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G Bruening

Publications and source records attributed to G Bruening.

At least 37 records · Page 2Linked to original sources
PubMed1992

Nucleotide sequence and genetic map of cowpea severe mosaic virus RNA 2 and comparisons with RNA 2 of other comoviruses.

We report the nucleotide sequence of cowpea severe mosaic comovirus (CPSMV) genomic RNA 2. The molecule is composed of 3732 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six reading frame registers has a long open reading frame, from nt 255 to nt 3260 in the polarity of encapsidated RNA and corresponding to a polyprotein of 1002 amino acid residues (aa). As has been reported for other comoviruses, a second in-frame AUG, at nt position 531, apparently also initiates translation, at least in vitro. Multiple alignments of the deduced CPSMV polyprotein aa sequence with those of bean pod mottle comovirus (BPMV), cowpea mosaic comovirus (CPMV), and red clover mottle comovirus (RCMV) were consistent with a similar size for each of the three genes: the putative movement protein, beginning at the second in-frame AUG, the large coat protein (L), and the small coat protein. Identical nucleotide sequences in the terminal noncoding regions of RNA 2 of the four viruses are limited to 9 nt at the 5' end and the 3' polyadenylate. However, extensive similarities in sequence and potential structure were found. For all three genes and the 5' untranslated region, CPSMV and BPMV are more similar to each other than either is to CPMV or RCMV, the last two being similar to each other. Observed similarities predict that both cleavage sites in the CPSMV RNA 2 polyprotein are at glutamine-serine dipeptides. A sequence of 16 aa at the amino terminus of L, determined by automated Edman degradation, matched a region of the deduced aa sequence in the polyprotein and is consistent with cleavage at the predicted glutamine-serine dipeptide.

Amino Acid Sequence↗
PubMed1991

Evidence for spontaneous circle formation in the replication of the satellite RNA of tobacco ringspot virus.

Replication of the satellite RNA of tobacco ringspot virus (sTobRV RNA) has been postulated to require rolling circle transcription. The expected product of rolling circle transcription, multimeric sTobRV RNA, is known to undergo self-cleavage in vitro to release unit-length sTobRV RNA. A spontaneous, efficient, not enzymically-catalyzed in vitro circularization reaction is characteristic of unit-length sTobRV RNA of the less abundant, (-) polarity. We mutated sTobRV RNA at two sites that are distant from each other in the polyribonucleotide chain. A third form of the sTobRV RNA was mutated at both sites. Multimeric forms of the one-site mutants of sTobRV(+)RNA and sTobRV(-)RNA showed, respectively, undiminished and slightly diminished self-cleavage, whereas the spontaneous circularization of each one-site-mutated, unit-length sTobRV(-)RNA was greatly reduced, compared to the reactions of wild-type sTobRV RNA and the two-site mutant. The two-site mutant and the wild-type sTobRV RNAs replicated with similar efficiency. They reduced the titer of, and severity of, symptoms induced by coinoculated tobacco ringspot virus (TobRV). When coinoculated with TobRV, neither one-site mutant increased or provided protection against TobRV. Rather, each induced a substantial accumulation of what is apparently an endogenous form of sTobRV RNA. Our results are consistent with the formation of circular sTobRV(-)RNA as an essential step in sTobRV RNA replication.

Base Sequence↗
PubMed1990

Identification of a non-junction phosphodiester that influences an autolytic processing reaction of RNA.

Oligoribonucleotides with specific sequences derived from the satellite RNA of tobacco ringspot virus undergo autolytic cleavage at the CpA phosphodiester that is the junction between unit sequences of multimeric satellite RNA. Buzayan et al. (Nucleic Acids Res., 16, 4009-4023 (1988)) showed that an oligoribonucleotide with 97 satellite RNA-derived nucleotide residues self-cleaved with greatly reduced efficiency when it was synthesized in vitro from adenosine-5'-O-(1-thiotriphosphate) (abbreviated rATP alpha S) and three rNTPs. No other substitution of one rNTP by the corresponding rNTP alpha S had this effect, suggesting that a phosphorothioate CpA junction inhibits self-cleavage. Here, we replaced the usual CpA junction of a small self-cleaving oligoribonucleotide with a CpU junction. Self-cleavage of this molecule was reduced not only by rUTP alpha S-substitution, as expected, but also by partial and complete rATP alpha S-substitution. By analysis of the locations of rAMPS residues in cleavage products derived from partially rATP alpha S-substituted oligoribonucleotides, we identified A26 as the residue contributing the non-junction phosphorothioate diester that most strongly inhibited self-cleavage. Manganese ions strongly stimulated the self-cleavage of the rATP alpha S-substituted, CpU-junction oligoribonucleotide but was less effective when the junction was CpA.

