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Tobacco mosaic virus assembly and disassembly: determinants in pathogenicity and resistance.

The structural proteins of plant viruses have evolved to self-associate into complex macromolecules that are centrally involved in virus biology. In this review, the structural and biophysical properties of the Tobacco mosaic virus (TMV) coat protein (CP) are addressed in relation to its role in host resistance and disease development. TMV CP affects the display of several specific virus and host responses, including cross-protection, systemic virus movement, hypersensitive disease resistance, and symptom development. Studies indicate that the three-dimensional structure of CP is critical to the control of these responses, either directly through specific structural motifs or indirectly via alterations in CP assembly. Thus, both the structure and assembly of the TMV CP function as determinants in the induction of disease and resistance responses.

Biological Transport↗

Role of ciliary motility in acute allergic mucociliary dysfunction.

The purpose of this investigation was to determine if abnormal ciliary function contributes to allergic mucociliary dysfunction. In conscious sheep with Ascaris suum hypersensitivity, ciliated cells were obtained with a cytology brush and tracheal mucous velocity (TMV) was determined before and serially for 2 h following antigen inhalation. The recovered cells (also containing mast cells) were suspended in a chamber, and ciliary activity was viewed microscopically and recorded on videotape for subsequent slow-motion analysis of ciliary beat frequency (CBF). One hour after A. suum challenge mean CBF (+/- SE) showed a slight increase from a base-line value of 630 +/- 16 to 716 +/- 30 beats/min (P less than 0.05) when mean TMV was decreased to 57% of base line (P less than 0.05). After 2 h, both mean CBF and TMV returned toward base line. Since possible in vivo actions of chemical mediators liberated by antigen challenge may have been lost after suspension of the brushed cells, we also assessed the effects of antigen on CBF in vitro. A. suum caused a dose-dependent increase in CBF that was blocked by cromolyn sodium. We conclude that 1) allergic mucociliary dysfunction is not caused by a decrease in CBF and 2) antigen-induced release of chemical mediators increases CBF.

Acute Disease↗

Estimation of the cell-to-cell spread of tobacco mosaic virus infection in mechanically inoculated leaves.

Procedures to estimate cell-to-cell spread of tobacco mosaic virus (TMV) infection in mechanically inoculated leaves are presented mathematically and experimentally. Based upon the mathematical estimation, it is demonstrated that resistance of TMV infection to 2-thiouracil and guanidine in mechanically inoculated leaves is correlated to the fraction of cells infected. The addition of 2-thiouracil or guanidine prevents cell-to-cell spread of TMV infection.

Antiviral Agents↗

Effect of supraoptimal temperatures upon tobacco mosaic virus RNA replicase.

An examination of the effect of supraoptimal temperatures upon tobacco mosaic virus (TMV) RNA replicase demonstrated that the enzyme activity rapidly decreased at 40 degrees whether incubated in vitro after extraction or in leaves at that temperature prior to extraction and assay. The loss of replicase activity paralled the decline in capacity for in vivo incorporation of 32P into TMV RNA at 25 degrees following different periods of incubation at 40 degrews. The optimal temperature for replicase activity in vitro 35 degrees, and greater activity occurred at 40 than at 25 degrees. After about 15 min incubation at 40 degrees, in vivo incorporation of 32P into double-stranded TMV RNA became temperature-sensitive. This function could not be reproduced with the in vitro replicase system.

Hot Temperature↗

Effect of 2-thiouracil on RNA and protein synthesis in synchronous and asynchronous infections of tobacco mosaic virus.

Examination of the effect of 2-thiouracil on tobacco mosaic virus (TMV) RNA and protein synthesis in synchronous and asynchronous systems of replication in tobacco leaves demonstrated that 2-thiouracil does not prevent synthesis of TMV RNA, as suggested by previous studies, but inhibits some earlier function. When added at different times after mechanical inoculation, 1 mM 2-thiouracil partially inhibited both viral RNA and protein synthesis, with greater inhibition when treatment began nearer the time of inoculation. In leaves systemically inoculated with TMV using a differential temperature inoculation procedure to synchronize the infection, 1 mM 2-thiouracil inhibited viral protein and RNA synthesis totally when treatment began within the first 4 h after initiation of replication, but not at all when treatment began at 12 h or later, even though earlier reports had shown that most RNA and protein synthesis occurred after 12 h.

