Publications

internal cleavage and trans-proteolytic activities of the vpg-proteinase (nia) of tobacco etch potyvirus in vivo.	the nia protein of plant potyviruses is a bifunctional protein containing an n-terminal vpg domain and a c-terminal proteinase region. the majority of tobacco etch potyvirus (tev) nia molecules are localized to the nucleus of infected cells, although a proportion of nia is attached covalently as vpg to viral rna in the cytoplasm. a suboptimal cleavage site that is recognized by the nia proteinase is located between the two domains. this site was found to be utilized in the vpg-associated, but no ...	1993	8230423
genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene.	three versions of the zucchini yellow mosaic virus (zymv) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version. these constructs were introduced into muskmelon (cucumis melo) and tobacco plants (nicotiana tabacum) via agrobacterium tumefaciens-mediated transformation; gene expression was verified by northern and western analysis. transgenic r0 and r1 muskmelon plants expressing the full ...	1993	8324251
spontaneous mutagenesis of a plant potyvirus genome after insertion of a foreign gene.	the rna genome of tobacco etch potyvirus (tev) was engineered to express bacterial beta-glucuronidase (gus) fused to the virus helper component proteinase (hc-pro). it was shown previously that prolonged periods (approximately 1 month) of tev-gus propagation in plants resulted in the appearance of spontaneous deletion variants. nine deletion mutants were identified by nucleotide sequence analysis of 40 cdna clones obtained after polymerase chain reaction amplification. the mutants were missing b ...	1993	8371351
nuclear transport of tobacco etch potyviral rna-dependent rna polymerase is highly sensitive to sequence alterations.	the putative rna-dependent rna polymerase (nib protein) of tobacco etch potyvirus accumulates primarily in the nucleus of infected cells, although viral rna replication is suggested to occur in the cytoplasm. to understand the possible relationship between nib nuclear localization and its function, we have studied translocation of nib using gene fusion and plant transformation techniques. when expressed as a fusion with a cytoplasmic reporter protein, beta-glucuronidase (gus), nib efficiently di ...	1993	8460496
rna-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in rna degradation.	transgenic nicotiana tabacum cv. burley 49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (tev) genome ligated to a mutated version of the tev coat protein gene sequence that rendered it untranslatable. eight different transgenic plant lines were analyzed for transgene expression and for resistance to tev. three different responses were noted when the transgenic plant lines were inoculated with tev: 1) some were highly resistant, and no virus replicat ...	1994	7949323
in vitro characterization of a cassette to accumulate multiple proteins through synthesis of a self-processing polypeptide.	the strategy for processing the polyprotein encoded by plant potyviruses has been mimicked by constructing an expression cassette based on the nuclear inclusion (nla) proteinase from tobacco etch virus (tev). this cassette (ppr01), includes the tev nla coding region flanked on each side by its heptapeptide cleavage sequence and cloning sites for the in frame insertion of two different open reading frames. ppr01 allows the synthesis, under the control of a single transcriptional promoter, of two ...	1994	8123791
the tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.	the tobacco etch potyvirus (tev) genome encodes a polyprotein that is processed by three virus-encoded proteinases. although replication of tev likely occurs in the cytoplasm, two replication-associated proteins, vpg-proteinase (nuclear inclusion protein a) (nia) and rna-dependent rna polymerase (nuclear inclusion protein b) (nib), accumulate in the nucleus of infected cells. the 6-kda protein is located adjacent to the n terminus of nia in the tev polyprotein, and, in the context of a 6-kda pro ...	1994	8139025
release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase.	an improved method for the production, cleavage, and purification of fusion proteins and peptides is described. the unique aspect of this method is dependent on the use of a proteinase from tobacco etch virus (tev). the proteinase used is a recombinant tev proteinase produced with a polyhistidine tract positioned at the amino terminus. the proteinase recognizes a specific, extended cleavage site sequence. the peptide or protein of interest is purified as a fusion protein with a tev proteinase cl ...	1994	8179197
specificity of replicase-mediated resistance to cucumber mosaic virus.	plants transformed with a nucleotide sequence coding for a truncated rna 2 replicase gene of the subgroup i strain of cucumber mosaic virus, fny-cmv, are resistant to cucumber mosaic disease. two resistant lines representing independent transformations in the original study have been propagated, and their progeny have been examined. resistance to fny-cmv was genetically integrated and was retained in the r4 generation. fourteen subgroup i cmv strains, 6 strains uncharacterized as to subgroup, an ...	1994	8184532
analysis of transgenic tobacco plants expressing a truncated form of a potyvirus coat protein nucleotide sequence.	transgenic nicotiana tabacum cv. burley 49 plants were generated which expressed a tobacco etch virus (tev) coat protein (cp) gene construct containing a stop codon positioned at codon 147. this gene construct was expected to produce a tev cp lacking the carboxy-terminal 118 amino acids of the full-length 264 amino acid cp. tev cp gene transcripts of the expected size could be detected in transgenic plants but the expected truncated cp could not be detected. ten independent transgenic lines expr ...	1994	8204829
distinct functions of capsid protein in assembly and movement of tobacco etch potyvirus in plants.	tobacco etch potyvirus engineered to express the reporter protein beta-glucuronidase (tev-gus) was used for direct observation and quantitation of virus translocation in plants. four tev-gus mutants were generated containing capsid proteins (cps) with single amino acid substitutions (r154d and d198r), a double substitution (dr), or a deletion of part of the n-terminal domain (delta n). each modified virus replicated as well as the parental virus in protoplasts, but was defective in cell-to-cell ...	1994	7511101
isolation and viral infection of capsicum leaf protoplasts.	a protocol for protoplast isolation was developed and tested with five capsicum genotypes representing two cultivated species, c. annuum and c. chinense. key variables included growth conditions for source plants and the concentration of mannitol used as osmoticum. protoplasts isolated from each of the genotypes became infected when inoculated via electroporation with viral rna from either pepper mottle potyvirus, tobacco etch potyvirus or cucumber mosaic cucumovirus.	1994	24193910
electron microscopic localization of atpase activity in tobacco cells infected by tobacco etch potyvirus and tobacco mosaic virus.	thin sections of leaves of plants infected by tobacco etch potyvirus (tev) or tobacco mosaic virus (tmv) were examined for the presence of atpase activity by electron microscopy. atpase activity was found as expected in mitochondria, chloroplasts and plasmalemma of both uninfected as well as cells infected by either tev or tmv. in the tev-infected cells, atpase activity was localized to virus-induced vesicles, endoplasmic reticulum and in some cells, to ribosomes attached to the er. in tmv-infec ...	1995	7646342
