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PubMed1998

Use of modified plum pox virus coat protein genes developed to limit heteroencapsidation-associated risks in transgenic plants.

Aphid transmission of a non-aphid-transmissible strain of zucchini yellow mosaic virus (ZYMV-NAT) occurs in transgenic plants expressing the plum pox potyvirus (PPV) coat protein (CP) gene. Heteroencapsidation has been shown to be responsible for this modification in the epidemiological characteristics of the infecting virus. In order to prevent this biological risk, several modified PPV CP constructs were produced that were designed to interfere with heteroencapsidation itself or to block aphid transmission of heteroencapsidated virions. These constructs were first expressed in Escherichia coli in order to check for the accumulation of pseudo-particles by electron microscopy. Virus-like particles (VLPs) were found with the full-length CP and with a PPV CP lacking the DAG amino acid triplet involved in aphid transmission. However, no VLPs were observed with CP lacking R220, Q221 or D264, amino acids known to be essential for the assembly of other potyvirus CPs. Transgenic Nicotiana benthamiana lines expressing the different PPV CP constructs were infected with ZYMV-NAT. Aphid transmission assays performed with these plants demonstrated that the strategies developed here provide an effective means of minimizing the biological risks associated with heteroencapsidation.

Capsid↗
PubMed2005

Studies on the mechanism of inactivation of the HIV-1 nucleocapsid protein NCp7 with 2-mercaptobenzamide thioesters.

The HIV-1 nucleocapsid protein (NCp7) is a small basic protein with two CysCysHisCys zinc-binding domains that specifically recognizes the Psi-site of the viral RNA. NCp7 plays a number of crucial roles in the viral lifecycle, including reverse transcription and RNA encapsidation. Several classes of potential anti-HIV compounds have been designed to inactivate NCp7 through zinc ejection, including a special class of thioester compounds. We have investigated the mechanism of action of two N-substituted-S-acyl-2-mercaptobenzamide compounds (compounds 1 and 2) that target NCp7. UV/Visible spectroscopy studies demonstrated that both thioesters were able to eject metal from NCp7. NMR and mass spectroscopy studies showed that the thioester compounds specifically ejected zinc from the carboxyl-terminal zinc-binding domain of NCp7 by covalent modification of Cys(39). Exposure of NCp7 to compounds 1 and 2 destroyed its ability to specifically bind RNA, whereas NCp7 already bound to RNA was protected from zinc ejection by the thioesters. The thiol component of the thioesters (compound 3, 2-mercaptobenzoyl-beta-alaninamide) did not eject zinc from NCp7, but when compound 3 was incubated with acetyl CoA prior to incubation with NCp7, we observed extensive metal ejection. Thus, the thiol released by the reaction of compounds 1 and 2 could be re-acylated in vivo by acyl CoA to form a new thioester compound that is able to react with NCp7. These studies provide a better understanding of the mechanism of action of thioester compounds, which is important for future design of anti-HIV-1 compounds that target NCp7.

Amino Acid Sequence↗
PubMed1986

Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry.

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.

Bacillus subtilis↗
PubMed1983

Studies on the in vitro uncoating of poliovirus. IV. Characteristics of solubilized membrane-modifying and -stabilizing factors.

Previous studies in our laboratory on the in vitro uncoating of poliovirus have shown that HeLa cell membrane contains a modifying factor which induces early modification of virus (the loss of VP4) and a stabilizing factor which protects virus against heat-induced degradation. It has now been found that membrane-modifying factor is dependent on the presence of phospholipid for activity. Modifying activity was lost after exposure of membrane (Mem) to phospholipase C. Triton X-100-solubilized modifying factor prepared from phospholipase C-treated Mem was reactivated by phospholipid. Lecithin, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin were found to exhibit a reactivating effect. Lecithin was the most effective individual phospholipid in terms of maximal reactivation. Partial purification of Triton X-100-solubilized modifying factor was achieved by concanavalin A-Sepharose chromatography. Membrane-stabilizing factor was extracted from HeLa cell membrane by solubilization with sodium deoxycholate (DOC). Some properties of DOC-solubilized stabilizing factor were studied. The solubilized stabilizing factor was inactivated by treatment with trypsin or chymotrypsin. Treatment of the solubilized stabilizing factor with certain lipid solvents, lipolytic enzymes, or lectins had no detectable effect on stabilizing activity.

Capsid↗
PubMed1997

Use of circular permutation to assess six bulges and four loops of DNA-packaging pRNA of bacteriophage phi29.

