| la autoantigen enhances and corrects aberrant translation of poliovirus rna in reticulocyte lysate. | translation initiation on poliovirus rna occurs by internal binding of ribosomes to a sequence within the 5' untranslated region. we have previously characterized a hela cell protein, p52, that binds to a fragment of the poliovirus 5' untranslated region (k. meerovitch, j. pelletier, and n. sonenberg, genes dev. 3:1026-1034, 1989). here we report the purification of the hela p52. protein microsequencing identified p52 as la autoantigen. the la protein is a human antigen that is recognized by ant ... | 1993 | 8389906 |
| evaluation of the use of antisense trna(met) as an inhibitor for eukaryotic protein synthesis. | we attempted to explore the use of antisense rnas against trna as an inhibitor of eukaryotic protein synthesis. for this purpose, antisense rna against the 5'-end half of the initiator trna of wheat germ was synthesized, and its effect on translation of the brome mosaic virus mrna was investigated in a wheat germ cell-free system. when the antisense rna against the 5'-end half of the initiator trna including the anticodon sequence was added at the concentration of 8 microm to the cell-free syste ... | 1993 | 8400802 |
| identification of domains in brome mosaic virus rna-1 and coat protein necessary for specific interaction and encapsidation. | even though many single-stranded rnas are present in the cytoplasm of infected cells, encapsidation by brome mosaic virus (bmv) coat protein is specific for bmv rna. although the highly conserved 3' region of each of the three bmv genomic rnas is an attractive candidate for the site of recognition by the coat protein, band shift and uv cross-linking assays in the presence of specific and nonspecific competitors revealed only nonspecific interactions. however, bmv rna-1 formed a retarded complex ... | 1993 | 8411342 |
| recombination and polymerase error facilitate restoration of infectivity in brome mosaic virus. | the trna-like structure present in the 3' noncoding region of each of the four virion rnas of brome mosaic virus possesses a conserved a-67-u-a-65 (67aua65) sequence. four mutations in this region (67uaa65, 67gaa65, and 67caa65, each with a double base change, and 67gua65, containing a single point mutation), previously shown in vitro to be defective in minus-strand promoter function, were introduced into full-length genomic rnas 2 and 3, and their replicative competence was analyzed in barley p ... | 1993 | 8419651 |
| characterization of a host protein associated with brome mosaic virus rna-dependent rna polymerase. | the association of host proteins with viral rna replication proteins has been reported for a number of (+)-strand rna viruses. however, little is known about the identity or function of these host proteins in viral replication. in this paper we report the characterization of a host protein associated with the rna-dependent rna polymerase (rdrp) from brome mosaic virus (bmv)-infected barley. a host protein was specifically and proportionally enriched with bmv rdrp activity through several purific ... | 1993 | 8434011 |
| rna-dependent replication, transcription, and persistence of brome mosaic virus rna replicons in s. cerevisiae. | this paper shows that yeast can be a host for the replication of a higher eukaryotic viral genome. brome mosaic virus (bmv) is a member of the alphavirus-like superfamily of animal and plant positive strand rna viruses. yeast expressing bmv rna replication genes 1a and 2a support rna-dependent replication and transcription of bmv rna3 derivatives. rna3 derivatives with the coat gene replaced by a reporter gene expressed that gene in a fashion dependent on 1a and 2a expression in trans and on bmv ... | 1993 | 8458084 |
| effect of tandem repeated aug codons on translation efficiency of eukaryotic mrna carrying a short leader sequence. | the effect on translation of multiple copies of the initiation codon aug at the initiation site in a eukaryotic mrna carrying a short leader sequence was tested in translation experiments in vitro. dna, corresponding to a chimeric mrna sequence consisting of the 5' leader region of brome mosaic virus (bmv) rna4 and the goat pre-alpha-lactalbumin mrna sequence, was prepared and transcribed in vitro using sp6 rna polymerase. site-directed mutagenesis was carried out to change the sequence around t ... | 1993 | 8479441 |
| reduction of tobacco mosaic virus accumulation in transgenic plants producing non-functional viral transport proteins. | transgenic plants producing the 30k temperature-sensitive transport protein (tp) of tobacco mosaic virus (tmv) mutant ni2519 (affecting cell-to-cell transport) were found to: (i) be susceptible to wild-type tmv u1 at 24 degrees c (a permissive temperature for ni2519 tp), (ii) acquire a certain level of resistance to tmv u1 accumulation when maintained at 33 degrees c (a non-permissive temperature for ni2519 tp) and (iii) lose the resistance to wild-type tmv after their transfer from 33 degrees c ... | 1993 | 8509764 |
| minus sense transcripts of brome mosaic virus rna-3 intercistronic region interfere with viral replication. | interference with virus replication through the use of defective viral sequences is providing new insight to replication strategies and novel approaches for induced resistance. because replication of brome mosaic virus (bmv) is potentiated by the intercistronic region of rna-3, we examined the effect of adding various (-)sense rnas corresponding to this region in co-transfections with wild type bmv rnas. progeny accumulation in barley protoplasts transfected with rnas 1+2 was decreased by 90% in ... | 1993 | 8517022 |
| homologous rna recombination in brome mosaic virus: au-rich sequences decrease the accuracy of crossovers. | brome mosaic virus, a tripartite positive-stranded rna virus of plants, was used for the determination of sequence requirements of imprecise (aberrant) homologous recombination. a 23-nucleotide (nt) region that included a 6-nt uuaaaa sequence (designated the au sequence) common between wild-type rna2 and mutant rna3 supported both precise and imprecise homologous recombination, though the latter occurred with lower frequency. doubling the length of the 6-nt au sequence in rna3 increased the inci ... | 1996 | 8523555 |
| biochemical and genetic analyses of the interaction between the helicase-like and polymerase-like proteins of the brome mosaic virus. | replication of the three positive-strand genomic rnas of brome mosaic virus requires the activities of the helicase-like 1a and the polymerase-like 2a proteins. one hundred fifteen amino acids of the 2a n-terminus and the 1a helicase-like region of over 50 kda are both necessary and sufficient for 1a-2a interaction. requirement of the large size of the 1a helicase-like domain suggests that higher order structures might be necessary for the protein's interaction with 2a. to explore the structural ... | 1995 | 8525639 |
| coding changes in the 3a cell-to-cell movement gene can extend the host range of brome mosaic virus systemic infection. | the m1 and m2 strains of brome mosaic virus (bmv) both systemically infect the monocot host barley, but only the m2 strain systemically infects the dicot cowpea line tvu-612. we have shown previously that this difference in host range maps primarily to rna3. to further characterize the role of rna3 in host specificity, a series of rna3 hybrids were tested, in inoculations with m1 rna1 and rna2, for ability to systemically infect tvu-612 cowpea. although all hybrids were amplified well in cowpea ... | 1995 | 8553548 |
| interaction of host proteins with the plus-strand promoter of brome mosaic virus rna-2. | the binding of five barley proteins (mr: 37, 36, 35, 34, and 30 kda) to the icr2 motif present at the 5' end of brome mosaic virus (bmv) rna-2 was identified using uv cross-linking. evidence that the interaction is specific included the observation that these proteins did not recognize a similar-size rna fragment transcribed from a nonviral (beta-glucuronidase) gene, nor did they bind to the 3' end of the plus strand of rna-3. replication-defective bmv rna-2 mutants bearing substitution mutation ... | 1995 | 8553568 |