evidence that the potyvirus p1 proteinase functions in trans as an accessory factor for genome amplification.	the tobacco etch potyvirus (tev) polyprotein is proteolytically processed by three viral proteinases (nia, hc-pro, and p1). while the nia and hc-pro proteinases each provide multiple functions essential for viral infectivity, the role of the p1 proteinase beyond its autoproteolytic activity is understood poorly. to determine if p1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire p1 coding region (delta p1 mutant) was produced with a modif ...	1995	7745715
requirement for hc-pro processing during genome amplification of tobacco etch potyvirus.	the helper component-proteinase (hc-pro) of tobacco etch potyvirus (tev) is a multifunctional protein with several known activities. the n-terminal region is required for aphid transmission and efficient genome amplification, the central region is required for long-distance movement in plants, and the c-terminal domain is a cysteine-type proteinase that autocatalytically cleaves between itself and the p3 protein. to investigate the requirement for hc-pro-mediated proteolysis during viral replica ...	1995	7747479
long-distance movement factor: a transport function of the potyvirus helper component proteinase.	transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. through genetic analysis of tobacco etch virus (tev; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. a mutation in ...	1995	7780307
expression and purification of a recombinant tobacco etch virus nia proteinase: biochemical analyses of the full-length and a naturally occurring truncated proteinase form.	the tobacco etch virus 27-kda nuclear inclusion a (nia) proteinase was expressed in escherichia coli as a recombinant fusion protein containing a seven-histidine tag at the amino-terminus. catalytically active and inactive (by virtue of a single amino acid change) forms of the proteinase were purified to homogeneity in a two-column chromatographic procedure. the active form of the proteinase was slowly converted to a lower molecular weight form, while the inactive form was not. this conversion w ...	1995	7793070
complementation of tobacco etch potyvirus mutants by active rna polymerase expressed in transgenic cells.	a genetic complementation system was developed in which tobacco etch virus (tev) polymerase (nib)-expressing transgenic plants or protoplasts were inoculated with nib-defective tev mutants. a beta-glucuronidase (gus) reporter gene integrated into the genomes of parental and four mutant viruses was used to assay rna amplification. two mutants (termed vnn and ede) contained substitutions affecting the conserved "gdd" polymerase motif or a nuclear localization signal sequence, respectively; one (ad ...	1995	7831310
5' proximal potyviral sequences mediate potato virus x/potyviral synergistic disease in transgenic tobacco.	the interaction of potato virus x (pvx) and potato virus y (pvy) in tobacco causes a synergistic disease characterized by a dramatic increase in symptom severity, a change in the regulation of pvx rna replication, and an increase in accumulation of pvx. in this study we demonstrate that pvx also interacts synergistically with three other members of the potyvirus group of plant viruses, tobacco vein mottling virus (tvmv), tobacco etch virus (tev), and pepper mottle virus. these synergisms resembl ...	1995	7831814
debilitation of plant potyvirus infectivity by p1 proteinase-inactivating mutations and restoration by second-site modifications.	tobacco etch virus (tev) encodes three proteinases that catalyze processing of the genome-encoded polyprotein. the p1 proteinase originates from the n terminus of the polyprotein and catalyzes proteolysis between itself and the helper component proteinase (hc-pro). mutations resulting in substitution of a single amino acid, small insertions, or deletions were introduced into the p1 coding sequence of the tev genome. deletion of the n-terminal, nonproteolytic domain of p1 had only minor effects o ...	1995	7853492
capsid protein determinants involved in cell-to-cell and long distance movement of tobacco etch potyvirus.	the tobacco etch potyvirus (tev) capsid protein (cp) is necessary for cell-to-cell and long distance transport of the virus in plants. in this study, the transport phenotypes of tev mutants containing cps with a substitution of the highly conserved ser122 (termed s122w) within the core domain, or with a deletion of sequences encoding 17 amino acid residues comprising most of the variable c-terminal domain (delta c), were analyzed. the s122w and delta c mutant genomes were amplified to levels com ...	1995	7856075
design of a new protease inhibitor by the manipulation of the bait region of alpha 2-macroglobulin: inhibition of the tobacco etch virus protease by mutant alpha 2-macroglobulin.	human alpha 2-macroglobulin (alpha 2m) inhibits a broad spectrum of proteases by changing its conformation and physically confining the enzyme. the inhibitory spectrum of alpha 2m is defined by a stretch of 39 amino acids, the bait region, located near the middle of the alpha 2m monomers. to investigate whether a new inhibitory specificity can be introduced by the manipulation of the bait region, recombinant alpha 2m (r alpha 2m) was produced in which the primary cleavage site was replaced by a ...	1995	7492312
the tobacco etch viral 5' leader and poly(a) tail are functionally synergistic regulators of translation.	the 5' cap (m7gpppn) and the poly(a) tail of eukaryotic mrnas work in concert to establish an efficient level of translation in vivo. nevertheless, several mrnas naturally lack a cap or a poly(a) tail. determining how these messages effectively compete for the translational machinery not only reveals alternative mechanisms for translational competence, but can also underscore similarities between alternative mechanisms and the standard cap/poly(a) tail interaction. the genomic rna of tobacco etc ...	1995	8522182
nucleotide sequence of a strain of tobacco etch virus that does not cause tabasco pepper wilt.		1995	8560790
rna-mediated resistance with nonstructural genes from the tobacco etch virus genome.	sense rna-mediated virus resistance has been described for transgenic plants expressing potyviral capsid protein sequences. this study was undertaken to determine if expression of other viral sequences could induce this type of virus resistance. plants showing highly resistant or 'recovery' phenotypes were generated by expressing the tobacco etch virus (tev) 6 kda/21 kda reading frames. expression of translatable or untranslatable versions of this tev sequence produced resistant lines. highly re ...	1995	8664491
roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.	the roles of the capsid protein (cp) and the cp coding sequence of tobacco etch potyvirus (tev) in genome amplification were analyzed. a series of frameshift-stop codon mutations that interrupted translation of the cp coding sequence at various positions were introduced into the tev genome. a series of 3' deletion mutants that lacked the cp coding sequence beyond each of the frameshift-stop codon mutations were also produced. in addition, a series of 5' cp deletion mutants were generated. amplif ...	1996	8676460