Base Sequence↗
PubMed1990

Two autolytic processing reactions of a satellite RNA proceed with inversion of configuration.

Both polarities of the satellite RNA of tobacco ringspot virus occur in infected cells in multimeric forms which are capable of autolytic processing, using different sequences and structures [Feldstein, P.A., et al., Proc. Nat. Acad. Sci. USA (1990) 87 (in press)]. These transesterification reactions generate a 2',3'-cyclophosphate and a 5'-hydroxyl as the two new end groups. Cleavage is at a CpA for the (+) polarity RNA and at an ApG for the (-) polarity RNA. We enzymically synthesized oligoribonucleotides with processing capability and with specific 35S-labeled phosphorothioate diesters in the Rp configuration. After processing had occurred, the terminal nucleoside-2',3'-cyclophosphorothioate diester residues were recovered from the appropriate product by digestion with nuclease and phosphatase. Comparisons with specially prepared endo- and exoisomer reference compounds by thin layer chromatography and autoradiography revealed that the [35S]cytidine- and [35S]adenosine-2',3'-cyclophosphorothioate both were endo-isomers. The results are consistent with transesterification occurring by an inline SN2(P) attack of the 2'-hydroxyl group in the autolytic processing reactions of both polarities of the satellite RNA.

Base Sequence↗
PubMed1990

Effect of actinomycin D on replication of satellite tobacco ringspot virus RNA in plant protoplasts.

We have developed a three-component system of host, tobacco ringspot virus (TobRV), and satellite tobacco ringspot virus RNA (sTobRV RNA) for investigating the specific contributions of host components or TobRV gene products to the propagative cycle of satellite RNA. Cowpea (Vigna unguiculata) protoplasts were inoculated with sTobRV and TobRV genomic RNAs by electroporation. An increase in sTobRV RNA was detected both by blot hybridization and by incorporation of [14C]uridine into material with the electrophoretic mobility of sTobRV RNA. DNA-dependent RNA synthesis in uninoculated protoplasts was effectively inhibited by 50 micrograms/ml actinomycin D (Act D) in the medium. Addition of Act D to protoplasts 24 or 48 hr after coinoculation with sTobRV RNA and TobRV genomic RNAs had little effect on accumulation of sTobRV RNA, whereas addition at 24 hr prior to coinoculation prevented any detected accumulation of sTobRV RNA of either polarity. Our results and previous findings of RNA complementary to encapsidated satellite RNA in extracts of infected tissue suggest that an RNA-dependent RNA polymerase is responsible for the synthesis of sTobRV RNA. The strongly inhibitory effect of Act D when added early implies a role for a host factor in the early phase of sTobRV RNA replication.

Dactinomycin↗
PubMed1990

Specific association between an endoribonucleolytic sequence from a satellite RNA and a substrate analogue containing a 2'-5' phosphodiester.

Both polarities of the satellite RNA of tobacco ringspot virus are sources of self-cleaving sequences. RNA of the less abundant, negative polarity, designated sTobRV-(-)RNA, has cleaving activity that was mapped previously to two noncontiguous regions of the polyribonucleotide chain. Endoribonucleolytic oligoribonucleotides (E) corresponding to the larger of the two regions cleaved smaller substrate oligoribonucleotides, at the ApG phosphodiester that is cleaved in sTobRV(-)RNA. An analogue of the substrate, which has a 2'-5' ApG phosphodiester, was not cleaved by E but acted as a competitive inhibitor of the cleavage of substrate. The analogue served as a primer, and E served as template, for reverse transcriptase-catalyzed copying of specific E sequences. The sequences transcribed suggest base pairing between the 5' region of E and a portion of the substrate that is located 3' to, but does not include, the ApG phosphodiester. Results from other experiments indicate this base pairing is a part of the functional cleavage complex. The association of the ends of E and substrate anticipates a second, 4-base-pair association between E and a portion of substrate that is 5' to, but does not include, the ApG phosphodiester. The effects of compensating mutations in E and substrate oligoribonucleotides support the existence of this second association in the active cleavage complex.

Base Sequence↗
PubMed1989

Two sequences participating in the autolytic processing of satellite tobacco ringspot virus complementary RNA.

