High-sensitivity express immunochromatographic method for detection of plant infection by tobacco mosaic virus

A highly sensitive express immunochromatography method for molecular diagnosis of plant virus infections was elaborated on the example of a model object — tobacco mosaic virus (TMV). The analysis time does not exceed 5 min, and the lower limit of TMV detection in non-clarified leaf extract (2–4 ng/ml) is comparable with the sensitivity of the enzyme-linked immunosorbent assay of the virus. A single measurement requires 0.1–0.2 ml tested solution (extract from 10–20 mg of leaf material). The sensitivity of TMV determination in the leaf tissue extract was increased by more than one order of magnitude using signal enhancement by silver and is 0.1 ng/ml. In this case, analysis time did not exceed 25 min. The simplicity of this method makes it especially convenient in express diagnosis of numerous analyzed specimens. The prototype of a diagnostic kit for serial analyses of plant viral infections both in laboratory and field conditions was elaborated.     

Inhibition of filovirus replication by the zinc finger antiviral protein

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.     

Interaction of tobacco mosaic virus and tobacco mosaic virus protein with bovine serum albumin.

Bovine serum albumin (BSA) causes tobacco mosaic virus (TMV) to crystallize at pH values where both have negative charges. The amount of albumin required to precipitate the virus varies inversely with ionic strength of added electrolyte. At pH values above 5, the precipitating power is greatest when BSA has the maximum total, positive plus negative, charge. Unlike early stages of the crystallization of TMV in ammonium sulfate-phosphate solutions, which can be reversed by lowering the temperature, the precipitation of TMV by BSA is not readily reversed by changes in temperature. The logarithm of the apparent solubility of TMV in BSA solutions, at constant ionic strength of added electrolyte, decreases linearly with increasing BSA concentration. This result and the correlation of precipitating power with total BSA charge suggest that BSA acts in the manner of a salting-out agent. The effect of BSA on the reversible entropy-driven polymerization of TMV protein (TMVP) depends on BSA concentration, pH, and ionic strength. In general, BSA promotes TMVP polymerization, and this effect increases with increasing BSA concentrations. The effect is larger at pH 6.5 than at pH 6. Even though increasing ionic strength promotes polymerization of TMVP in absence of BSA, the effect of increasing ionic strength from 0.08 to 0.18 at pH 6.5 decreases the polymerization-promoting effect of BSA. Likewise, the presence of BSA decreases the polymerization-promoting effect of ionic strength. The polymerization-promoting effect of BSA can be interpreted in terms of a process akin to salting-out. The mutual suppression of the polymerization-promoting effects of BSA and of electrolytes by each other can be partially explained in terms of salting-in of BSA.     

Inhibition of virus DNA replication by artificial zinc finger proteins

Prevention of virus infections is a major objective in agriculture and human health. One attractive approach to the prevention is inhibition of virus replication. To demonstrate this concept in vivo, an artificial zinc finger protein (AZP) targeting the replication origin of the Beet severe curly top virus (BSCTV), a model DNA virus, was created. In vitro DNA binding assays indicated that the AZP efficiently blocked binding of the viral replication protein (Rep), which initiates virus replication, to the replication origin. All of the transgenic Arabidopsis plants expressing the AZP showed phenotypes strongly resistant to virus infection, and 84% of the transgenic plants showed no symptom. Southern blot analysis demonstrated that BSCTV replication was completely suppressed in the transgenic plants. Since the mechanism of viral DNA replication is well conserved among plants and mammals, this approach could be applied not only to agricultural crop protection but also to the prevention of virus infections in humans.     

Electroporation-mediated infection of tobacco leaf protoplasts with tobacco mosaic virus RNA and cucumber mosaic virus RNA

Conditions were established for the introduction of both tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV) RNAs into tobacco mesophyll protoplasts by electroporation. The proportion of infected protoplasts was quantified by staining with viral coat protein-specific antibodies conjugated to fluorescein isothiocyanate. Approximately 30–40% of the protoplasts survived electroporation. Under optimal conditions, up to 75% of these were infected with TMV-RNA. Successful infection was demonstrated in 19 out of 20 experiments. Optimal infection was achieved with several direct current pulses of 90 sec at a field strength of 5 to 10 kV/cm. Changing the position of the protoplasts within the chamber between electric pulses was essential for achievement of high rates of infection. Optimal viral RNA concentration was about 10 g/ml in a solution of 0.5 M mannitol without buffer salts.     

The tobacco mosaic virus 126-kilodalton protein, a constituent of the virus replication complex, alone or within the complex aligns with and traffics along microfilaments

Virus-induced cytoplasmic inclusion bodies (referred to as virus replication complexes [VRCs]) consisting of virus and host components are observed in plant cells infected with tobacco mosaic virus, but the components that modulate their form and function are not fully understood. Here, we show that the tobacco mosaic virus 126-kD protein fused with green fluorescent protein formed cytoplasmic bodies (126-bodies) in the absence of other viral components. Using mutant 126-kD:green fluorescent fusion proteins and viral constructs expressing the corresponding mutant 126-kD proteins, it was determined that the size of the 126-bodies and the corresponding VRCs changed in synchrony for each 126-kD protein mutation tested. Through colabeling experiments, we observed the coalignment and intracellular trafficking of 126-bodies and, regardless of size, VRCs, along microfilaments (MFs). Disruption of MFs with MF-depolymerizing agents or through virus-induced gene silencing compromised the intracellular trafficking of the 126-bodies and VRCs and virus cell-to-cell movement, but did not decrease virus accumulation to levels that would affect virus movement or prevent VRC formation. Our results indicate that (1) the 126-kD protein modulates VRC size and traffics along MFs in cells; (2) VRCs traffic along MFs in cells, possibly through an interaction with the 126-kD protein, and the negative effect of MF antagonists on 126-body and VRC intracellular movement and virus cell-to-cell movement correlates with the disruption of this association; and (3) virus movement was not correlated with VRC size.     

Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain

The Cucumber mosaic virus (CMV)-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, we used the yeast two-hybrid system using tobacco cDNA library. One of the cDNA clones encoded a protein homologous to the Arabidopsis putative protein kinase and was designated as Tcoi2 (Tobacco CMV 1a interacting protein 2). Tcoi2 specifically interacted with methyltransferase (MT) domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum. These results suggest Tcoi2 as a novel host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein.     

A non-coat protein synthesized in tobacco mesophyll protoplasts infected by tobacco mosaic virus

Summary A high molecular weight protein unrelated to the viral coat was detected in tobacco mesophyll protoplasts infected by tobacco mosaic virus.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

Selective repression of translation by the brome mosaic virus 1a RNA replication protein

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.     

A novel proteinase inhibitor gene transiently induced by tobacco mosaic virus infection

A gene (NgPI) encoding a novel proteinase inhibitor (PI) has been isolated from tobacco leaves. Protein encoded by the gene consists of 241 amino acid residues having a predicted molecular mass of 26.7 kDa and a calculated pI of 8.7. A predicted N-terminal signal sequence followed by a vacuolar targeting signal and a peptide conserved in the Kunitz type PIs were identified. The deduced NgPI protein has sequence homology with aspartic and cysteine protease inhibitors. The gene is present as double copies in the Nicotiana glutinosa genome. Expression of the NgPI gene is rapidly and transiently induced by tobacco mosaic virus infection at a time earlier than apparent lesions of hypersensitive responses appear on the leaves.