| phosphorylation of plant eukaryotic initiation factor-2 by the plant-encoded double-stranded rna-dependent protein kinase, ppkr, and inhibition of protein synthesis in vitro. | regulation of protein synthesis by eukaryotic initiation factor-2alpha (eif-2alpha) phosphorylation is a highly conserved phenomenon in eukaryotes that occurs in response to various stress conditions. protein kinases capable of phosphorylating eif-2alpha have been characterized from mammals and yeast. however, the phenomenon of eif2-alpha-mediated regulation of protein synthesis and the presence of an eif-2alpha kinase has not been demonstrated in higher plants. we show that plant eif-2alpha (pe ... | 1996 | 8626809 |
| thermodynamic prediction of conserved secondary structure: application to the rre element of hiv, the trna-like element of cmv and the mrna of prion protein. | an algorithm for prediction of conserved secondary structure of single-stranded rna is presented. for each rna of a set of homologous rnas optimal and suboptimal secondary structures are calculated and stored in a base-pair probability matrix. a multiple sequence alignment is performed for the set of rnas. the resulting gaps are introduced into the individual probability matrices. these homologous probability matrices are summed to give a consensus probability matrix emphasizing the conserved se ... | 1996 | 8637012 |
| the polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in rna replication. | brome mosaic virus (bmv), a member of the alphavirus-like super-family of positive-strand rna viruses, encodes two proteins required for viral rna replication: 1a and 2a. 1a contains m7g methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by n- and c-terminal extensions. genetic studies show that bmv rna replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. in vitro, la interacts with the n-terminal 125-am ... | 1996 | 8676500 |
| usefulness of functional and structural solution data for the modeling of trna-like structures. | structures of large rnas are not easily solved by x-ray crystallography or by nmr spectroscopy. this paper reviews the alternate methodology based on enzymatic and chemical mapping data collected on rnas combined with graphical modeling for the construction of three-dimensional models. the different steps that lead to the establishment of the models are critically discussed. it is shown how the correctness of an rna model can be strengthened by establishing correlations between the structure and ... | 1996 | 8786997 |
| initiation of minus-strand rna synthesis by the brome mosaicvirus rna-dependent rna polymerase: use of oligoribonucleotide primers. | various dna- and rna-dependent rna polymerases have been reported to use oligoribonucleotide primers to initiate nucleic acid synthesis. for the brome mosaic virus rna-dependent rna polymerase (rdrp), we determined that in reactions performed with limited gtp concentrations, minus-strand rna synthesis can be stimulated by the inclusion of guanosine monophosphate or specific oligoribonucleotides. furthermore, guanylyl-3',5'-guanosine (gpg) was incorporated into minus-strand rna and increased the ... | 1996 | 8794323 |
| a single codon change in a conserved motif of a bromovirus movement protein gene confers compatibility with a new host. | brome mosaic virus (bmv) and cowpea chlorotic mottle virus (ccmv) are closely related bromoviruses with tripartite rna genomes, but distinct host ranges: bmv systemically infects the monocot barley, while ccmv systemically infects the dicot cowpea. we have previously shown that in approximately 10% of inoculated cowpea plants, a ccmv hybrid [ccmv(b3a)] with the 3a cell-to-cell movement protein gene replaced by that of cowpea-nonadapted bmv directs systemic infections, which are caused by seconda ... | 1996 | 8806564 |
| the mode of action of hirsutellin a on eukaryotic cells. | a 16-kda protein toxin was purified from hirsutella thompsonii var thompsonii and named hirsutellin a (hta). at 0.5 and 5.0 microm concentrations, hta caused detectable cytopathic effects on spodoptera frugiperda cells (sf-9) within 2-4 hr and completely inhibited sf-9 cell growth at 4 days posttreatment. electron microscope data showed that the hta treated sf-9 cells became hypotrophied and internal organelles and cell membranes were disrupted. at the same concentration, hta effectively inhibit ... | 1996 | 8812603 |
| rotavirus vp1 alone specifically binds to the 3' end of viral mrna, but the interaction is not sufficient to initiate minus-strand synthesis. | recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsrna from viral mrna in a cell-free system (d. chen, c. q.-y. zeng, m. j. wentz, m. gorziglia, m. k. estes, and r. f. ramig, j. virol. 68:7030-7039, 1994). to determine which of the core proteins, vp1, vp2, or vp3, recognizes the template mrna during rna replication, sa11 open cores were incubated with 32p-labeled rna probes of viral and nonviral origin and the r ... | 1996 | 8892917 |
| initiation of (-)-strand rna synthesis catalyzed by the bmv rna-dependent rna polymerase: synthesis of oligonucleotides. | rna replication, a process of fundamental importance for pathogenesis by many viruses, remains poorly understood at the mechanistic level because relatively few of the responsible enzymes have been purified and characterized biochemically. partially purified rna-dependent rna polymerase (rdrp) from brome mosaic virus (bmv)-infected barley leaves is able to synthesize (-)-strand rnas from input (+)-strand templates. in resolving rdrp products generated during (-)-strand bmv rna synthesis, we foun ... | 1996 | 8941317 |
| in vitro translation and translocation of apolipoprotein b in a cell-free system from hepg2 cells. | an mrna-dependent cell-free system has been developed from hepg2 cells by hydrolysis of endogenous mrna with micrococcal nuclease. when supplied with rna extracted from hepg2 cells, the system synthesized liver specific proteins such as albumin and apolipoprotein b100. significant amounts of microsomes were also detected in the lysate by measuring nadh-cytochrome c reductase activity and ultracentrifugation. protease protection assays showed the capability of the hepg2 lysate to translocate newl ... | 1996 | 8941365 |
| molecular studies on bromovirus capsid protein. i. characterization of cell-to-cell movement-defective rna3 variants of brome mosaic virus. | to evaluate the extent to which brome mosaic virus (bmv) coat protein (cp) gene is involved in the process of cell-to-cell movement. chenopodium quinoa plants were coinoculated with bmv wild-type rnas 1 and 2 and a rna3 variant containing either the beta-glucuronidase (gus) gene in place of cp (b3/cp-gus), to be subjected to gus analysis, or a deletion in the cp gene to be analyzed by fluorescence in situ hybridization (fish). irrespective of time postinoculation, gus expressed from the subgenom ... | 1996 | 8955048 |
| molecular studies on bromovirus capsid protein. ii. functional analysis of the amino-terminal arginine-rich motif and its role in encapsidation, movement, and pathology. | the n-terminal region of the brome mosaic bromovirus (bmv) coat protein (cp) contains an arginine-rich motif that is conserved among plant and nonplant viruses and implicated in binding the rna during encapsidation. to elucidate the functional significance of this conserved motif in the bmv cp, a series of deletions encompassing the arginine-rich motif was introduced into a biologically active clone of bmv rna3, and their effect on replication, encapsidation, and infection in plants was examined ... | 1996 | 8955049 |
| brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral rna synthesis. | the helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (bmv) are required for viral rna replication in vivo, are present in membrane-bound viral rna polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. to better understand bmv rna replication and bmv-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral rna accumulation in bmv-inf ... | 1996 | 8971020 |
| deletion of the c-terminal 33 amino acids of cucumber mosaic virus movement protein enables a chimeric brome mosaic virus to move from cell to cell. | the movement protein (mp) gene of brome mosaic virus (bmv) was precisely replaced with that of cucumber mosaic virus (cmv). infectivity tests of the chimeric bmv on chenopodium quinoa, a permissive host for cell-to-cell movement of both bmv and cmv, showed that the chimeric bmv failed to move from cell to cell even though it replicated in protoplasts. a spontaneous mutant of the chimeric bmv that displayed cell-to-cell movement was subsequently obtained from a local lesion during one of the expe ... | 1997 | 9032362 |
| structure-based rationale for the rescue of systemic movement of brome mosaic virus by spontaneous second-site mutations in the coat protein gene. | we describe spontaneous second-site reversions within the coat protein open reading frame that rescue the systemic-spread phenotype and increase virion stability of a mutant of brome mosaic virus. based on the crystal structure of the related cowpea chlorotic mottle virus, we show that the modified residues are spatially clustered to affect the formation of hexamers and pentamers and therefore virion stability. | 1997 | 9032390 |
| a mutation in the putative rna polymerase gene inhibits nonhomologous, but not homologous, genetic recombination in an rna virus. | brome mosaic bromovirus (bmv), a positive-stranded rna virus, supports both homologous and nonhomologous rna recombinations. two bmv (temperature-sensitive) mutants with alterations in the 2a protein, the putative rna polymerase component of the viral replicase, were tested for their ability to support both types of recombination. here we report that one of these mutants with the leu-486 substituted by phe did not support nonhomologous recombination. effect on homologous recombination was mainly ... | 1997 | 9050907 |
| engineering of homologous recombination hotspots with au-rich sequences in brome mosaic virus. | previously, we observed that crossovers sites of rna recombinants clustered within or close to au-rich regions during genetic recombination in brome mosaic bromovirus (bmv) (p. d. nagy and j. j. bujarski. j. virol. 70:415-426, 1996). to test whether au-rich sequences can facilitate homologous recombination, au-rich sequences were introduced into parental bmv rnas (rna2 and rna3). these insertions created a homologous rna2-rna3 recombination hotspot. two other au-rich sequences also supported hig ... | 1997 | 9094655 |
| minimal templates directing accurate initiation of subgenomic rna synthesis in vitro by the brome mosaic virus rna-dependent rna polymerase. | short regions of (-)-strand brome mosaic virus (bmv) rna3, proscripts, were shown to direct accurate in vitro synthesis of (+)-strand subgenomic rna by the bmv rna-dependent rna polymerase (rdrp), facilitating characterization of the sequences and/or structures directing subgenomic rna synthesis. proscripts retaining fewer than 8-nt of an 18-nt polyuridylate tract located just upstream of the core promoter sequence (french r, ahlquist p, 1988, j virol 62:2411-2420; marsh le, dreher tw, hall tc, ... | 1997 | 9174098 |
| molecular studies on bromovirus capsid protein. iii. analysis of cell-to-cell movement competence of coat protein defective variants of cowpea chlorotic mottle virus. | to determine whether the role of coat protein (cp) in cell-to-cell movement of dicot-adapted cowpea chlorotic mottle bromovirus (ccmv) is distinct from that of monocot-adapted brome mosaic bromovirus (bmv), two reporter genes, beta-glucuronidase (gus) and enhanced green fluorescent protein (egfp), were substituted for the cp in a biologically active clone of ccmv rna3 (c3). primary leaves of nicotiana benthamiana, chenopodium quinoa, and cowpea were co-inoculated with wild-type (wt) ccmv rna 1 a ... | 1997 | 9191853 |
| rna synthesis by the brome mosaic virus rna-dependent rna polymerase: transition from initiation to elongation. | the initiation and elongation phases of (-)-strand rna synthesis in vitro by the brome mosaic virus rna-dependent rna polymerase (rdrp) are differentially sensitive to inhibitors. in an attempt to characterize further the transition rdrp makes from initiation to elongation, we determined the conditions needed to pause the ternary complex and complete only one round of rna synthesis. during the transition we were able to discern step-wise increases in the affinity of rdrp for rna by measuring sen ... | 1997 | 9201217 |
| complementation of a potato virus x mutant mediated by bombardment of plant tissues with cloned viral movement protein genes. | microprojectile bombardment was used to examine the transport function of the 25 kda movement protein (mp) encoded in the triple gene block of potato virus x (pvx). a 25 kda mp-defective full-length cloned pvx genome carrying a beta-glucuronidase (gus) reporter gene was co-bombarded with 35s promoter constructs containing either the 25 kda mp gene of wild-type pvx, the mp gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (r ... | 1997 | 9267010 |
| tubule-forming capacity of the movement proteins of alfalfa mosaic virus and brome mosaic virus. | the structural phenotype of the movement proteins (mps) of two representatives of the bromoviridae, alfalfa mosaic virus (amv) and brome mosaic virus (bmv), was studied in protoplasts. immunofluorescence microscopy showed that the mps of these viruses, for which there has been no evidence of a tubule-guided mechanism, assemble into long tubular structures at the surface of the infected protoplast. electron microscopy and immunogold analysis confirmed the presence of both mp and virus particles i ... | 1997 | 9267012 |
| effects of aspirin on nitric oxide formation and de novo protein synthesis by rinm5f cells and rat islets. | aspirin and aspirin-like drugs are the most commonly indicated agents for the treatment of inflammation. mechanisms of action for these drugs, however, are not clearly understood. in this study, we examined the effects of aspirin on production of nitric oxide (no), a proinflammatory mediator, and show that aspirin inhibits no production by transformed pancreatic beta cells (rinm5f) and rat islets in a concentration-dependent manner with an ic50 value of approximately 3 mm. therapeutic concentrat ... | 1997 | 9281601 |
| analysis of the interaction of viral rna replication proteins by using the yeast two-hybrid assay. | the yeast two-hybrid system has been a useful tool in the genetic evaluation of protein-protein interactions. however, the biological relevance of these two-hybrid interactions to viral positive-strand rna replication has not been demonstrated. the brome mosaic virus (bmv) system has been characterized extensively both genetically and biochemically, providing numerous mutations in the bmv 1a helicase-like and 2a polymerase-like proteins. we have tested wild-type 1a and 18 insertion mutations of ... | 1997 | 9311832 |
| in vivo dna expression of functional brome mosaic virus rna replicons in saccharomyces cerevisiae. | to facilitate manipulation of brome mosaic virus (bmv) rna replicons in saccharomyces cerevisiae and for yeast genetic analysis of bmv rna replication, gene expression, and host interactions, we constructed dna plasmids from which bmv rna3 and rna3 derivatives can be transcribed in vivo from the galactose-inducible yeast gal1 promoter and terminated by a self-cleaving ribozyme at or near their natural 3' ends. in galactose-induced yeast harboring such plasmids, expression of bmv rna replication ... | 1997 | 9311863 |