Circular and multimeric forms of the satellite RNA of tobacco ringspot virus and their autolytic processing reactions are well known. They suggest replication models in which key elements are rolling circle transcription and the processing of the resulting multimeric RNA to generate the unit, 'monomeric' satellite RNA sequence. We prepared plasmids bearing two distinct sequences of the satellite RNA. Each was arranged to allow transcription of an oligoribonucleotide (r-oligo) of the polarity that is complementary to encapsidated satellite RNA. One sequence has the autolytic processing phosphodiester bond, ApG, and the other is located at a distance of about 150 nucleotide (nt) residues. The second r-oligo accomplished cleavage of the first, in a catalytic fashion. Analysis of truncated forms showed that 10 nt of the ApG junction-containing r-oligo and 46 of the endoribonucleolytic r-oligo were sufficient for recognition in the cleavage reaction. These results map the sequences involved in autolytic processing of the complementary polarity satellite RNA to two regions.

Base Sequence↗
PubMed1988

RNA mediated formation of a phosphorothioate diester bond.

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

Base Sequence↗
PubMed1988

Autolytic processing of a phosphorothioate diester bond.

A small satellite RNA of tobacco ringspot virus replicates in tissues infected with tobacco ringspot virus and accumulates in virus capsids, forming virus-like particles. Previous research showed that multimeric forms of this satellite RNA have tandem repeats of the "monomeric" satellite RNA sequence of 359 or 360 nucleotide residues. The multimeric RNAs undergo autolytic processing at a specific CpA phosphodiester bond, the junction, to generate the monomeric RNA. We substituted phosphorothioate diester bonds for various sets of phosphodiester bonds, in dimeric and truncated forms of the satellite RNA. The degree of reduction in autolytic cleavage varied both with the sites of substitution and the size of the RNA molecules. Analyses of a product of the autolysis reaction suggest that one phosphorothioate diester bond most strongly interferes with processing, the one introduced at the CpA junction during its synthesis from adenosine-5'-0-(1-thiotriphosphate). However, extensive introduction of phosphorothioate diester bonds elsewhere in the molecule also decreased processing, possibly by altering conformation.

Base Sequence↗
PubMed1987

A nucleotide sequence rearrangement distinguishes two isolates of satellite tobacco ringspot virus RNA.

Several strains of tobacco ringspot virus (TobRV) support the replication and encapsidation of satellite tobacco ringspot virus RNA (STobRV RNA). We have compared the nucleotide sequences of four STobRV RNAs, each initially associated with a different isolate of TobRV. A STobRV RNA from a geranium isolate of TobRV and STobRV RNA from the previously analyzed budblight isolate (J.M. Buzayan, W.L. Gerlach, G. Bruening, P. Keese, and A.R. Gould, 1986, Virology 151, 186-199) differed by a single nucleotide residue substitution. STobRV RNAs from TobRV isolates 62L and NC-87 have the same 360-residue nucleotide sequence. This sequence differs from that of the 359-nucleotide residue budblight STobRV RNA principally at locations 100 through 140. The differences between the two sequences in this region are consistent with a rearrangement of blocks of nucleotide residues. The two sequences can be folded with similar patterns of base pairing. All four STobRV RNAs share a sequence of eighty 5'-terminal and of twenty 3'-terminal residues, including the 5' hydroxyl group and 2':3'-cyclic phosphodiester group.

Base Sequence↗
PubMed1986

Nucleotide sequence and newly formed phosphodiester bond of spontaneously ligated satellite tobacco ringspot virus RNA.

The satellite RNA of tobacco ringspot virus (STobRV RNA) replicates and becomes encapsidated in association with tobacco ringspot virus. Previous results show that the infected tissue produces multimeric STobRV RNAs of both polarities. RNA that is complementary to encapsidated STobRV RNA, designated as having the (-) polarity, cleaves autolytically at a specific ApG bond. Purified autolysis products spontaneously join in a non-enzymic reaction. We report characteristics of this RNA ligation reaction: the terminal groups that react, the type of bond in the newly formed junction and the nucleotide sequence of the joined RNA. The nucleotide sequence of the ligated RNA shows that joining of the reacting RNAs restored an ApG bond. The junction ApG has a 3'-to-5' phosphodiester bond. Thus the net ligation reaction of STobRV (-)RNA is the precise reversal of autolysis. We discuss this new type of RNA ligation reaction and its implications for the formation of multimeric STobRV RNAs during replication.

Base Sequence↗
PubMed1986

Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing.