RNA, Viral↗

Examination of the mode of action of ribavirin against tobacco mosaic virus.

Ribavirin inhibited a specific step of tobacco mosaic virus (TMV) replication. Time-courses of sensitivity of TMV multiplication in asynchronous and synchronous infections demonstrated that ribavirin inhibited an early function that occurs prior to the period of viral RNA and protein synthesis. In the synchronous infection, ribavirin inhibited the synthesis of both single-stranded genomic RNA and double-stranded replicative form RNA when treatment began during the first 4 h, but there was no inhibition when treatment began at 12 h or later, even though most RNA synthesis occurred after 12 h. Viral protein synthesis also was inhibited when treatment began during the first 4 h and not when treatment began later. Unlike the mechanisms proposed against animal viruses, ribavirin appears to inhibit TMV replication by preventing an early function that is necessary to initiate viral RNA synthesis.

Kinetics↗

Specific inhibition of tobacco mosaic virus protein and single-stranded RNA synthesis by arabinofuranosyladenine.

9-beta-D-Arabinofuranosyladenine (ara A) was shown to inhibit a specific step of tobacco mosaic virus (TMV) multiplication. The time course of the ara A-sensitive function occurred 'late' coinciding with the period of viral RNA and protein synthesis. When treatment began after viral RNA synthesis was established, 1.0 mM ara A inhibited viral single-stranded RNA and viral protein synthesis, but not viral double-stranded RNA synthesis. Viral double-stranded RNA synthesis was inhibited totally only when treatment with ara A began within the first 4 h (before any RNA synthesis was detected), and the degree of inhibition decreased rapidly when treatments began at later times. In contrast, single-stranded TMV RNA synthesis and viral protein synthesis were inhibited throughout the infection. The mode of action of ara A against TMV involves selective inhibition of a function required for single-stranded RNA synthesis and viral protein synthesis without detectable inhibition of RNA or protein synthesis of the host or ongoing viral double-stranded RNA synthesis.

RNA↗

Translation in vitro of artificially produced fragments of a tobamovirus genome.

Particles of the U2 strain of tobacco mosaic virus (TMV) were partly disassembled by SDS, treated with RNases and then phenol, and yielded RNA molecules one quarter to half the size of the intact virus genome. These molecules, when translated in vitro, produced the coat protein of the virus. Reassembly experiments indicated that the active messenger molecules were those that most rapidly reassembled with coat protein; the rate of reassembly was greatly diminished by treatment with spleen phosphodiesterase. Particles of sunnhemp mosaic virus (the bean strain of TMV) resist disassembly by detergent much more than those of the U2 strain of TMV.

Cell-Free System↗

Elastase contributes to antigen-induced mucociliary dysfunction in ovine airways.

Antigen-induced bronchoconstriction is associated with impairment of mucociliary clearance with a time course that is consistent with the initial influx of neutrophils into the airway. In this study we tested the hypothesis that elastase released from activated neutrophils contributes to the acute (0 to 6-hr) antigen-induced mucociliary dysfunction. Tracheal mucous velocity CTMV), an index of mucociliary function, was measured with a roentgenographic technique before and serially after airway challenge with Ascaris suum antigen alone, or after pretreatment with aerosolized alpha1-protease inhibitor (alpha1-PI, 10 mg) or the specific neutrophil elastase inhibitor ICI 200,355 (10 mg). Antigen alone significantly decreased TMV. Treatment with either alpha1-PI or ICI 200,355, given either at 30 min before antigen challenge or 1 h after challenge, significantly attenuated the antigen-induced reduction in TMV at 6 h after challenge, whereas sheep treated with inactivated alpha1-PI were not protected from this antigen-induced event. Inhalation of ovine elastase (obtained from stimulated neutrophils) significantly decreased TMV, and this effect was also blocked by pretreatment with alpha1-PI. Both alpha1-PI and ICI 200,355 inhibited the activity of elastase obtained from stimulated ovine neutrophils. To verify that the neutrophil numbers and elastase activity increased in sheep airways after antigen challenge, nine animals underwent bronchoalveolar lavage (BAL) at 2 h and 4 h after instillation of A. summ antigen. Four hours after challenge, the number of neutrophils had increased by 50-fold, and free elastase activity in lavage fluid had increased. These data indicate that the antigen-induced impairment of mucociliary clearance is partly dependent on increased elastase activity, and that elastase inhibitors may be useful in protecting against mucociliary dysfunction.