| sequence-specific recognition of a subgenomic rna promoter by a viral rna polymerase. | rna templates of 33 nucleotides containing the brome mosaic virus (bmv) core subgenomic promoter were used to determine the promoter elements recognized by the bmv rna-dependent rna polymerase (rdrp) to initiate rna synthesis. nucleotides at positions -17, -14, -13, and -11 relative to the subgenomic initiation site must be maintained for interaction with the rdrp. changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for rdr ... | 1997 | 9326593 |
| yeast mutations in multiple complementation groups inhibit brome mosaic virus rna replication and transcription and perturb regulated expression of the viral polymerase-like gene. | brome mosaic virus (bmv), a member of the alphavirus-like superfamily of positive-strand rna viruses, encodes two proteins, 1a and 2a, that interact with each other, with unidentified host proteins, and with host membranes to form the viral rna replication complex. yeast expressing 1a and 2a support replication and subgenomic mrna synthesis by bmv rna3 derivatives. using a multistep selection and screening process, we have isolated yeast mutants in multiple complementation groups that inhibit bm ... | 1997 | 9391109 |
| molecular studies on bromovirus capsid protein. iv. coat protein exchanges between brome mosaic and cowpea chlorotic mottle viruses exhibit neutral effects in heterologous hosts. | two members of the bromovirus group, brome mosaic virus (bmv) and cowpea chlorotic mottle virus (ccmv), selectively infect barley and cowpea, respectively, and also differ in their ability to systemically infect a common permissive host, chenopodium quinoa. ccmv is confined to inoculated leaves of c. quinoa, whereas bmv causes rapid systemic mottling. to examine whether host-specific determinants for systemic movement of bmv and ccmv in each of these hosts are localized in the coat protein (cp), ... | 1997 | 9400617 |
| silencing homologous rna recombination hot spots with gc-rich sequences in brome mosaic virus. | it has been observed that au-rich sequences form homologous recombination hot spots in brome mosaic virus (bmv), a tripartite positive-stranded rna virus of plants (p. d. nagy and j. j. bujarski, j. virol. 71:3799-3810, 1997). to study the effect of gc-rich sequences on the recombination hot spots, we inserted 30-nucleotide-long gc-rich sequences downstream of au-rich homologous recombination hot spot regions in parental bmv rnas (rna2 and rna3). although these insertions doubled the length of s ... | 1998 | 9445008 |
| transfer rna identity rules and conformation of the tyrosine trna-like domain of bmv rna imply additional charging by histidine and valine. | this paper reports the first example of a triple aminoacylation specificity of a viral trna-like domain. these findings were based on structural studies on the brome mosaic virus (bmv) trna-like domain (felden et al., 1994, j. mol. biol. 235, 508-531) together with knowledge on trna aminoacylation identity rules suggesting potential histidinylation and valylation capacities of the viral rna in addition to its already known tyrosylation ability. here, both predictions are demonstrated by in vitro ... | 1998 | 9480825 |
| brome mosaic virus rna replication protein 1a dramatically increases in vivo stability but not translation of viral genomic rna3. | brome mosaic virus (bmv), a positive-strand rna virus in the alphavirus-like superfamily, encodes two rna replication proteins: 1a, which contains a helicase-like domain and a domain implicated in rna capping, and 2a, which contains a polymerase-like domain. to further explore their functions, we expressed 1a and 2a individually and together in yeast also expressing replicatable transcripts of bmv genomic rna3. complementing prior results that 1a and 2a are required jointly for positive-strand r ... | 1998 | 9482867 |
| characterization of the brome mosaic virus movement protein expressed in e. coli. | the biochemical and functional properties of the movement protein (mp) of brome mosaic virus (bmv) were investigated. expression and purification of the bmv mp from escherichia coli resulted in a pure and soluble protein preparation. sucrose gradient centrifugation revealed that bmv mp forms oligomers consisting of two or more copies but no higher order multimers even when different ionic strengths and phs were applied. nitro-cellulose filter binding and gel retardation studies showed that in vi ... | 1998 | 9514964 |
| nucleic acid-binding properties and subcellular localization of the 3a protein of brome mosaic bromovirus. | brome mosaic bromovirus (bmv) 3a protein is required for cell-to-cell movement of the virus in host plants. the bmv 3a protein (b3a) was produced in escherichia coli using an expression vector. gel retardation analysis and uv cross-linking experiments demonstrated that b3a bound single-stranded rna cooperatively without sequence specificity. binding competition analysis showed that b3a bound to single-stranded nucleic acids more strongly than to double-stranded nucleic acids. deletion mutagenesi ... | 1998 | 9603343 |
| the preparation of rna-dependent rna polymerase complex from virus infected plants. | cucumber mosaic virus (cmv) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic rnas. the very 3' ends of each of the genomic rnas can fold into a trna-like structure. based on the structural analysis of the 3' trna-like structure of the brome mosaic virus (bmv), we superimposed and redrew the 3' trna-like structure of cmv. we homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra- ... | 1998 | 9615471 |
| mechanistic analysis of rna synthesis by rna-dependent rna polymerase from two promoters reveals similarities to dna-dependent rna polymerase. | the brome mosaic virus (bmv) rna-dependent rna polymerase (rdrp) directs template-specific synthesis of (-)-strand genomic and (+)-strand subgenomic rnas in vitro. although the requirements for (-)-strand rna synthesis have been characterized previously, the mechanism of subgenomic rna synthesis has not. mutational analysis of the subgenomic promoter revealed that the +1 cytidylate and the +2 adenylate are important for rna synthesis. unlike (-)-strand rna synthesis, which required only a high g ... | 1998 | 9630251 |
| the effect of histone-mrna interaction in protein synthesis. | the effect of lysine-rich and arginine-rich histones on selective inhibition of translation of mrnas of tobacco mosaic virus (tmv), brome mosaic virus (bmv) and luciferase, in rabbit reticulocyte (rrl) was evaluated. nuclease-treated rrl were supplemented with an appropriate mrna and incubated with lysine-rich or arginine-rich histones at 5 and 2.5 micrograms/100 microliters lysate at 30 degrees c. protein synthesis was measured as incorporation of 3h-leucine into protein for 24 min. the lysine- ... | 1997 | 9693888 |
| interactions between the structural domains of the rna replication proteins of plant-infecting rna viruses. | brome mosaic virus (bmv), a positive-strand rna virus, encodes two replication proteins: the 2a protein, which contains polymerase-like sequences, and the 1a protein, with n-terminal putative capping and c-terminal helicase-like sequences. these two proteins are part of a multisubunit complex which is necessary for viral rna replication. we have previously shown that the yeast two-hybrid assay consistently duplicated results obtained from in vivo rna replication assays and biochemical assays of ... | 1998 | 9696810 |
| resected rna pseudoknots and their recognition by histidyl-trna synthetase. | duplexes constituted by closed or open rna circles paired to single-stranded oligonucleotides terminating with 3'-ccaoh form resected pseudoknots that are substrates of yeast histidyl-trna synthetase. design of this rna fold is linked to the mimicry of the pseudoknotted amino acid accepting branch of the trna-like domain from brome mosaic virus, known to be charged by tyrosyl-trna synthetases, with rna minihelices recapitulating accepting branches of canonical trnas. prediction of the histidylat ... | 1998 | 9724720 |
