA ability to assemble with the homologous protein is rapidly inactivated by the UV-irradiation, PVX RNA ability to be encapsidated by the PVX coat protein is quite resistant to the irradiation. More than that, the irradiation of TMV RNA with the dose strongly inhibiting its assembly with the homologous protein, did not result in any significant inhibition of this RNA ability to be coated with the PVX protein. The results testify to the profound differences in the mechanisms of RNA-protein interactions in the processes of self-assembly in vitro of tobamoviruses and potexviruses.

Radial distributions of density within macromolecular complexes determined from dark-field electron micrographs.

A procedure has been developed for direct determination of radial distributions of density in filamentous and spheroidal particles by analyzing dark-field scanning transmission electron micrographs of unstained freeze-dried specimens. Unlike electron microscopic methods based on staining or shadowing with heavy atoms, this approach can be used to probe the internal structure of macromolecular complexes. As an experimental proving ground, we have applied the procedure to tobacco mosaic virus (TMV) and to RNA-free helical polymers of TMV coat protein. Both structures are found to project outermost diameters of 17.6 +/- 0.4 nm, to have empty axial holes approximately equal to 3.5 nm in diameter, and to have density peaks at radii of 2.5 +/- 0.5 and 6.7 +/- 0.3 nm. Thus visualized, the only significant difference between them is the presence in the virion of an additional density peak at 4.1 +/- 0.5 nm contributed by its internalized RNA molecule. We have also used the procedure to monitor the structural expression of radiation damage in the low electron dose regime prior to the onset of significant mass loss. Changes in the radial density profiles are detected at average doses as low as approximately equal to 400 electrons per nm2: the trend is for the internal structure of these particles to fuse toward a state of uniform density, although the values of their outermost diameters remain unaffected.

Cross-correlation laser scattering.

Cross-correlation between two detectors was applied to analyze laser light-scattering fluctuations. Laser scattering from random concentration fluctuations is spatially coherent over small angular areas that are inversely proportional in size to the dimension of the scattering volume. By cross-correlating scattering intensity fluctuations in different angles, the correlation due to relaxation of concentration fluctuations is practically eliminated, and correlations reflecting changes in the scattering from the individual particles can be enhanced. Rotational diffusion of assymetric particles, conformational relaxation of random coils, and association-dissociation dynamics are determined here using the above approach.

Sensitivity to ultraviolet light of tobacco mosaic virus modified by cetyltrimethylammonium bromide.

Cetyltrimethylammonium bromide (CTAB) modified tobacco mosaic virus (TMV) virions so that the intrinsic fluorescence changed, viral infectivity decreased, sensitivity to RNase or UV irradiation increased, and coat protein subunits were released by the addition of Triton X-100. The change in fluorescence emission at 320 nm shifted to 340 nm was observed at 100 micrograms of CTAB per ml. This represents a change in the tryptophan environment inside the virion. At a lower concentration of CTAB, intersubunit contact was weakened, resulting in the release of coat protein subunits and an increase in RNase sensitivity. The release of coat protein took place gradually and two relatively stable intermediates were observed. Increase in UV sensitivity was observed at a lower concentration of CTAB and formation of pyrimidine hydrate was involved in this inactivation. The nature of the minor structural change leading to UV inactivation is discussed.