a mammalian 2-5a system functions as an antiviral pathway in transgenic plants.	resistance to virus infections in higher vertebrates is mediated in part through catalysis of rna decay by the, interferon-regulated 2-5a system. a functional 2-5a system requires two enzymes, a 2-5a synthetase that produces 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5a) in response to double-stranded rna, and the 2-5a-dependent rnase l. we have coexpressed these human enzymes in transgenic tobacco plants by using a single plasmid containing the cdnas for both human rnase l and a low mol ...	1996	8692895
analysis of the vpg-proteinase (nia) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification.	a mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus vpg-proteinase (nia) protein in vivo. the nia n-terminal domain contains the vpg attachment site, whereas the c-terminal domain contains a picornavirus 3c-like proteinase. cleavage at an internal site separating the two domains occurs in a subset of nia molecules. the majority of nia molecules in tev-infected cells accumulate within the nucleus. by using a reporter fusion strategy, the nia nuclear loca ...	1996	8794348
genetic and biochemical dissection of transgenic rna-mediated virus resistance.	rna-mediated virus resistance has been observed in transgenic plants at varying frequencies, suggesting that a nuclear requirement or other pre-condition must be met. this study was undertaken to characterize genetically transgenes that confer a highly resistant state to infection by tobacco etch virus (tev). transgenic tobacco line 2rc-6.13, expressing an untranslatable mrna containing the tev coat protein open reading frame, had three distinct transgene integration events that segregated as tw ...	1996	8597662
loss of potyvirus transmissibility and helper-component activity correlate with non-retention of virions in aphid stylets.	the hypothesis that loss of aphid transmissibility of potyvirus mutants is due to non-retention of virions in the mouthparts was tested by feeding aphids through membranes on purified virions of aphid transmissible (at or hat) and non-aphid-transmissible (nat) tobacco vein mottling virus (tvmv) or tobacco etch virus (tev), in the presence of functional [potato virus y (pvy) hc or tvmv hc] or non-functional (pvc hc) helper component (hc). tvmv virions were detected, by electron microscopic examin ...	1996	8609482
site-specific proteolysis of the escherichia coli seca protein in vivo.	a seven-amino-acid cleavage site specific for tobacco etch virus (tev) protease was introduced into seca at two separate positions after amino acids 195 and 252. chromosomal wild-type seca was replaced by these seca constructs. simultaneous expression of tev protease led to cleavage of both seca derivatives. in the functional seca dimer, proteolysis directly indicated surface exposure of the tev protease cleavage sites. cleavage of seca near residue 195 generated an unstable proteolysis product ...	1996	8631693
suppression of long-distance movement of tobacco etch virus in a nonsusceptible host.	to investigate host functions involved in the tobacco etch potyvirus (tev) infection process, a tobacco line (v20) with a strain-specific defect in supporting systemic infection was analyzed. using a modified tev encoding a reporter protein, beta-glucuronidase (gus), genome amplification, cell-to-cell movement, and long-distance movement were measured in v20 and a susceptible line, havana425. comparable levels of tev-gus genome amplification were measured in inoculated protoplasts from both toba ...	1996	8642685
potyvirus transmission is not increased by pre-acquisition fasting of aphids reared on artificial diet.	aphids (myzus persicae), fasted after removal from healthy rearing plants, transmitted tobacco etch potyvirus (tev) more efficiently than unfasted aphids whether virus acquisition was from infected leaves or through membranes. there was no difference in uptake of 125i-labelled tev by fasted or unfasted aphids as measured by liquid scintillation counting. when aphids acquired 125i-labelled tev, label was retained in the stylets (as determined by autoradiographic light microscopy) by 51 % of 272 f ...	1996	9000109
genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase.	the tobacco etch potyvirus (tev) helper component-proteinase (hc-pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. to investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the hc-pro coding sequence. the resulting mutants were analyzed with respect to hc-pro proteolytic ...	1997	9123832
mutations in the region encoding the central domain of helper component-proteinase (hc-pro) eliminate potato virus x/potyviral synergism.	coinfection of tobacco plants with potato virus x (pvx) and any of several members of the potyvirus group causes a synergistic disease characterized by a dramatic increase in symptom severity correlated with a 3- to 10-fold increase in the accumulation of pvx in the first systemically infected leaves. we have recently shown that pvx/potyviral synergistic disease is mediated by expression of potyviral 5'-proximal sequences encoding p1, helper component-proteinase (hc-pro), and a fraction of p3 (t ...	1997	9143300
two separate regions in the genome of the tobacco etch virus contain determinants of the wilting response of tabasco pepper.	infection of tabasco pepper by the tobacco etch virus (tev) typically causes wilting associated with root necrosis. however, a strain of tev, designated tev nonwilting (tev nw), is able to infect tabasco pepper plants but does not cause wilting. in order to locate the genetic determinants responsible for the wilting response, a full-length cdna clone of tev nw from which infectious transcripts can be derived was made. a number of chimeric constructs were prepared by substituting cdna fragments b ...	1997	9150596
immunocytology shows the presence of tobacco etch virus p3 protein in nuclear inclusions.	intracellular localization studies of various potyvirus proteins have been made in hope of finding clues to their function(s). immunocytological studies localized many of the tobacco etch virus (tev)-encoded proteins in infected cells. we used antiserum against the nonstructural p3 protein of tev to determine the subcellular location of the p3 protein in ultrathin sections of virus-infected cells. immunogold labeling with the antiserum showed labels associated with nucleoli, nuclei, or nis, abso ...	1997	9169234
transgenic accumulation of two plant virus coat proteins on a single self-processing polypeptide.	an expression cassette based on the highly specific tobacco etch potyvirus (tev) nuclear inclusion (nia) proteinase has been developed to produce multiple proteins through the translation of a single self-processing polypeptide. gene constructs encoding tev nia, the tobacco mosaic tobamovirus (tmv) coat protein (cp) and the soybean mosaic potyvirus (smv) cp were used to develop transgenic tobacco plants. proper processing of the multifunctional polypeptide was demonstrated, leading to accumulati ...	1997	9225054
formation of plant rna virus replication complexes on membranes: role of an endoplasmic reticulum-targeted viral protein.	the mechanisms that direct positive-stranded rna virus replication complexes to plant and animal cellular membranes are poorly understood. we describe a specific interaction between a replication protein of an rna plant virus and membranes in vitro and in live cells. the tobacco etch virus (tev) 6 kda protein associated with membranes as an integral protein via a central 19 amino acid hydrophobic domain. in the presence or absence of other viral proteins, fluorescent fusion proteins containing t ...	1997	9233814