Allergens↗

Correlation of clinical and genetic findings in Hungarian patients with Stargardt disease.

PURPOSE: Autosomal recessive Stargardt disease (arSTGD) presents with substantial clinical and genetic heterogeneity. This study was conducted to correlate foveolar thickness (FT) and total macular volume (TMV), measured by optical coherence tomography (OCT), with other clinical characteristics and with specific genetic variation in Hungarian patients with arSTGD. METHODS: After a standard ophthalmic workup, both eyes of 35 patients with STGD from Hungary and of 25 age-matched healthy control subjects were tested with OCT. FT and TMV were measured automatically with the OCT mapping software in the nine Early Treatment Diabetic Retinopathy Study areas of 3500 microm in diameter. All patients were screened for mutations by a combination of the ABCR400 microarray and direct sequencing. RESULTS: The patients with STGD presented with markedly thinned retina in the foveola and decreased macular volume, 72 microm and 1.69 mm3, respectively, compared with 169 microm and 2.48 mm3 in the normal subjects, respectively. Statistically significant correlation was observed between visual acuity (VA) and TMV and between VA and FT. Disease-associated mutations were detected in 23 (65.7%) of 35 patients, including 48.5% with both alleles and 17.2% with one allele. The most frequent ABCA4 alleles in Hungarian patients with STGD were L541P/A1038V (in 28% of all patients), G1961E (20%) and IVS40+5G-->A (17%). Specific genotypes correlated with some phenotypic features and allowed for predictions of the disease progression. CONCLUSIONS: Hungarian patients with STGD presented with extensive foveolar thinning and macular volume loss. Genetic analysis detected several ABCA4 alleles at high frequency in the cohort of patients, suggesting founder effect(s). Unusually homogeneous distribution of disease-associated mutations aided genotype-phenotype correlation analyses in this population.

ATP-Binding Cassette Transporters↗

Leukocyte adhesion in angiogenic blood vessels. Role of E-selectin, P-selectin, and beta2 integrin in lymphotoxin-mediated leukocyte recruitment in tumor microvessels.

Interaction of circulating leukocytes with tumor microvasculature is a critical event in the recruitment of effector cells into the tumor stroma. We have examined the ability of lymphotoxin (TNF-beta), to stimulate rolling, adhesion, and transmigration of leukocytes in angiogenic blood vessels induced by tumor spheroids of Lewis lung carcinoma (LLC) implanted in dorsal skinfold chambers of nude mice. In the absence of cytokine stimulation, circulating leukocytes failed to appreciably interact with tumor microvessels (TMV), although significant rolling and adhesion was observed in normal vessels. However, stimulation with lymphotoxin (LT) resulted in a rapid increase in the number of fast and slow rolling leukocytes in TMV. Treatment with anti-P-selectin mAb 5H1 resulted in inhibition of fast rollers alone, while combination treatment with anti-P-selectin and anti-E-selectin (9A9) mAbs effectively blocked slow rolling of leukocytes. Superfusion of the lymphotoxin-stimulated neovasculature with leukotriene B4 (LTB4) resulted in stable cell adhesion followed by emigration of leukocytes into the tumor stroma. LTB4-mediated adhesion and transmigration was significantly inhibited by treatment with anti-beta2 mAb 2E6. These studies delineate a multistep cascade of leukocyte adhesion in TMV and demonstrate that stimulation of the neovasculature with cytokines and chemoattractants can result in P- and E-selectin-dependent rolling and beta2-dependent stable adhesion followed by transmigration into the tumor stroma.