| moieties in an rna promoter specifically recognized by a viral rna-dependent rna polymerase. | rnas 33 nucleotides in length can direct accurate initiation of subgenomic rna synthesis by the brome mosaic virus rna-dependent rna polymerase (rdrp), provided that the native sequences are maintained at five positions: -17, -14, -13, -11, and the +1 initiation site. the functional groups in the bases of these essential nucleotides required to interact with rdrp were examined by using chemically synthesized rnas containing base analogs at each of the five positions. analysis using a template co ... | 1998 | 9751714 |
| mutations in the n terminus of the brome mosaic virus polymerase affect genetic rna-rna recombination. | previously, we have observed that mutations in proteins 1a and 2a, the two virally encoded components of the brome mosaic virus (bmv) replicase, can affect the frequency of recombination and the locations of rna recombination sites (p. d. nagy, a. dzianott, p. ahlquist, and j. j. bujarski, j. virol. 69:2547-2556, 1995; m. figlerowicz, p. d. nagy, and j. j. bujarski, proc. natl. acad. sci. usa 94:2073-2078, 1997). also, it was found before that the n-terminal domain of 2a, the putative rna polyme ... | 1998 | 9765466 |
| properties of a virus causing mosaic and leaf curl disease of celosia argentea l. in nigeria. | a sap transmissible virus, causing mosaic and leaf curl disease of celosia argentea, was isolated at vegetable farms in amuwo odofin, tejuoso, and abule ado, lagos, nigeria. the virus had a restricted host range confined to a few species of the amaranthaceae, chenopodiaceae and solanaceae families. it failed to infect several other species of the aizoaceae, brassicaceae, cucurbitaceae, fabaceae, lamiaceae, malvaceae, poaceae and tiliaceae families. the virus was transmitted in a non-persistent m ... | 1998 | 9842442 |
| spatial perturbations within an rna promoter specifically recognized by a viral rna-dependent rna polymerase (rdrp) reveal that rdrp can adjust its promoter binding sites. | rna synthesis during viral replication requires specific recognition of rna promoters by the viral rna-dependent rna polymerase (rdrp). four nucleotides (-17, -14, -13, and -11) within the brome mosaic virus (bmv) subgenomic core promoter are required for rna synthesis by the bmv rdrp (r. w. siegel et al., proc. natl. acad. sci. usa 94:11238-11243, 1997). the spatial requirements for these four nucleotides and the initiation (+1) cytidylate were examined in rnas containing nucleotide insertions ... | 1999 | 9847322 |
| subgenomic rna promoters dictate the mode of recognition by bromoviral rna-dependent rna polymerases. | both the brome mosaic virus (bmv) and cowpea chlorotic mottle virus (ccmv) rna-dependent rna polymerases (rdrps) were found to recognize the bmv core subgenomic promoter in the same manner, requiring specific functional groups at positions -17, -14, -13, and -11 relative to the subgenomic initiation site (+1). for ccmv subgenomic rna synthesis, both rdrps required the same nucleotides and four additional nucleotides at positions -20, -16, -15, and -10. the -20 nucleotide is partially responsible ... | 1998 | 9875310 |
| sequences 5' of the conserved trna-like promoter modulate the initiation of minus-strand synthesis by the brome mosaic virus rna-dependent rna polymerase. | each of the brome mosaic virus (bmv) genomic rnas contains a conserved trna-like structure that is sufficient to direct minus-strand rna synthesis in vitro. the trna-like promoters, tb1 and tb3, direct approximately equal amounts of synthesis in vitro. however, 5' sequences were found to affect the amount of minus-strand synthesis, suggesting that sequences beyond the trna-like structure are important in moderating minus-strand synthesis. consistent with this, sequences upstream the trna-like st ... | 1998 | 9878626 |
| a core promoter hairpin is essential for subgenomic rna synthesis in alfalfa mosaic alfamovirus and is conserved in other bromoviridae. | the nucleotide sequence immediately in front of the initiation site for subgenomic rna 4 synthesis on rna 3 minus strand, which has been proved to function as a core promoter, was inspected for secondary structure in 26 species of the plant virus family bromoviridae. in 23 cases a stable hairpin could be predicted at a distance of 3 to 8 nucleotides from the initiation site of rna 4. this hairpin contained several conserved nucleotides that are essential for core promoter activity in brome mosai ... | 1998 | 9926399 |
| mapping sequences active in homologous rna recombination in brome mosaic virus: prediction of recombination hot spots. | the mechanism of homologous recombination has been studied previously in brome mosaic virus (bmv), a tricomponent, positive-stranded rna virus of plants, by using artificial sequences (reviewed by j. j. bujarskip. d. nagy (1996). semin. virol. 7, 363-372). here we extend these studies over bmv-derived sequences to obtain clues on prediction of homologous recombination hot spots. first, mismatch mutations, which reduced the au content, were introduced into the common 60-nt recombination hot-spot ... | 1999 | 9927577 |
| a minimal rna promoter for minus-strand rna synthesis by the brome mosaic virus polymerase complex. | the approximately 150 nt trna-like structure present at the 3' end of each of the brome mosaic virus (bmv) genomic rnas is sufficient to direct minus-strand rna synthesis. rnas containing mutations in the trna-like structure that decrease minus-strand synthesis were tested for their ability to interact with rdrp (rna-dependent rna polymerase) using a template competition assay. mutations that are predicted to disrupt the pseudoknot and stem b1 do not affect the ability of the trna-like structure ... | 1999 | 10024445 |
| a brome mosaic virus intergenic rna3 replication signal functions with viral replication protein 1a to dramatically stabilize rna in vivo. | brome mosaic virus (bmv), a positive-strand rna virus in the alphavirus-like superfamily, encodes two rna replication proteins. the 1a protein has putative helicase and rna-capping domains, whereas 2a contains a polymerase-like domain. saccharomyces cerevisiae expressing 1a and 2a is capable of replicating a bmv rna3 template produced by in vivo transcription of a dna copy of rna3. although insufficient for rna3 replication, the expression of 1a protein alone results in a dramatic and specific s ... | 1999 | 10074107 |
| genotypic and phenotypic analysis of bromovirus adaptive mutants derived from a single plant. | eight adaptive mutant clones have been made from the total rna extracted from uninoculated upper leaves of a single cowpea plant exhibiting systemic infection after inoculation with a hybrid cowpea chlorotic mottle bromovirus (ccmv) with the 3a movement protein gene of ccmv replaced by that of cowpea-nonadapted brome mosaic bromovirus (bmv). sequence and mutational analyses of these clones showed genotypic and phenotypic diversity of the cloned virus population, but all examined clones had the a ... | 1999 | 10229274 |
| the n-terminal half of the brome mosaic virus 1a protein has rna capping-associated activities: specificity for gtp and s-adenosylmethionine. | the n-terminal half of the brome mosaic virus (bmv) 1a replication-associated protein contains sequence motifs found in rna methyltransferases. we demonstrate that recombinant bmv methyltransferase-like (mt) domain expressed in escherichia coli forms an adduct with a guanine nucleotide in a reaction that requires s-adenosylmethionine (adomet) and divalent cations. moieties in gtp and adomet required for adduct formation were determined using a competition assay and chemical analogues. in the gua ... | 1999 | 10364504 |