suppression of potyvirus infection by coexpressed closterovirus protein.	a tobacco etch virus (tev)-based expression vector has been used for insertion of several orfs derived from the unrelated beet yellows virus (byv). hybrid tev variants expressing the byv capsid protein, 20-kda protein, or hsp70 homolog systemically infected nicotiana tabacum and stably retained byv sequences. in contrast, insertion of the orf encoding byv leader proteinase (l-pro) resulted in severely impaired systemic transport and accumulation of recombinant tev. progeny of this virus underwen ...	1997	9268155
vpg of tobacco etch potyvirus is a host genotype-specific determinant for long-distance movement.	the v20 cultivar of nicotiana tabacum was shown previously to exhibit a strain-specific restriction of long-distance movement of tobacco etch potyvirus (tev). in v20, both tev-hat and tev-oxnard strains are capable of genome amplification and cell-to-cell movement, but only tev-oxnard is capable of systemic infection by vasculature-dependent long-distance movement. to investigate the basis for host-specific movement of tev, chimeric virus genomes were assembled from tev-hat and tev-oxnard. virus ...	1997	9343220
rna binding activity of nia proteinase of tobacco etch potyvirus.	the c-terminal domain of nia protein (niapro) from tobacco etch potyvirus (tev) is a sequence-specific proteinase required for processing of the viral polyprotein. this proteinase also interacts with nib, the tev rna-dependent rna polymerase. niapro and two niapro-containing polyproteins (nia and 6/nia) were analyzed from extracts of recombinant escherichia coli. using rna-protein blot and uv-crosslinking assays, niapro and the niapro-containing polyproteins were shown to possess rna-binding act ...	1997	9356344
tntin and tntap: mini-transposons for site-specific proteolysis in vivo.	tobacco etch virus (tev) protease recognizes a 7-aa consensus sequence, glu-xaa-xaa-tyr-xaa-gln-ser, where xaa can be almost any amino acyl residue. cleavage occurs between the conserved gln and ser residues. because of its distinct specificity, tev protease can be expressed in the cytoplasm without interfering with viability. polypeptides that are not natural substrates of tev protease are proteolyzed if they carry the appropriate cleavage site. thus, this protease can be used to study target p ...	1997	9371808
functions of the tobacco etch virus rna polymerase (nib): subcellular transport and protein-protein interaction with vpg/proteinase (nia).	the nib protein of tobacco etch potyvirus (tev) possesses several functions, including rna-dependent rna polymerase and nuclear translocation activities. using a reporter protein fusion strategy, nib was shown to contain two independent nuclear localization signals (nls i and nls ii). nls i was mapped to a sequence within amino acid residues 1 to 17, and nls ii was identified between residues 292 and 316. clustered point mutations resulting in substitutions of basic residues within the nlss were ...	1997	8995687
characterization of post-transcriptionally suppressed transgene expression that confers resistance to tobacco etch virus infection in tobacco.	tobacco lines expressing transgenes that encode tobacco etch virus (tev) coat protein (cp) mrna with or without nonsense codons give rise to tev-resistant tissues that have reduced levels of tev cp mrna while maintaining high levels of transgene transcriptional activity. two phenotypes for virus resistance in the lines containing the transgene have been described: immune (no virus infection) and recovery (initial systemic symptoms followed by gradual recovery over several weeks). here, we show t ...	1997	12237389
charge changes near the n terminus of the coat protein of two potyviruses affect virus movement.	mutants of tobacco vein mottling virus (tvmv) with substitutions of lys or arg for asp in the dag motif at position 5 in the coat protein (cp) failed to infect tobacco plants systemically, but replicated and produced virions in protoplasts. occasional systemic infections occurred when nicotiana benthamiana or transgenic tobacco plants expressing wild-type tvmv cp were inoculated with these mutants, but viral progeny contained reversions to negatively or non-charged amino acids at position 5 or s ...	1998	9460938
a hypersensitive response-like mechanism is involved in resistance of potato plants bearing the ry(sto) gene to the potyviruses potato virus y and tobacco etch virus.	potato plants carrying the ry(sto) gene from solanum stoloniferum are extremely resistant to a number of potyviruses, but it is not known at what stage of infection the resistance is expressed. the resistance may be due to ry(sto) or to a closely linked gene. in this investigation, we used potato virus y (pvy) and a tobacco etch virus construct that encodes beta-glucuronidase (tev-gus) to monitor virus infections of potato plants. systemic spread of either virus in resistant potato plants was no ...	1998	9460939
simultaneous accumulation of multiple viral coat proteins from a tev-nia based expression vector.	we previously described an expression cassette that relies on the tobacco etch virus (tev) nuclear inclusion a (nia) protease and leads to the coordinated accumulation of multiple proteins through self processing of a polyprotein [21]. however, low levels of proteins accumulated when the full-length protease was encoded within the polyprotein [22]. studies were conducted to evaluate whether the disruption of nia nuclear localization would affect the levels of proteins produced via the cassette. ...	1998	9484436
secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome.	the 3'-terminal 350 nucleotides of the tobacco etch potyvirus (tev) genome span the end of the capsid protein (cp)-coding sequence and the 3' nontranslated region (ntr). the cp-coding sequence within this region contains a 105-nucleotide cis-active element required for genome replication (s. mahajan, v. v. dolja, and j. c. carrington, j. virol. 70:4370-4379, 1996). to investigate the sequence and secondary structure requirements within the cp cis-active region and the 3' ntr, a systematic linker ...	1998	9557696
identification and characterization of a locus (rtm1) that restricts long-distance movement of tobacco etch virus in arabidopsis thaliana.	screens of arabidopsis thaliana for susceptibility to tobacco etch virus (tev) revealed that each of 10 ecotypes were able to support genome replication and cell-to-cell movement in inoculated leaves. however, only four ecotypes, including c24 and la-er, supported complete infections in which tev was able to replicate and move from cell to cell and long distances through the vasculature. the rates of cell-to-cell movement of a reporter-tagged tev strain (tev-gus) in inoculated leaves of c24 and ...	1998	9628015
role of the helper component in vector-specific transmission of potyviruses.	four aphid species were tested for their ability to transmit tobacco etch (tev) and turnip mosaic (tumv) potyviruses. myzus persicae and aphis gossypii transmitted both viruses efficiently from infected plants, whereas lipaphis erysimi transmitted only tumv and myzus ascalonicus was a poor or non-transmitter of either virus. similar electrically monitored probing patterns were produced by m. persicae, l. erysimi and m. ascalonicus, ruling out behavioural differences as the cause of differential ...	1998	9634096