Animals↗

Actions of pokeweed antiviral protein on virus-infected protoplasts.

Pokeweed antiviral protein (PAP) belongs to a group of ribosome-inactivating proteins (RIPs) that inactivate ribosomes by depurinating rRNA at a specific site. To study the mechanism for the antiviral activity of PAP, the actions of PAP on TMV-infected and uninfected tobacco protoplasts were investigated. The addition of 0.33 microM PAP to TMV-inoculated protoplasts caused a complete inhibition of TMV production. The same concentration of PAP was found to inhibit protein synthesis in the virus-infected protoplasts and to kill the cells, but it had no effect on the uninfected protoplasts. The concentration dependence of protein synthesis-inhibition by PAP was related to that of inhibition of viral multiplication. Furthermore, two other RIPs (ricin A-chain and luffin-a), which showed 240 and 430-fold less activity on tobacco ribosomes than PAP in a cell-free system, did not inhibit viral multiplication even at a concentration of 3.3 microM. The analysis of RNAs from the virus-infected and PAP-treated protoplasts demonstrated that 25S rRNA was depurinated by PAP in the infected cells. These results suggest that PAP, which is normally unable to penetrate the plasma membrane of uninfected protoplasts, gains entrance to the cytosol of infected cells and prevents viral multiplication by inactivating ribosomes.

Animals↗

A longitudinal study on the nutritional state of elderly women at a nursing home in Japan.

OBJECTIVE: Elderly people at nursing homes often suffer from malnutrition, which is characterized by a loss of muscle mass and hypoalbuminemia. This malnourished state is closely associated with an impaired activity of daily living (ADL). We analyzed the nutritional state of such elderly individuals longitudinally over 3 years by anthropometry, serum albumin, and muscle and fat volume as estimated by MRI. PATIENTS AND METHODS: The subjects consisted of 16 elderly women aged 83 +/- 7 (mean +/- SD) years who resided at a nursing home in an urban area of central Japan. We determined their ADL levels using the Barthel Index (BI) at entry. Seven women belonged to group A (BI; 65-100), thus implying either a mild or no decline in ADL, while the other 9 were in group B (BI; 0-60) and they demonstrated a severe decline in ADL. We measured the following parameters every year from 2000 to 2003; anthropometry including height, body weight, arm circumference (AC), and arm muscle circumference (AMC), thigh muscle and fat volume as estimated by MRI [thigh muscle volume (TMV) and thigh fat volume (TFV)], serum albumin, and plasma amino acid levels by blood biochemistry. The anthropometric values were converted into percentages of the age- and sex-matched reference values for Japanese. RESULTS: In all subjects, the TMV, %AMC, and serum albumin level decreased significantly during the three-year period (p<0.05, respectively). The change in TMV correlated significantly with those in the %AC and %AMC (p<0.05, respectively). Group B showed significantly larger decreases in the %AMC and serum albumin level than group A. CONCLUSION: Both the muscular and visceral protein levels were found to decrease with aging in the subjects at the nursing home. This decrease depends partly on the ADL level of each subject.

Activities of Daily Living↗

[Differences in the spatial structure of an envelope protein from tobacco mosaic virus and its mutant, detected by tritium planigraphy].

Mutant ts21-66 of the tobacco mosaic virus (TMV) differs from the wild-type TMV-U1 by two mutations (Ile-21-->Thr and Asp-66-->Gly) in the coat protein (CP) gene and in symptoms produced in infected N' plants. The CP structure in TMV-U1 and ts21-66 virions was probed by tritium planigraphy. Compared with the wild-type CP, labeling of the N-terminal region of mutant CP was half as high and suggested its greater shielding. A role of this CP region in virus interactions with the N' resistance system is discussed.

Amino Acid Sequence↗

Temporal evolution of the sensitivity to tetranitromethane of the active site of anti-Tobacco mosaic virus antibodies.