| rna recombination in brome mosaic virus, a model plus strand rna virus. | studies on the molecular mechanism of genetic recombination in rna viruses have progressed at the time when experimental systems of efficient recombination crossovers were established. the system of brome mosaic virus (bmv) represents one of the most useful and most advanced tools for investigation of the molecular aspects of the mechanism of rna-rna recombination events. by using engineered bmv rna components, the occurrence of both homologous and nonhomologous crosses were demonstrated among t ... | 1998 | 10397334 |
| initiation of genomic plus-strand rna synthesis from dna and rna templates by a viral rna-dependent rna polymerase. | in contrast to the synthesis of minus-strand genomic and plus-strand subgenomic rnas, the requirements for brome mosaic virus (bmv) genomic plus-strand rna synthesis in vitro have not been previously reported. therefore, little is known about the biochemical requirements for directing genomic plus-strand synthesis. using dna templates to characterize the requirements for rna-dependent rna polymerase template recognition, we found that initiation from the 3' end of a template requires one nucleot ... | 1999 | 10400734 |
| use of dna, rna, and chimeric templates by a viral rna-dependent rna polymerase: evolutionary implications for the transition from the rna to the dna world. | all polynucleotide polymerases have a similar structure and mechanism of catalysis, consistent with their evolution from one progenitor polymerase. viral rna-dependent rna polymerases (rdrp) are expected to have properties comparable to those from this progenitor and therefore may offer insight into the commonalities of all classes of polymerases. we examined rna synthesis by the brome mosaic virus rdrp on dna, rna, and hybrid templates and found that precise initiation of rna synthesis can take ... | 1999 | 10400735 |
| brome mosaic virus defective rnas generated during infection of barley plants. | brome mosaic virus (bmv) purified from systemically infected barley leaves 8 weeks post-inoculation (p.i.) contained defective rnas (d-rnas). the d-rnas were detected in total and virion rnas extracted from infected plants at 8 weeks p.i. or later, but not before, when barley plants had been inoculated with virions either containing or lacking d-rna. the d-rnas were derived from genomic rna3 by double or mainly single deletions in the 3a protein orf, and formed a heterogeneous population. by usi ... | 1999 | 10501508 |
| putative rna capping activities encoded by brome mosaic virus: methylation and covalent binding of guanylate by replicase protein 1a. | brome mosaic virus (bmv) rna replication is directed by two virus-encoded proteins, 1a and 2a. the amino-terminal half of 1a is a distant homolog of alphavirus nonstructural protein nsp1, which has been implicated in capping viral rnas. in this study, we examined the enzymatic activities of bmv 1a expressed in yeast, where the protein is fully functional in rna replication. 1a methylated gtp, dgtp, and the cap analogs gpppg and gpppa, using s-adenosylmethionine (adomet) as the methyl donor. prod ... | 1999 | 10559320 |
| brome mosaic virus rna replication proteins 1a and 2a colocalize and 1a independently localizes on the yeast endoplasmic reticulum. | the universal membrane association of positive-strand rna virus rna replication complexes is implicated in their function, but the intracellular membranes used vary among viruses. brome mosaic virus (bmv) encodes two mutually interacting rna replication proteins: 1a, which contains rna capping and helicase-like domains, and the polymerase-like 2a protein. in cells from the natural plant hosts of bmv, 1a and 2a colocalize on the endoplasmic reticulum (er). 1a and 2a also direct bmv rna replicatio ... | 1999 | 10559348 |
| rna-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein. | repeated, specific interactions between capsid protein (cp) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions. the results presented here reveal a striking in vivo switch in cp assembly. using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (bmv) cp can assemble in vivo two remarkably distinct capsids that selectively package bmv-derived rnas in the absence of bmv rna replication: a ... | 1999 | 10570127 |
| in vitro transcription by the turnip yellow mosaic virus rna polymerase: a comparison with the alfalfa mosaic virus and brome mosaic virus replicases. | recently, we showed that the main determinant in the trna-like structure of turnip yellow mosaic virus rna to initiate minus-strand synthesis in vitro is the 3' acca end. by mutational analysis of the 3'-terminal hairpin, we show here that only a non-base-paired acca end is functional and that the stability of the wild-type 3'-proximal hairpin is the most favorable, in that it has the lowest deltag value and a high transcription efficiency. with a nested set of rna fragments, we show that the mi ... | 2000 | 10590114 |
| rt-pcr detection and identification of three species of cucumoviruses with a genus-specific single pair of primers. | reverse transcription and polymerase chain reaction (rt-pcr) was used for detection and identification of three cucumoviruses (cucumber mosaic virus, cmv; peanut stunt virus, psv; tomato aspermy virus, tav) in various plants sources with a single pair of primers, designed as cptall-3 and cptall-5. the pair of cucumovirus genus-specific primers that flank the coat protein gene were designed and used to amplify a dna fragment of approximately ranging from 938 to 966 bp. the rt-pcr with the set of ... | 1999 | 10598084 |
| the cognate coat protein is required for cell-to-cell movement of a chimeric brome mosaic virus mediated by the cucumber mosaic virus movement protein. | cucumber mosaic cucumovirus (cmv) and brome mosaic bromovirus (bmv) have many similarities, including the three-dimensional structure of virions, genome organizations, and requirement of the coat protein (cp) for cell-to-cell movement. we have shown that a chimeric bmv with the cmv 3a movement protein (mp) gene instead of its own cannot move from cell to cell in chenopodium quinoa, a common permissive host for both bmv and cmv. another chimeric bmv was constructed by replacing both mp and cp gen ... | 1999 | 10600595 |
| rna sequence and secondary structural determinants in a minimal viral promoter that directs replicase recognition and initiation of genomic plus-strand rna synthesis. | viral rna replication provides a useful system to study the structure and function of rnas and the mechanism of rna synthesis from rna templates. previously we demonstrated that a 27 nt rna from brome mosaic virus (bmv) can direct correct initiation of genomic plus-strand rna synthesis by the bmv replicase. in this study, using biochemical, nuclear magnetic resonance, and thermodynamic analyses, we determined that the secondary structure of this 27 nt rna can be significantly altered and retain ... | 1999 | 10610788 |
| conformational changes preceding decapsidation of bromegrass mosaic virus under hydrostatic pressure: a small-angle neutron scattering study. | the stability of bromegrass mosaic virus (bmv) and empty shells reassembled in vitro from purified bmv coat protein was investigated under hydrostatic pressure, using solution small-angle neutron scattering. this technique allowed us to monitor directly the dissociation of the particles, and to detect conformational changes preceding dissociation. significant dissociation rates were observed only if virions swelled upon increase of pressure, and pressure effects became irreversible at very high- ... | 2000 | 10698634 |