functional characterization and localization of protein phosphatase type 2c from paramecium.	we cloned a protein phosphatase 2c gene from paramecium (ptpp2c), which codes for one of the smallest pp2c isoforms (klumpp, s., hanke, c., donella-deana, a., beyer, a., kellner, r., pinna, l. a., and schultz, j. e. (1994) j. biol. chem. 269, 32774-32780). after mutation of 9 ciliate q codons (taa) to caa ptpp2c was expressed as an active protein in escherichia coli. the catalytic core region contains 284 amino acids as defined by c- and n-terminal deletions. the c terminus from amino acid 200-3 ...	1998	9668103
genetic evidence for an essential role for potyvirus ci protein in cell-to-cell movement.	the potyvirus cylindrical inclusion (ci) protein, an rna helicase required for genome replication, was analyzed genetically using alanine-scanning mutagenesis. thirty-one mutations were introduced into the ci protein coding region of modified tobacco etch virus (tev) genomes expressing either beta-glucuronidase or green fluorescent protein reporters. twelve of the mutants were replication-defective in protoplast inoculation assays. among the 19 replication-competent mutants, several possessed ce ...	1998	9670556
non-toxic concentrations of cadmium inhibit systemic movement of turnip vein clearing virus by a salicylic acid-independent mechanism.	systemic movement of plant viruses is a central event in viral infection. to better understand this process, the heavy metal cadmium was used to inhibit systemic spread of turnip vein clearing virus (tvcv), a tobamovirus, in tobacco plants. study of the mechanism by which cadmium exerts this inhibitory effect may provide insights into the essential steps of the tvcv systemic movement pathway. our results demonstrated that cadmium treatment did not affect tvcv transport from the inoculated non-va ...	1998	9807823
a counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing.	posttranscriptional gene silencing (ptgs) in plants inactivates some aberrant or highly expressed rnas in a sequence-specific manner in the cytoplasm. a silencing mechanism similar to ptgs appears to function as an adaptive antiviral response. we demonstrate that the p1/hc-pro polyprotein encoded by tobacco etch virus functions as a suppressor of ptgs. a locus comprised of a highly expressed beta-glucuronidase (gus) transgene was shown to exhibit ptgs. genetic crosses and segregation analyses re ...	1998	9827799
ultrastructural localization of nonstructural and coat proteins of 19 potyviruses using antisera to bacterially expressed proteins of plum pox potyvirus.	antisera to the bacterially expressed nonstructural proteins (nsp) hc-pro, ci, nia, and nib and the coat protein (cp) of plum pox potyvirus (ppv) were used for analysing the composition of virus-induced cytoplasmic and nuclear inclusions by electron microscopy. the antisera reacted with nsp and cp of ppv on immunogold-labelled ultrathin sections. antiserum to cp reacted with virions of seven out of 18 other potyviruses. cp was distributed throughout the cytoplasm of infected cells. antisera to p ...	1998	9856098
isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.	a system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (tev)-derived gene vectors. the vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' n or c termini. these vectors were utilized for expression of proteins encoded by beet yellows closterovirus (byv). approximately 4 micrograms/g of 20-kda byv protein was readily isolated from plants systemically infected by hybr ...	1998	9875335
mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets.	mutations of k --> e in the highly conserved 'kitc' motif of the potyvirus helper component (hc) protein result in loss of hc function in aphid transmission, presumably because of inability to interact with virions, stylets or both. in this study we show that hc of potato virus c (pvc), a naturally occurring variant of potato virus y (pvy) that has the k --> e mutation, lacks the ability to be retained in stylets, whereas pvy hc is retained. the k --> e mutation in either pvc or a site-directed ...	1998	9880030
application of tev protease in protein production.	in many cases, the analysis of a specific protein is impeded by the inability to purify large amounts of it from a native source. proteins of interest may be present in minute quantities and/or purification may be plagued with technical problems. recombinant dna methodologies have enabled researchers to circumvent some of these limitations by producing and purifying large quantities of protein in a nonnative system. various systems and strategies have been successfully employed, depending on the ...	1998	21390844
selectable viruses and altered susceptibility mutants in arabidopsis thaliana.	the genetic basis for susceptibility or nonsusceptibility of plants to viruses is understood poorly. two selectable tobacco etch virus (tev) strains were developed for identification of arabidopsis thaliana mutants with either gain-of-susceptibility or loss-of-susceptibility phenotypes. these strains conferred a conditional-survival phenotype to arabidopsis based on systemic expression of herbicide resistance or proherbicide sensitivity genes, thereby facilitating mass selections and screens for ...	1999	9892709
histidine-tagging and purification of tobacco etch potyvirus helper component protein.	the coding sequence for a series of six histidines (his-tag) was inserted near the 5' terminus of the helper component (hc) coding region of tobacco etch potyvirus (tev). full length genomic clones containing the his-tag coding sequence were infectious and produced symptoms in tobacco (nicotiana tabacuma) similar to those induced by wild-type tev. the modified virus was genetically stable and the his-tag sequence was maintained through at least four cycles of aphid transmission. a protocol for p ...	1999	10029320
imaging fluorescence resonance energy transfer between two green fluorescent proteins in living yeast.	we show that fluorescence resonance energy transfer between two mutants of the green fluorescent protein (gfp) can be monitored by imaging microscopy in living yeast. this work is based on the constitutive expression of a gfp-containing fusion protein and the inducible expression of the tobacco etch virus (tev) protease. in the fusion protein, the p4.3 gfp mutant is linked to the ys65t gfp mutant by a spacer bearing the tev protease-specific cleavage site.	1999	10218581
a novel motif mediates the targeting of the arabidopsis cop1 protein to subnuclear foci.	the constitutive photomorphogenesis 1 (cop1) protein of arabidopsis thaliana accumulates in discrete subnuclear foci. to better understand the role of subnuclear architecture in cop1-mediated gene expression, we investigated the structural motifs of cop1 that mediate its localization to subnuclear foci using mutational analysis with green fluorescent protein as a reporter. in a transient expression assay, a subnuclear localization signal consisting of 58 residues between amino acids 120 and 177 ...	1999	10480941