A 120-base phage phi29 encoded RNA (pRNA) has a novel role in DNA packaging. This pRNA possesses five single-base bulges, one three-base bulge, one bifurcation bulge, one bulge loop, and two stem loops. Circularly permuted pRNAs (cpRNA) were constructed to examine the function of these bulges and loops as well as their adjacent sequences. Each of the five single-base bulges was nonessential. The bifurcation bulge could be deleted and replaced with a new opening to provide flexibility for maintaining an overall correct folding in three-way junction. All of these nonessential bulges or their adjacent bases could be used as new termini for cpRNAs. The three-base (C18C19A20) bulge was dispensable for procapsid binding, but was indispensable for DNA packaging. The secondary structure around this CCA bulge and the phylogenetically conserved bases within or around it were investigated. Bases A14C15U16 were confirmed, by compensatory modification, to pair with U103G102A101. A99 was needed only to allow the proper folding of CCA bulge in the appropriate sequence order and distance constraints. Beyond these, the seemingly phylogenetic conservation of other bases has little role in pRNA activity. Each of the three stem loops was essential for procapsid binding, DNA packaging, and phage assembly. Disruption of the middle of any one of the loops resulted in dramatic reductions in procapsid binding, subsequent DNA packaging, and phage assembly activities. However, disruption of the loops at sequences that were close to double-stranded regions of the RNA did not interfere with pRNA activity significantly. Our results suggest that double-stranded helical regions near these loops were most likely not involved in interactions with components of the DNA-packaging machinery. Instead, these regions appear to be merely present to serve as a scaffolding to display the single-stranded loops that are important for pRNA tertiary structure or for interaction with the procapsid or other packaging components.

Bacillus Phages↗
PubMed2002

Modifications in adenoviral coat fiber proteins and transcriptional regulatory sequences enhance transgene expression.

OBJECTIVE: To characterize the adenoviral properties required to enhance intracellular transgene expression for gene therapy. METHODS: Primary human fibroblasts and macrophages were infected with standard replication-defective adenoviruses, adenoviral vectors containing modified fiber coat proteins expressing Arg-Gly-Asp (RGD) or heparin sulfate binding moieties, or a tetracycline-regulatable transgene transcription system. Each of these vectors expressed the beta-galactosidase gene (beta-Gal), which was quantified by flow cytometry. Ankle joints from rats with adjuvant induced arthritis were transduced intraarticularly with each of the vectors and B-Gal expression was quantified by flow cytometry. RESULTS: Primary human fibroblasts and macrophages displayed marked increases in transgene expression from both modified fiber protein vectors and from the tetracycline-regulatable vector, compared to an unmodified vector expressing the transgene from the cytomegalovirus promoter/enhancer. In the rat model, the modified fiber protein vectors and the tetracycline-regulatable vector system also displayed increased transgene expression in inflamed rat joints. CONCLUSION: Adenovirus attachment and uptake by cells and promoter strength limit transgene expression from conventional adenoviral vectors in models of rheumatoid arthritis.

Adenoviridae↗
PubMed1995

The role of 2'-hydroxyl groups in an RNA-protein interaction.

The role of the 2'-hydroxyl group in RNA--protein interaction has been investigated using MS2 coat protein and its hairpin RNA operator as a model system. Derivatives of the MS2 translational operator were prepared where individual riboses were replaced by deoxyribose and their binding affinities to MS2 coat protein were determined. Only 1 (U-5) out of 15 positions tested reduced protein affinity by 1.6 kcal/mol. A variety of other 2'-modifications were tested at this position to understand the role of this particular 2'-hydroxyl group. Normal binding of the U-NH2 variant and weaker binding of the U-O-methyl variant are consistent with the ability of these functional groups to provide a hydrogen bond donor. This is also supported by recent crystallographic data which indicate a possible interaction between the 2'-hydroxyl of U-5 and the carboxylate group of glutamate 63 [Valegård et al. (1994) Nature 371, 623-626]. Complementary experiments introducing riboses into a DNA hairpin confirm the putative protein contact, and also identify a requirement for riboses in the two upper base pairs of the hairpin. Several arguments suggest these riboses are required to maintain an A-form helix in this region of the binding site. A minimum requirement of four 2'-hydroxyl groups for wild-type coat protein binding has been determined, one of which is at the -5 position and other three in the upper stem in any combination.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2003

Prediction of tyrosine sulfation sites in animal viruses.

Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand-receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-Specific-Scoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized.

Algorithms↗
PubMed2006

Adenovirus fiber shaft contains a trimerization element that supports peptide fusion for targeted gene delivery.

Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

Adenoviridae↗
PubMed2006

A type IV modification dependent restriction nuclease that targets glucosylated hydroxymethyl cytosine modified DNAs.

The Escherichia coli CT596 prophage exclusion genes gmrS and gmrD were found to encode a novel type IV modification-dependent restriction nuclease that targets and digests glucosylated (glc)-hydroxymethylcytosine (HMC) DNAs. The protein products GmrS (36 kDa) and GmrD (27 kDa) were purified and found to be inactive separately, but together degraded several different glc-HMC modified DNAs (T4, T2 and T6). The GMR enzyme is able to degrade both alpha-glucosy-HMC T4 DNA and beta-glucosyl-HMC T4 DNA, whereas no activity was observed against non-modified DNAs including unmodified T4 cytosine (C) DNA or non-glucosylated T4 HMC DNA. Enzyme activity requires NTP, favors UTP, is stimulated by calcium, and initially produces 4 kb DNA fragments that are further degraded to low molecular mass products. The enzyme is inhibited by the T4 phage internal protein I* (IPI*) to which it was found to bind. Overall activities of the purified GmrSD enzyme are in good agreement with the properties of the cloned gmr genes in vivo and suggest a restriction enzyme specific for sugar modified HMC DNAs. IPI* thus represents a third generation bacteriophage defense against restriction nucleases of the Gmr type.

5-Methylcytosine↗
PubMed2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

3T3-L1 Cells↗
PubMed2001

Chemical modification patterns of active and inactive as well as procapsid-bound and unbound DNA-packaging RNAof bacterial virus Phi29.

During replication, the lengthy genome of dsDNA viruses is translocated with remarkable velocity into the limited space within the preformed procapsid. We previously found that a viral-encoded RNA (pRNA) played a key role in bacterial virus phi29 DNA translocation. Design of mutant pRNA sets containing two and three inactive mutant pRNAs, respectively, led to the conclusion that the stoichiometry of pRNA in DNA packaging is the common multiple of 2 and 3. Together with studies using binomial distribution of mutant and wild-type pRNA, it has been confirmed that six pRNAs of phi29 form a hexagonal complex to drive the DNA translocating machine. These findings have brought about commonality between viral DNA packaging and other universal DNA/RNA-riding processes including DNA replication and RNA transcription. Chemical modification was used to compare the structures of active and inactive as well as free and procapsid-bound pRNA. Our results explain why certain pRNA mutants are inactive in DNA packaging while remaining competent in procapsid binding, since the mutations were located in a domain involved in DNA translocation that is dispensable for procapsid binding. A mutant pRNA that had reduced procapsid binding was revealed to have a structural alteration within the procapsid-binding region that may account for the binding deficiency. Chemical probing of procapsid-bound pRNA revealed a large area of protection, while a 3-base bulge, C(18)C(19)A(20), was accessible to chemicals. A pRNA with a deletion of this 3-base bulge was fully competent to form dimers, bind procapsids, and inhibit phi29 virion assembly in vitro; however, its activity in DNA packaging and virion assembly was completely lost. The results suggest that this bulge is not involved in procapsid binding but may interact with other DNA-packaging components. A computer model showing the location of the CCA bulge was presented.

Bacillus Phages↗
PubMed2002

Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line.

In microglia, it is difficult to introduce exogenous genes of interest even by recombinant adenovirus vectors (Ad) which can infect with high efficiency only to the cells expressing coxackievirus and adenovirus receptors (CAR). We found a lack of CAR expression in primary cultured murine microglia (PCMG) and its immortalized cell line MG5 by reverse transcription-polymerase chain reaction. In order to improve the efficiency of gene transfer, we generated a novel Ad (Ad-RGD) by an incorporation of the Arg-Gly-Asp motif (RGD) containing peptide in the HI loop of the viral fiber knob domain, which enables the virus to contact target cells through alpha V integrins which are known to be ubiquitously expressed on the surface of mammalian cells. Ad-RGD showed a remarkable improvement (13-18-fold) in the delivery of Escherichia coli LacZ gene in MG5 cells and a moderate increase in PCMG cells under the treatment with granulocyte-macrophage colony stimulating factor. These results suggest that Ad-RGD may be a potent tool for the delivery of genes to microglia activated by optimum stimulation, and thus analyzing the function of microglia with utilization of MG5 and PCMG cells.

Adenoviridae↗
PubMedDate not supplied

Evaluation of cotton as a carrier for solid-phase peptide synthesis.