| virus recovery is induced in brome mosaic virus p2 transgenic plants showing synchronous complementation and rna-2-specific silencing. | nicotiana benthamiana plants expressing brome mosaic virus (bmv) p2 protein complemented replication of rnas1 + 3 but, surprisingly, supported little or no replication of rna-2. despite this, the p2 transgenic plants were able to support systemic migration of rnas-1 and -3. kinetic analyses showed identical degradation rates for rnas-2 and -3, greatly detracting from the concept of an induction of an rna-2-specific degradation system. deletion analysis identified a 200-nucleotide sequence that m ... | 2000 | 10707350 |
| role of rna structure in non-homologous recombination between genomic molecules of brome mosaic virus. | brome mosaic virus (bmv) is a tripartite genome, positive-sense rna virus of plants. previously it was demonstrated that local hybridization between bmv rnas (rna-rna heteroduplex formation) efficiently promotes non-homologous rna recombination. in addition, studies on the role of the bmv polymerase in rna recombination suggested that the location of non-homologous crossovers depends mostly on rna structure. as a result, a detailed analysis of a large number of non-homologous recombinants genera ... | 2000 | 10734190 |
| rna motifs mediating in vivo site-specific nonhomologous recombination in (+) rna virus enforce in vitro nonhomologous crossovers with hiv-1 reverse transcriptase. | there are several lines of evidence that both rna viruses and retroviruses recombine according to a copy choice mechanism. using the brome mosaic virus (bmv)-based system, we recognized elements in the rna structure that enhance nonhomologous crossovers within or near the local heteroduplex formed by recombining molecules. the same structural motifs were employed in vitro to test the ability of human immunodeficiency virus reverse transcriptase (hiv-rt) to switch templates during dna synthesis. ... | 2000 | 10744019 |
| a novel mechanism for inhibition of translation by pokeweed antiviral protein: depurination of the capped rna template. | pokeweed antiviral protein (pap) is known to inactivate ribosomes by removal of a specific adenine from the sarcin/ricin (s/r) loop of the large rrna, thereby inhibiting translation. we demonstrate here that in addition to the previously identified adenine (a4324), pap removes another adenine (a4321) and a guanine (g4323) from the eukaryotic large rrna. recent results indicate that the antiviral activity of pap may not be due to depurination of host ribosomes. using pap mutants that do not depur ... | 2000 | 10744021 |
| frequent homologous recombination events between molecules of one rna component in a multipartite rna virus. | brome mosaic bromovirus (bmv), a tripartite plus-sense rna virus, has been used as a model system to study homologous rna recombination among molecules of the same rna component. pairs of bmv rna3 variants carrying marker mutations at different locations were coinoculated on a local lesion host, and the progeny rna3 in a large number of lesions was analyzed. the majority of doubly infected lesions accumulated the rna3 recombinants. the distribution of the recombinant types was relatively even, i ... | 2000 | 10756034 |
| brome mosaic virus polymerase-like protein 2a is directed to the endoplasmic reticulum by helicase-like viral protein 1a. | brome mosaic virus (bmv), a positive-strand rna virus in the alphavirus-like superfamily, encodes rna replication proteins 1a and 2a. 1a contains a c-terminal helicase-like domain and an n-terminal domain implicated in viral rna capping, and 2a contains a central polymerase-like domain. 1a and 2a colocalize in an endoplasmic reticulum (er)-associated replication complex that is the site of bmv-specific rna-dependent rna synthesis in plant and yeast cells. 1a also localizes to the er in the absen ... | 2000 | 10756046 |
| identification and characterization of a host protein required for efficient template selection in viral rna replication. | biochemical studies suggest that positive-strand rna virus replication involves host as well as viral functions. brome mosaic virus (bmv) is a member of the alphavirus-like superfamily of animal and plant positive-strand rna viruses. yeast expressing the bmv rna replication proteins 1a and 2a supports bmv rna replication and mrna synthesis. using the ability of bmv to replicate in yeast, we show that efficient bmv rna replication requires lsm1p, a yeast protein related to core rna splicing facto ... | 2000 | 10759565 |
| molecular studies on bromovirus capsid protein. | specific interactions are likely to occur between the highly conserved n-proximal arginine-rich motif (arm) of brome mosaic virus (bmv) coat protein (cp) and each of three genomic rnas and a single subgenomic rna during in vivo encapsidation. to characterize these interactions, three independent deletions were engineered into a biologically active clone of bmv rna3 (b3) such that the matured cp of each b3 variant precisely lacks either the entire arm (b3/delta919) or two consecutive arginine res ... | 2000 | 10792997 |
| rna motifs that determine specificity between a viral replicase and its promoter. | the 3' end of brome mosaic virus rna contains a trna-like sequence that directs its rna synthesis. a stem loop structure in this sequence, stem loop c (slc), was investigated using nmr, and correlated with its ability to direct rna synthesis by its replicase. slc consists of two discrete domains, a flexible stem with an internal loop and a rigid stem containing a 5'-aua-3' triloop. efficient rna synthesis requires the sequence on only one side of the flexible stem and a specific compact conforma ... | 2000 | 10802741 |
| a conserved hairpin structure in alfamovirus and bromovirus subgenomic promoters is required for efficient rna synthesis in vitro. | the coat protein gene in rna 3 of alfalfa mosaic virus (amv; genus alfamovirus, family bromoviridae) is translated from the subgenomic rna 4. analysis of the subgenomic promoter (sgp) in minus-strand rna 3 showed that a sequence of 37 nt upstream of the rna 4 start site (nt +1) was sufficient for full sgp activity in an in vitro assay with the purified viral rna-dependent rna-polymerase (rdrp). the sequence of nt -6 to -29 could be folded into a potential hairpin structure with a loop represente ... | 2000 | 10836792 |
| demonstration of functional requirement of polypyrimidine tract-binding protein by selex rna during hepatitis c virus internal ribosome entry site-mediated translation initiation. | polypyrimidine tract-binding protein (ptb) has been previously shown to physically interact with the hepatitis c virus (hcv) rna genome at its 5'- and 3'-noncoding regions. using high affinity selex rna molecules, we present evidence for the functional requirement of ptb during hcv internal ribosome entry site (ires)-controlled translation initiation. this study was carried out in rabbit reticulocyte translation lysates in which the hcv ires-driven reporter rna was introduced along with the ptb- ... | 2000 | 10938288 |
| the 3a cell-to-cell movement gene is dispensable for cell-to-cell transmission of brome mosaic virus rna replicons in yeast but retained over 10(45)-fold amplification. | in yeast expressing the rna replication proteins encoded by brome mosaic virus (bmv), b3ura3, a bmv rna3 derivative that harbours the 3a cell-to-cell movement protein gene and the yeast uracil biosynthesis gene ura3, was replicated and maintained in 85-95% of progeny at each cell division. transmission of the b3ura3 rna replicon from mother to daughter yeast did not require the 3a gene. nevertheless, even after passaging for 165 cycles of rna replication and yeast cell division, each of 40 indep ... | 2000 | 10950990 |
| exploiting polymerase promiscuity: a simple colorimetric rna polymerase assay. | we developed a convenient colorimetric assay for monitoring rna synthesis from dna-dependent rna polymerases (ddrp) and viral rna-dependent rna polymerases (rdrp). atp and gtp with a p-nitrophenyl moiety attached to the gamma-phosphate were synthesized (pnp-ntps). these pnp-ntps can be used for rna synthesis by several rna polymerases, including the rdrps from brome mosaic virus and bovine viral diarrhea virus and the ddrps from bacteriophage t7 and sp6. when the polymerase reactions were perfor ... | 2000 | 10964785 |