regulation of closterovirus gene expression examined by insertion of a self-processing reporter and by northern hybridization.	a reporter open reading frame (orf) coding for a fusion of bacterial beta-glucuronidase (gus) with a proteinase domain (pro) derived from tobacco etch potyvirus was utilized for tagging individual genes of beet yellows closterovirus (byv). insertion of this reporter orf between the first and second codons of the byv orfs encoding the hsp70 homolog (hsp70h), a major capsid protein (cp), and a 20-kda protein (p20) resulted in the expression of the processed gus-pro reporter from corresponding subg ...	1999	10482546
functional analysis of the interaction between vpg-proteinase (nia) and rna polymerase (nib) of tobacco etch potyvirus, using conditional and suppressor mutants.	the tobacco etch potyvirus (tev) rna-dependent rna polymerase (nib) has been shown to interact with the proteinase domain of the vpg-proteinase (nia). to investigate the significance of this interaction, a saccharomyces cerevisiae two-hybrid assay was used to isolate conditional nia mutant proteins with temperature-sensitive (ts) defects in interacting with nib. thirty-six unique tsnia mutants with substitutions affecting the proteinase domain were recovered. most of the mutants coded for protei ...	1999	10482627
aids vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but not following intravenous challenge with cell-free virus.	the feline immunodeficiency virus (fiv) provides an excellent model system for aids vaccination studies. in the present experiments we investigated the immunogenicity and the protective activity of two inactivated vaccines prepared from a primary virus isolate. one vaccine was composed of whole virus inactivated with paraformaldehyde and then purified (wiv) and the other of viral proteins extracted with tween-ether (tev). both vaccines elicited robust antiviral responses, but neither conferred a ...	1999	10501242
targeted rnases: a feasibility study for use in hiv gene therapy.	a targeted rnase would be ideal for gene therapy of several acquired and inherited disorders. such an rnase may be engineered to contain a ribonucleolytic domain and a specific target rna binding domain. to demonstrate the feasibility of this approach, an rnase targeted against human immunodeficiency virus (hiv) rna--tev-rnase t1--was designed and tested for its use in hiv-1 gene therapy. a human cd4+ t lymphoid (mt4) cell line and human peripheral blood lymphocytes (pbls) were transduced with r ...	1999	10505117
identification and characterization of the functional elements within the tobacco etch virus 5' leader required for cap-independent translation.	translation in plants is highly cap dependent, and the only plant mrnas known to naturally lack a cap structure (m(7)gpppn) are viral in origin. the genomic rna of tobacco etch virus (tev), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mrna that is naturally uncapped and yet is a highly competitive mrna during translation. the 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mrnas. we have carried out a deletion a ...	1999	10516014
context of the coat protein dag motif affects potyvirus transmissibility by aphids.	previous work with tobacco vein mottling virus (tvmv) has established that a highly conserved three amino acid motif, asp-ala-gly (dag), located near the n terminus of the coat protein (cp), is important for aphid transmission. however, several other potyviruses which have motifs other than dag are aphid-transmissible. creation of these motifs in tvmv through site-directed mutagenesis failed to render tvmv aphid-transmissible from infected plants, and the creation of a putative complementary mot ...	1999	10567662
the use of cysteine proteinase inhibitors to engineer resistance against potyviruses in transgenic tobacco plants.	as the processing mechanism of all known potyviruses involves the activity of cysteine proteinases, we asked whether constitutive expression of a rice cysteine proteinase inhibitor gene could induce resistance against two important potyviruses, tobacco etch virus (tev) and potato virus y (pvy), in transgenic tobacco plants. tobacco lines expressing the foreign gene at varying levels were examined for resistance against tev and pvy infection. there was a clear, direct correlation between the leve ...	1999	10585723
a modular set of prokaryotic and eukaryotic expression vectors.	a modular series of versatile expression vectors is described for improved affinity purification of recombinant fusion proteins. special features of these vectors include (i) serial affinity tags (hexahistidine-gst) to yield extremely pure protein even with very low expression rates, (ii) highly efficient proteolytic cleavage of affinity tags under a variety of conditions by hexahistidine-tagged tobacco etch virus (tev) protease, (iii) pcr cloning design that results in a product of proteolytic ...	2000	10610695
cloning of the arabidopsis rtm1 gene, which controls restriction of long-distance movement of tobacco etch virus.	the locus rtm1 is necessary for restriction of long-distance movement of tobacco etch virus in arabidopsis thaliana without causing a hypersensitive response or inducing systemic acquired resistance. the rtm1 gene was isolated by map-based cloning. the deduced gene product is similar to the alpha-chain of the artocarpus integrifolia lectin, jacalin, and to several proteins that contain multiple repeats of a jacalin-like sequence. these proteins comprise a family with members containing modular o ...	2000	10618445
expression, purification, and initial structural characterization of yadq, a bacterial homolog of mammalian clc chloride channel proteins.	yadq of escherichia coli is a homolog of the mammalian chloride channels of the clc family. the yadq gene was cloned as a fusion protein with a hexahistidine tag and tobacco etch virus protease site for the removal of the tag. the protein was expressed in the membrane of e. coli and extracted with decylmaltoside. purification was achieved by metal affinity chromatography followed by cation exchange. circular dichroism revealed a high alpha-helical content. size exclusion chromatography suggests ...	2000	10648805
synthesis of (-)-strand rna from the 3' untranslated region of plant viral genomes expressed in transgenic plants upon infection with related viruses.	when expressed in transgenic tobacco plants, transgene mrna that includes the 3' untranslated region (3' utr) of lettuce mosaic virus served as template for synthesis of complementary (-)-strand rna following an infection by tobacco etch virus, tobacco vein mottle virus or pepper mottle virus, but not when infected with cucumber mosaic virus. deletion of the 3' utr from the transgene abolished the synthesis of (-)-strand transcripts. similar results were obtained in transgenic tobacco plants exp ...	2000	10725441
arabidopsis rtm2 gene is necessary for specific restriction of tobacco etch virus and encodes an unusual small heat shock-like protein.	arabidopsis plants have a system to specifically restrict the long-distance movement of tobacco etch potyvirus (tev) without involving either hypersensitive cell death or systemic acquired resistance. at least two dominant genes, rtm1 and rtm2, are necessary for this restriction. through a series of coinfection experiments with heterologous viruses, the rtm1/rtm2-mediated restriction was shown to be highly specific for tev. the rtm2 gene was isolated by a map-based cloning strategy. isolation of ...	2000	10760245