The satellite RNA of tobacco ringspot virus depends upon tobacco ringspot virus for its replication and source of coat protein. The satellite RNA reduces virus accumulation and the severity of virus-induced symptoms. Repetitive sequence, dimeric, and higher forms of the satellite RNA are known to autolytically process to form biologically active monomeric RNA of 359 nucleotide residues [Prody, G. A., Bakos, J. T., Buzayan, J. M., Schneider, I. R. & Bruening, G. (1986) Science 231, 1577-1580], with a 5'-hydroxyl and a 2',3'-cyclic phosphodiester as the new terminal groups. We show here that transcripts of full-length and truncated DNA clones of the satellite RNA sequence also process in a nonenzymic reaction. One such transcript was an RNA that has about one-fourth of the satellite RNA sequence, representing the 3'-terminal and 5'-terminal portions of monomeric RNA joined in the junction that is cleaved in dimeric RNA. This RNA autolytically processed more efficiently than molecules with a larger proportion of the satellite RNA nucleotide sequence.

Journal Article↗
PubMed1984

RNA and capsid accumulation in cowpea protoplasts that are resistant to cowpea mosaic virus strain SB.

Leaf protoplasts from the Arlington line of cowpea (Vigna unguiculata) support only a limited increase of cowpea mosaic virus strain SB (CPMV-SB), whereas cowpea severe mosaic virus, another member of the comovirus group, replicates efficiently in Arlington cowpea protoplasts. CPMV-SB replicates efficiently in protoplasts of cowpea line Blackeye 5. Some characteristics of the virus-specific resistance of Arlington protoplasts to CPMV-SB are reported. Differences between progeny CPMV-SB from Arlington and Blackeye 5 protoplasts were not detected. Inoculation with CPMV-SB RNA, rather than virions, did not make Arlington protoplasts fully susceptible. These results favor, for likely involvement in the CPMV-SB restriction phenomenon, events in the virus life cycle that occur after exposure of virion RNA to the cytoplasm and before assembly of particles is completed. The accumulation of CPMV-SB RNAs of both polarities was found to be depressed in inoculated Arlington protoplasts. However, (+)RNA (virion RNA polarity) accumulated to no lesser extent, per unit of (-)RNA, in Arlington protoplasts than in Blackeye 5 protoplasts. Capsid antigen accumulation, per unit of (+)RNA, was reduced in Arlington protoplasts as compared to Blackeye 5 protoplasts. A working hypothesis consistent with the above and other observations is that Arlington protoplasts have an inhibitory substance that interferes with the production or/and function of CPMV-SB specified proteins.

Capsid↗
PubMed1983

An absolute requirement for the 5' cap structure for mRNA translation in sea urchin eggs.

Translation of a variety of RNAs was studied in a cell-free translation system derived from sea urchin eggs. While RNAs such as globin or tobacco mosaic virus are efficiently translated, viral RNAs which do not contain the 5' cap structure, such as cow pea mosaic virus (CPMV) and poliovirus, are not translated. Mixing experiments with reticulocyte lysates indicated that the lack of translation of uncapped viral RNAs is not due to the presence of a potent inhibitor or the absence of an activating agent. RNA competition experiments between capped and uncapped RNAs indicated that uncapped RNAs do not interact with the sea urchin egg initiation machinery. Proteolytic removal of the 5' viral protein did not allow the translation of CPMV RNA. However, chemical decapping of vesicular stomatitis virus mRNA completely inhibited the translation of this mRNA in the sea urchin cell-free system. We conclude that the sea urchin egg lacks the initiation pathway used to initiate uncapped mRNAs in mammalian cells and thus has an absolute requirement for the 5' cap structure for initiation. In addition we discuss the implications of these findings for the control of protein synthesis after fertilization of the sea urchin egg.

Animals↗
PubMed1979

Tumor induction by Agrobacterium tumefaciens prevented in Vigna sinensis seedlings systemically infected by ribonucleic acid viruses.

Cowpea (Vigna sinensis) seedlings failed to develop tumors after being inoculated with crown gall bacteria (Agrobacterium tumefaciens) if, at times earlier than 1 day later, they were inoculated on the primary leaves with a cowpea mosaic virus that systemically infects them. Inoculation with buffer or with a virus that is restricted to a localized infection, or to which the cowpea is immune, did not interfere with the subsequent development of tumors. The virus infection did not appear to affect directly the titer of A. tumefaciens in the inoculation sites. Nor did mixing of virus particles with A. tumefaciens prevent subsequent appearance of tumors. The influence of virus infection extended across grafts (into tissue that is not susceptible to the virus) and there prevented tumor formation. The sap from infected plants, but not purified virus, decreased tumor formation on carrot disks. Systemic virus infection may induce in cowpeas a translocated substance that prevents tumor induction by A. tumefaciens.

Mosaic Viruses↗