| helicase and capping enzyme active site mutations in brome mosaic virus protein 1a cause defects in template recruitment, negative-strand rna synthesis, and viral rna capping. | brome mosaic virus (bmv) encodes two rna replication proteins: 1a, which contains rna capping and helicase-like domains, and 2a, which is related to polymerases. bmv 1a and 2a can direct virus-specific rna replication in the yeast saccharomyces cerevisiae, which reproduces the known features of bmv replication in plant cells. we constructed single amino acid point mutations at the predicted capping and helicase active sites of 1a and analyzed their effects on bmv rna3 replication in yeast. the h ... | 2000 | 10982322 |
| electron microscopy of two viruses of deadly nightshade (atropa belladonna l.). | deadly nightshade plants showing severe necrotic lesions on leaves were observed in southern bohemia. in negatively stained preparations of spontaneously infected deadly nightshade, artificially inoculated host plants and purified preparations two types of virus particles, isometric ones of about 26 nm in diameter and flexuous ones with length of 765 nm were seen by electron microscopy. the virus with isometric particles was identified as belladonna mottle virus (bmv), indistinguishable serologi ... | 2000 | 10989692 |
| packaging of tobacco mosaic virus subgenomic rnas by brome mosaic virus coat protein exhibits rna controlled polymorphism. | the coat protein (cp) of icosahedral brome mosaic virus (bmv) was expressed from a genetically engineered rod-shape tobacco mosaic virus. molecular characterization of the progeny recovered from symptomatic plants revealed that bmv cp selectively packaged the three subgenomic rnas of the hybrid virus into two differently sized icosahedral virus-like particles (vlps). the smaller vlps packaged only the two smaller subgenomic rnas. additional in vitro reassembly assays with bmv cp subunits and tra ... | 2000 | 10998324 |
| molecular studies on bromovirus capsid protein. vii. selective packaging on bmv rna4 by specific n-terminal arginine residuals. | an arginine-rich rna-binding motif (arm) found at the n-proximal region of brome mosaic virus (bmv) coat protein (cp) adopts alpha-helical conformation and shares homology with cps of plant and insect rna viruses, hiv-rev and tat proteins, bacterial antiterminators, and ribosomal splicing factors. the arm of bmv cp, consisting of amino acids 9 through 21 with six arginine residues, is essential for rna binding and subsequent packaging. in this study analysis of the alpha-helical contents of wild ... | 2000 | 11017800 |
| recognition of the core rna promoter for minus-strand rna synthesis by the replicases of brome mosaic virus and cucumber mosaic virus. | replication of viral rna genomes requires the specific interaction between the replicase and the rna template. members of the bromovirus and cucumovirus genera have a trna-like structure at the 3' end of their genomic rnas that interacts with the replicase and is required for minus-strand synthesis. in brome mosaic virus (bmv), a stem-loop structure named c (slc) is present within the trna-like region and is required for replicase binding and initiation of rna synthesis in vitro. we have prepare ... | 2000 | 11044076 |
| bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea. | previously, we reported that ccmv(b3a), a hybrid of bromovirus cowpea chlorotic mottle virus (ccmv) with the 3a cell-to-cell movement protein (mp) gene replaced by that of cowpea-nonadapted bromovirus brome mosaic virus (bmv), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. to determine whether the lack of systemic movement of ccmv(b3a) is due to restriction of local spread at specific leaf tissue interfaces, w ... | 2000 | 11059486 |
| a mutant allele of essential, general translation initiation factor ded1 selectively inhibits translation of a viral mrna. | positive-strand rna virus genomes are substrates for translation, rna replication, and encapsidation. to identify host factors involved in these functions, we used the ability of brome mosaic virus (bmv) rna to replicate in yeast. we report herein identification of a mutation in the essential yeast gene ded1 that inhibited bmv rna replication but not yeast growth. ded1 encodes a dead (asp-glu-ala-asp)-box rna helicase required for translation initiation of all yeast mrnas. inhibition of bmv rna ... | 2000 | 11069307 |
| efficient and specific initiation of subgenomic rna synthesis by cucumber mosaic virus replicase in vitro requires an upstream rna stem-loop. | we defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (cmv) subgenomic rna4. the necessary sequence maps to positions -28 to +15 relative to the initiation cytidylate used to initiate rna synthesis in vivo. positions -28 to -5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. considerable changes in the stem and the loop are tolerated for rna synthesis, including replacement with a different stem-loop. in a template compe ... | 2000 | 11070017 |
| characterization of the adomet-dependent guanylyltransferase activity that is associated with the n terminus of bamboo mosaic virus replicase. | bamboo mosaic virus (bamv), a member of the potexvirus group, infects primarily members of the bambusoideae. open reading frame 1 (orf1) of bamv encodes a 155-kda polypeptide that has long been postulated to be a replicase involved in the replication and formation of the cap structure at the 5' end of the viral genome. to identify and characterize the enzymatic activities associated with the n-terminal domain of the bamv orf1 protein, the intact replicase and two c-terminally truncated proteins ... | 2001 | 11134291 |
| site-specific single amino acid changes to lys or arg in the central region of the movement protein of a hybrid bromovirus are required for adaptation to a nonhost. | a hybrid cowpea chlorotic mottle virus (ccmv) [ccmv(b3a)] in which the ccmv 3a movement protein gene is replaced by the 3a (b3a) gene of brome mosaic virus cannot infect cowpea systemically. previously, analysis of rna3 cdna clones constructed from cowpea-adapted mutants derived from ccmv(b3a) revealed that a single codon change in the b3a gene allowed ccmv(b3a) to infect cowpea systemically. in this study, to extend the analysis of the ccmv(b3a) adaptation mechanism, we directly sequenced b3a g ... | 2001 | 11145888 |
| alfalfa mosaic virus replicase proteins p1 and p2 interact and colocalize at the vacuolar membrane. | replication of alfalfa mosaic virus (amv) rnas depends on the virus-encoded proteins p1 and p2. p1 contains methyltransferase- and helicase-like domains, and p2 contains a polymerase-like domain. coimmunoprecipitation experiments revealed an interaction between in vitro translated-p1 and p2 and showed that these proteins are present together in fractions with rna-dependent rna polymerase activity. a deletion analysis in the yeast two-hybrid system showed that in p1 the c-terminal sequence of 509 ... | 2001 | 11160687 |
| mutation of host delta9 fatty acid desaturase inhibits brome mosaic virus rna replication between template recognition and rna synthesis. | all positive-strand rna viruses assemble their rna replication complexes on intracellular membranes. brome mosaic virus (bmv) replicates its rna in endoplasmic reticulum (er)-associated complexes in plant cells and the yeast saccharomyces cerevisiae. bmv encodes rna replication factors 1a, with domains implicated in rna capping and helicase functions, and 2a, with a central polymerase-like domain. factor 1a interacts independently with the er membrane, viral rna templates, and factor 2a to form ... | 2001 | 11160714 |