production and characterization of biologically active human gm-csf secreted by genetically modified plant cells.	human granulocyte-macrophage colony-stimulating factor (gm-csf), a hemopoietic growth factor, was produced and secreted from tobacco cell suspensions. the gm-csf cdna was carried by a binary vector under the control of the camv 35s promoter and the t7 terminator. in addition, a 5'-nontranslated region from the tobacco etch virus (tev leader sequence) was fused to the n-terminal end of the gm-csf transgene. for ease of purification, a 6-his tag was added to the 3' end of the gm-csf cdna. addition ...	2000	10833400
a trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression.	we prepared a short (29 nucleotides) 5' utr that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' utr. the trimmed sequence, designated as te(37-65), was obtained from a conserved region among several potyviruses. the productivities of uncapped reporter mrnas carrying the te(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 ml of translation reaction mixture. the rib ...	2000	10835258
molecular cloning, expression, and purification of nuclear inclusion a protease from tobacco vein mottling virus.	the gene encoding the c-terminal protease domain of the nuclear inclusion protein a (nia) of tobacco vein mottling virus (tvmv) was cloned from an isolated virus particle and expressed as a fusion protein with glutathione s-transferase in escherichia coli xl1-blue. the 27-kda protease was purified from the fusion protein by glutathione affinity chromatography and mono s chromatography. the purified protease exhibited the specific proteolytic activity towards the nonapeptide substrates, ac-glu-as ...	2000	10850655
controlled intracellular processing of fusion proteins by tev protease.	here we describe a method for controlled intracellular processing (cip) of fusion proteins by tobacco etch virus (tev) protease. a fusion protein containing a tev protease recognition site is expressed in escherichia coli cells that also contain a tev protease expression vector. the fusion protein vector is an iptg-inducible cole1-type plasmid, such as a t7 or tac promoter vector. in contrast, the tev protease is produced by a compatible p15a-type vector that is induced by tetracyclines. not onl ...	2000	10873547
novel topological features of fhac, the outer membrane transporter involved in the secretion of the bordetella pertussis filamentous hemagglutinin.	many pathogenic gram-negative bacteria secrete virulence factors across the cell envelope into the extracellular milieu. the secretion of filamentous hemagglutinin (fha) by bordetella pertussis depends on the pore-forming outer membrane protein fhac, which belongs to a growing family of protein transporters. protein alignment and secondary structure predictions indicated that fhac is likely to be a beta-barrel protein with an odd number of transmembrane beta-strands connected by large surface lo ...	2000	10906141
strain-specific interaction of the tobacco etch virus nia protein with the translation initiation factor eif4e in the yeast two-hybrid system.	the nia protein of potyviruses provides vpg and proteolytic functions during virus replication. it has also been shown to confer host genotype-specific movement functions in plants. specifically, nia from tobacco etch virus (tev)-oxnard, but not from most other strains, confers the ability to move long distances in nicotiana tabacum cultivar "v-20." this led to the hypothesis that all or part of nia may interact with one or more cellular factors. to identify cellular proteins that interact with ...	2000	10915600
conversion of compatible plant-pathogen interactions into incompatible interactions by expression of the pseudomonas syringae pv. syringae 61 hrma gene in transgenic tobacco plants.	the hrma gene from pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium p. syringae pv. tabaci in all examined tobacco cultivars. we expressed this gene in tobacco plants under the control of the tobacco delta0. 3 tobrb7 promoter, which is induced upon nematode infection in tobacco roots (opperman et al. 1994, science, 263, 221-223). a basal level of hrma expression in leaves of transgenic plants activated the expression of pathogenesis-relat ...	2000	10929114
an ry-mediated resistance response in potato requires the intact active site of the nia proteinase from potato virus y.	ry confers extreme resistance to all strains of potato virus y (pvy). to identify the elicitor of the ry-mediated resistance against pvy in potato, we expressed each of the pvy-encoded proteins in leaves of pvy-resistant (ry) and -susceptible (ry) plants. for most of the proteins tested, there was no evident response. however, when the nia proteinase was expressed in leaves of ry plants, there was a hypersensitive response (hr). proteinase active site mutants failed to induce the ry-mediated res ...	2000	10972891
reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells.	the p53 tumor-suppressor gene has been implicated in the inducible activation of excision repair of ultraviolet (uv)-induced cyclobutane pyrimidine dimers (cpds) in human cells. because the large t antigen (ltag) of the simian virus 40 (sv40) binds p53 protein and can interfere with its function, it was of interest to study dna repair in normal human fibroblasts that had been transformed by sv40 compared with that in their nontransformed parental counterparts and to determine whether such transf ...	2000	11020243
expression, purification, refolding, and characterization of recombinant human interleukin-13: utilization of intracellular processing.	interleukin-13 (il-13) is a pleiotropic cytokine that elicits both proinflammatory and anti-inflammatory immune responses. recent studies underscore its role in several diseases, including asthma and cancer. solution studies of il-13 and its soluble receptors may facilitate the design of antagonists/agonists which would require milligram quantities of specifically labeled protein. a synthetic gene encoding human il-13 (hil-13) was inserted into the pmal-c2 vector with a cleavage site for the tob ...	2000	11049743
virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway.	certain plant viruses encode suppressors of posttranscriptional gene silencing (ptgs), an adaptive antiviral defense response that limits virus replication and spread. the tobacco etch potyvirus protein, helper component-proteinase (hc-pro), suppresses ptgs of silenced transgenes. the effect of hc-pro on different steps of the silencing pathway was analyzed by using both transient agrobacterium tumefaciens-based delivery and transgenic systems. hc-pro inactivated ptgs in plants containing a pree ...	2000	11078509
a mammalian expression vector for expression and purification of secreted proteins for structural studies.	a mammalian expression vector with features optimized for simple expression and purification of secreted proteins has been developed. this vector was constructed to facilitate x-ray crystallographic studies of cysteine-rich glycoproteins that are difficult to express by other means. proteins expressed with this vector possess an n-terminal human growth hormone domain and an octahistidine tag separated from the desired polypeptide sequences by a tobacco etch virus protease recognition site. advan ...	2000	11087690
identification of the genome-linked protein in virions of potato virus a, with comparison to other members in genus potyvirus.	viruses of the genus potyvirus, the largest genus of plant-infecting viruses, have a messenger-polarity ssrna genome encapsidated by approximately 2000 units of the viral coat protein (cp), resulting in filamentous virions. only few studies have examined potyvirus virions for the presence of other structural proteins. a protein linked covalently to the 5'-end of the genome has been identified in tobacco vein mottling virus (tvmv) and tobacco etch virus (tev). in tev, it is either the viral nia p ...	2001	11172914