Cotton as a new support for the solid-phase synthesis of peptides was evaluated under the conditions of both Boc/Bzl and Fmoc/But strategy. Syntheses of peptides containing only bifunctional amino acids gave comparable results, but peptides containing trifunctional amino acids are clearly advantageously synthesized using the Fmoc approach. The comparison of cotton with other planar carriers (paper or polypropylene membranes) and with classical pellicular ones has shown practical applicability of cotton as the replacement of more sophisticated carriers. N-Methylimidazole was shown to be the optimal acylation catalyst for the modification of the cellulose carrier. Stability of the ester bond to the cotton was studied and shown to be adequate for the syntheses of small to medium size peptides. A relatively low level of substitution of cotton carrier in connection with its ability to soak the solvent was used for the complete elimination of a synthetic vessel. Couplings can be performed by wetting the carrier with the coupling solution, and all solvents can be removed either by squeezing it together with a porous material (paper) or by centrifugation. This leads to a significant decrease of solvent consumption. The efficiency of solvent elimination by centrifugation was demonstrated by the synthesis of model peptide ACP 65-74 in which only one wash between coupling and deprotection and between deprotection and coupling was used and which afforded the product of the same quality as the product synthesized using the standard protocol. Bromophenol blue monitoring has been proven as the optimal way of monitoring the acylation reaction progress. Peptides directly bound to cotton were shown to be useful in enzyme-linked immunosorbent assay tests.

Acylation↗
PubMed2002

Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

Adenoviridae↗
PubMed1997

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

Alteration of the natural tropism of adenovirus (Ad) will permit gene transfer into specific cell types and thereby greatly broaden the scope of target diseases that can be treated by using Ad. We have constructed two Ad vectors which contain modifications to the Ad fiber coat protein that redirect virus binding to either alpha(v) integrin [AdZ.F(RGD)] or heparan sulfate [AdZ.F(pK7)] cellular receptors. These vectors were constructed by a novel method involving E4 rescue of an E4-deficient Ad with a transfer vector containing both the E4 region and the modified fiber gene. AdZ.F(RGD) increased gene delivery to endothelial and smooth muscle cells expressing alpha(v) integrins. Likewise, AdZ.F(pK7) increased transduction 5- to 500-fold in multiple cell types lacking high levels of Ad fiber receptor, including macrophage, endothelial, smooth muscle, fibroblast, and T cells. In addition, AdZ.F(pK7) significantly increased gene transfer in vivo to vascular smooth muscle cells of the porcine iliac artery following balloon angioplasty. These vectors may therefore be useful in gene therapy for vascular restenosis or for targeting endothelial cells in tumors. Although binding to the fiber receptor still occurs with these vectors, they demonstrate the feasibility of tissue-specific receptor targeting in cells which express low levels of Ad fiber receptor.

Adenoviridae↗
PubMed2000

Inactivation of HIV-1 nucleocapsid protein P7 by pyridinioalkanoyl thioesters. Characterization of reaction products and proposed mechanism of action.

The synthesis and antiviral properties of pyridinioalkanoyl thioester (PATE) compounds that target nucleocapsid p7 protein (NCp7) of the human immunodeficiency virus type 1 (HIV-1) have been described previously (Turpin, J. A., Song, Y., Inman, J. K., Huang, M., Wallqvist, A., Maynard, A., Covell, D. G., Rice, W. G., and Appella, E. (1999) J. Med. Chem. 42, 67-86). In the present study, fluorescence and electrospray ionization-mass spectrometry were employed to determine the mechanism of modification of NCp7 by two lead compounds, N-[2-(5-pyridiniovaleroylthio)benzoyl]sulfacetamide bromide and N-[2-(5-pyridiniovaleroylthio)benzoyl]-4-(4-nitrophenylsulfonyl )anili ne bromide (compounds 45 and 47, respectively). Although both compounds exhibit antiviral activity in cell-based assays, we failed to detect appreciable ejection of zinc from NCp7 under conditions in which previously described NCp7-active disulfides readily eject zinc. However, upon "activation" by Ag(+), compound 45 reacted with NCp7 resulting in the zinc ejection from both zinc fingers. The reaction followed a two-step mechanism in which zinc was ejected from the carboxyl-terminal zinc finger faster than from the amino-terminal zinc finger. Both compounds covalently modified the protein with pyridinioalkanoyl groups. Compound 45 modified cysteines 36 and 49 of the carboxyl-terminal zinc finger. The results obtained herein demonstrate that PATE compounds can be constructed that selectively target only one of the two zinc fingers of NCp7, thus providing an impetus to pursue development of highly selective zinc finger inhibitors.

Amino Acid Sequence↗
PubMed1987

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was approximately 0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector.

Blotting, Northern↗