Rabbit anti-TMV antibody taken 9 days after antigen injection is inactivated by much lower concentrations of tetranitomethane (TNM) than anti-body taken after 30 days. This change in sensitivity to TNM occurs in a very heterogeneous population of molecules, including IgM and IgG classes, but the affinity of the antibody remains unchanged. Thus, the phenomenon is not the result of selection of antibody-producing cells under the influence of antigen. Inactivation of early anti-TMV antibody appears to result from the nitration of one tyrosyl residue per Fab fragment. This tyrosine is probably located in the heavy chain and most probably in the variable portion. The moderate sensitivity of late anti-body to TNM is attributed to the lower reactivity of a tyrosine in the active site, and to the fact that nitration of this tyrosine does not inactivate the binding site. The suggested interpretation is that the majority of molecules of a very heterogeneous anti-TMV antibody pool may have a common structure in their active site during the early stage of the antibody response. This may later be replaced in most molecules by different structures.

Animals↗

A diterpene as an endogenous signal for the activation of defense responses to infection with tobacco mosaic virus and wounding in tobacco.

In pathogen-infected or wounded tobacco plants, the activation of wound-induced protein kinase (WIPK), a tobacco mitogen-activated protein kinase, has been implicated in the defense response. However, no endogenous signal responsible for the activation has been identified. A WIPK-activating substance was isolated from tobacco leaves and identified as (11E,13E)-labda-11,13-diene-8alpha,15-diol, designated WAF-1. When applied in nanomolar concentrations to leaves, either natural WAF-1 or chemically synthesized WAF-1 activated WIPK as well as salicylic acid-induced protein kinase, a tobacco mitogen-activated protein kinase, and enhanced the accumulation of transcripts of wound- and pathogen-inducible defense-related genes. Quantitative analysis of endogenous WAF-1 revealed that levels increased rapidly in leaves during a hypersensitive response to Tobacco mosaic virus (TMV) and after wounding. Furthermore, treatment of leaves with WAF-1 resulted in enhanced resistance to TMV infection. These results suggest that WAF-1 functions as an endogenous signal to mediate the defense responses of tobacco plants to TMV infection and wounding.

Cyclopentanes↗

Effect of different proteolytic enzymes on the nature of subunit composition of arachins from groundnut (Arachis hypogaea L.).

Arachin, the major protein from groundnut, was isolated from three varieties of groundnut (Spanish Improved, TMV-2 and DH-3-30) using a modified procedure involving precipitation with 18% ammonium sulphate to obtain homogeneous protein. The homogeneity was judged by polyacrylamide gel electrophoresis, gel filtration and sedimentation velocity techniques as well as correlation with amino acid composition. Rates of hydrolysis of arachins by trypsin (pH 7.6) and alpha-chymotrypsin (pH 7.8) were significantly different between the three varieties. Arachin from the Spanish Improved variety contained higher amounts of alanine and phenylalanine and lower amounts of carbohydrate and phosphorus as compared to TMV-2 and DH-3-30. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis pattern of arachin from TMV-2 showed only seven bands of which the ones with low molecular weight were more intense than those of the other two varieties. The far-ultraviolet circular dichroic spectra showed no significant differences among the three varieties in respect of alpha-helix content (5 +/- 2%), beta-structure (19 +/- 2%) and the aperiodic structure. The observed differences in hydrolysis rates have been explained as due to the differences in the acidic and basic subunits of arachins.

Amino Acids↗

Interactions of plant viral RNAs and tRNA nucleotidyl transferase.

Ribonucleic acid from TMV and BMV can accept AMP and CMP when digested with VPD. This incorporation is catalyzed by E. coli and yeast tRNA nucleotidyl transferases. Complex formation is obtained between TYMV RNA and tRNA nucleotidyl transferase in sucrose gradients while TMV and BMV RNAs failed to form a complex in the same conditions. The affinity of the enzyme for viral RNAs is lower than the affinity for tRNA as shown by complex formation on nitrocellulose filters and competition with tRNA. Coat protein from TMV particles enhances AMP and CMP incorporation onto tRNA catalyzed by the E. coli tRNA nucleotidyl transferase.

Adenosine Monophosphate↗