expression, purification and characterization of the structure and disulfide linkages of insulin-like growth factor binding protein-4.	insulin-like growth factor binding protein-4 (igfbp-4), like the other five igfbps, is a critical regulator of the activity of insulin-like growth factor (igf)-i and igf-ii. however igfbp-4 seems to be the only igfbp with no potential to enhance the mitogenic actions of the igfs. igfbp-1 to -3 and -5 each contain 18 conserved cysteine residues, igfbp-6 lacks two of the twelve n-terminal cysteines, while igfbp-4 has two additional cysteines in the central region. a plasmid was constructed to expr ...	2001	11182766
system for cleavable fc fusion proteins using tobacco etch virus (tev) protease.	we describe a novel fc fusion protein system that can be cleaved by tobacco etch virus (tev) protease. this system is desirable because it takes advantage of the high specificity of tev protease and its activity at 4 degrees c. we produced two tev-fc fusion proteins that contain the first three ig domains and all six ig domains of the cell adhesion molecule l1. both proteins were efficiently cleaved by tev protease at 4 degrees c. functional analysis of the cleavage products in neurite outgrowth ...	2001	11196321
a new rt-pcr method for the identification of reoviruses in seawater samples.	the frequent occurrence of reoviruses in environmental samples could be a potential source of interference with enterovirus detection, especially when enterovirus isolation on cell culture is required. in order to evaluate new virus-based criteria for enforcing recreational water quality standards, a new method based on a broad reverse transcribed polymerase chain reaction (rt-pcr) was set up to detect reoviruses. two primers were engineered to amplify a 538 base pair fragment of the sigma 2 gen ...	2001	11229010
the venus's-flytrap and cysteine-rich domains of the human ca2+ receptor are not linked by disulfide bonds.	the extracellular n-terminal domain of the human ca(2+) receptor (hcar) consists of a venus's-flytrap (vft) domain and a cysteine-rich (cys-rich) domain. we have shown earlier that the cys-rich domain is critical for signal transmission from the vft domain to the seven-transmembrane domain. the vft domain contains 10 cysteines: two of them (cys(129) and cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (cys(60)-cys(101), cys(35 ...	2001	11238442
large-scale purification of a stable form of recombinant tobacco etch virus protease.	tobacco etch virus nia proteinase (nia-pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. we have designed a mutant nia-pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. histidine-tagged forms of both wild-type and mutant nia-pro were overexpressed in e. coli under conditions in which greater than 95% of the protease ...	2001	11252791
virus-specific spatial differences in the interference with silencing of the chs-a gene in non-transgenic petunia.	potyviruses, such as potato virus y and tobacco etch virus, as well as cucumber mosaic cucumovirus, interfere with post-transcriptional gene silencing (ptgs). when redstar-type petunia hybrida cultivars, whose flowers have alternating white and pigmented sectors, were infected with these viruses, each virus induced a different pattern of restoration of floral anthocyanin pigmentation. local reversion to coloured phenotypes in the white sectors, which occurred through interference with ptgs of th ...	2001	11297699
functional role of the hiv-1 rev exon 1 encoded region in complex formation and trans-dominant inhibition.	to study functional aspects of the exon 1 encoded region of the human immunodeficiency virus type 1 rev protein, the viral tev protein which exhibits low rev activity but lacks the rev exon 1 encoded region was examined. neither rev-tev heteromer complex formation nor inhibition of rev by an export deficient tev mutant was observed. insertion of the rev exon 1 encoded region into the tev mutant allowed it to oligomerize with rev and act as a trans-dominant negative mutant. this showed that the e ...	2001	11322956
a novel method to determine the topology of peroxisomal membrane proteins in vivo using the tobacco etch virus protease.	most proteins essential for the biogenesis of peroxisomes (peroxins) that are identified to date are associated with or are integral components of the peroxisomal membrane. a prerequisite in elucidating their function is to determine their topology in the membrane. we have developed a novel tool to analyze the topology of peroxisomal membrane proteins in the yeast hansenula polymorpha in vivo using the 27-kda nia protease subunit from the tobacco etch virus (tevp). tevp specifically cleaves pept ...	2001	11443138
analysis of the p1 gene sequences and the 3'-terminal sequences and secondary structures of the single-stranded rna genome of potato virus v.	the immunocapture reverse transcriptase pcr method (ic-rt-pcr) was used to selectively amplify specific genome sequences of an isolate of potato virus v (pvv, genus potyvirus) from a potato plant infected by multiple viruses in the field in finland. the sequences of the 5'- and 3'-non-translated regions (ntr) and the p1-and coat protein (cp)-encoding sequences were determined because they are the most variable genomic regions in potyviruses. the sequences of the new finnish pvv isolate obtained ...	2001	11450952
silencing on the spot. induction and suppression of rna silencing in the agrobacterium-mediated transient expression system.	the agrobacterium-mediated transient expression assay in intact tissues has emerged as a rapid and useful method to analyze genes and gene products in plants. in many cases, high levels of active protein can be produced without the need to produce transgenic plants. in this study, a series of tools were developed to enable strong or weak induction of rna silencing and to suppress rna silencing in the absence of stable transgenes. transient delivery of a gene directing production of a double-stra ...	2001	11457942
production and characterization of the recombinant sphingomonas chlorophenolica pentachlorophenol 4-monooxygenase.	pentachlorophenol 4-monooxygenase (pcp4mo) from sphingomonas chlorophenolica is a flavoprotein that hydroxylates pcp in the presence of nadph and oxygen. in order to investigate the structure and function of active site, recombinant pcp4mo (repcp4mo) was produced in escherichia coli as a glutathione s-transferase (gst) fusion protein. moreover, a tobacco etch virus (tev) protease cleavage site (eklyfqg) was introduced into gst-pcp4mo and a his-tagged tev protease was employed. hence, a two-step ...	2001	11708794
cap-independent translation conferred by the 5' leader of tobacco etch virus is eukaryotic initiation factor 4g dependent.	the 5' leader of tobacco etch virus (tev) genomic rna directs efficient translation from the naturally uncapped viral mrna. two distinct regions within the tev 143-nucleotide leader confer cap-independent translation in vivo even when present in the intercistronic region of a discistronic mrna, indicating that the tev leader contains an internal ribosome entry site (ires). in this study, the requirements for tev ires activity were investigated. the tev ires enhanced translation of monocistronic ...	2001	11711605