PubMed Health⌕ Search

Biomedical subjects

R Goldbach

Publications and source records attributed to R Goldbach.

At least 37 records · Page 2Linked to original sources
PubMed1997

Multiplication of tomato spotted wilt virus in primary cell cultures derived from two thrips species.

Primary cell cultures prepared from embryos of the thrips species Frankliniella occidentalis and Thrips tabaci were tested for their potential to support replication of tomato spotted wilt virus (TSWV). Using polyclonal antibodies against the viral nucleocapsid protein (N) and indirect immunofluorescent staining, discrete spots with strong signals were observed in the cytoplasm at 48 h post-inoculation in the cell cultures of a F. occidentalis, and a T. tabaci population which failed to transmit the virus. The infection was found in approximately 40% of the monolayer cells. Using antibodies against a nonstructural protein (NSs) of TSWV, uniform and more diffused staining was observed throughout the cytoplasm of these cells, underlying active genome replication. The NSs protein accumulated slower than the N protein in the cells of both thrips species. No multiplication of TSWV was observed in a heterologous insect cell line, i.e. from Spodoptera frugiperda, suggesting the existence of specific host factors in the thrips-derived cells.

Animals↗
PubMed1997

Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants expressing NS(M) gene sequences.

Transgenic Nicotiana tabacum plants expressing RNA sequences of the tomato spotted wilt virus NS(M) gene, which encodes the putative viral movement protein, were found to be highly resistant to infection with the virus. Expression of untranslatable as well as anti-sense RNA of the NS(M) gene resulted in resistance levels as high as those in plants expressing translatable RNA sequences. For all three types of transgenic plants resistance levels of up to 100% were reached in the S2 progeny. These results indicate that the resistance mediated by the NS(M) gene is accomplished by expression of transcripts rather than protein in transgenic plants, similar to previously observed N gene-mediated resistance. Protoplast inoculations revealed that resistant plants expressing NS(M) are, in contrast to N transgenic resistant plants, not resistant at the cellular level. This suggests the RNA-mediated resistance mechanism against TSWV targets viral mRNAs rather than the viral genome.

Genes, Plant↗
PubMed1997

Expression of the potato leafroll virus ORF0 induces viral-disease-like symptoms in transgenic potato plants.

The role of the open reading frame 0 (ORF0) of luteoviruses in the viral infection cycle has not been resolved, although the translation product (p28) of this ORF has been suggested to play a role in host recognition. To investigate the function of the potato leafroll luteovirus (PLRV) p28 protein, transgenic potato plants were produced containing the ORF0. In the lines in which the ORF0 transcripts could be detected by Northern (RNA) analysis, the plants displayed an altered phenotype resembling virus-infected plants. A positive correlation was observed between levels of accumulation of the transgenic transcripts and severity of the phenotypic aberrations observed. In contrast, potato plants transformed with a modified, untranslatable ORF0 sequence were phenotypically indistinguishable from wild-type control plants. These results suggest that the p28 protein is involved in viral symptom expression. Southern blot analysis showed that the transgenic plants that accumulated low levels of ORF0 transcripts detectable only by reverse transcription-polymerase chain reaction, contained methylated ORF0 DNA sequences, indicating down-regulation of the transgene provoked by the putatively unfavorable effects p28 causes in the plant cell.

Amino Acid Sequence↗
PubMed1997

Completion of the impatiens necrotic spot virus genome sequence and genetic comparison of the L proteins within the family Bunyaviridae.

The nucleotide sequence of the large (L) genome segment of impatiens necrotic spot virus (INSV) has been determined, and herewith the complete nucleotide sequence of the whole, tripartite genome of this important tospovirus has been elucidated. The L RNA is 8776 nucleotides long and of negative polarity, containing one large ORF on the viral complementary strand. Comparison of the deduced amino acid sequence of the INSV L RNA primary translation product (330.3 kDa) with those of the L RNAs of other members of the family Bunyaviridae reveals that this protein represents the putative viral RNA-dependent RNA polymerase. A cluster dendrogram of the (putative) RNA polymerases indicates that the genera Tospovirus and Tenuivirus, though both encompassing ambisense plant-infecting viruses, have different affinities to the animal-infecting Bunyaviridae, tospoviruses being most closely related to the genus Bunyavirus, and tenuiviruses to the genus Phlebovirus.

Amino Acid Sequence↗
PubMed1997

A protoplast system for studying tomato spotted wilt virus infection.

A plant protoplast system for studying tomato spotted wilt tospovirus (TSWV) infection was established and tested. Using polyethylene glycol-mediated inoculation with highly infectious TSWV particles, generally 50% or more of Nicotiana rustica protoplasts were infected. In these cells viral RNA and viral protein synthesis became detectable at 16 h post-inoculation (p.i.) and continued at least until 90 h p.i. Both the structural viral proteins [nucleoprotein (N) and the envelope glycoproteins G1 and G2] and the nonstructural viral proteins NSs and NSm accumulated to amounts sufficient for detection and immunocytological analysis. Local lesion tests on petunia leaves and electron microscopical analysis confirmed the production of mature, infectious virus particles, underlining the conclusion that a full infection cycle was completed in this system. Upon inoculation of Vigna unguiculata (cowpea) protoplasts with TSWV particles, comparable proportions of infected cells and amounts of NSs, NSm and N protein were obtained, but much lower amounts of viral glycoproteins were detected than in N. rustica protoplasts, and progeny virus particles were less abundant. With the N. rustica-based protoplast system, a powerful synchronized single-cell infection system has now become available for more precise in vivo studies of the processes occurring during tospovirus infection.

Animals↗
PubMed1997

Characterization of the in vitro activity of the RNA-dependent RNA polymerase associated with the ribonucleoproteins of rice hoja blanca tenuivirus.

An RNA-dependent RNA polymerase (RdRp) activity associated with the ribonucleoproteins of rice hoja blanca tenuivirus (RHBV) was detected and analyzed. Conditions for in vitro RNA synthesis and for coupled RNA synthesis-translation of RHBV were established. In both cases, synthesis of the viral and viral complementary genomic and subgenomic RNA3 and RNA4 were observed, demonstrating that both transcription and replication occurred. Though coupling of RNA synthesis to translation allowed efficient translation of the newly synthesized subgenomic RNAs, studies of the effect of various inhibitors of protein synthesis revealed that RNA synthesis was independent of translation. Primer extension experiments demonstrated that in the presence of capped exogenous RNAs, a stretch of 10 to 16 nonviral nucleotides was added to the 5' end of a population of newly synthesized viral complementary RNA4. It appears that in addition to RdRp activity, RHBV-associated protein(s) also possessed cap-snatching capacity.

Blotting, Northern↗
PubMed1996

Sequence analysis of the 5' ends of tomato spotted wilt virus N mRNAs.

Messenger RNAs transcribed from the tomato spotted wilt virus (TSWV) RNA genome have characteristic extra non-templated heterogeneous sequences at their 5' ends which may be the result of a cap-snatching event involving cellular mRNAs. In order to investigate the genetic origin of these extra sequences and to gain more insight in the process of cap-snatching as performed by TSWV, nucleocapsid protein (N) mRNAs derived from the TSWV S RNA were cloned and sequenced. Twenty clones were obtained which contained 5'-proximal sequences of non-viral origin, ranging in length from 12 to 21 nucleotides. None of the sequences analyzed were identical and no base preference at the endonucleolytic site was observed.

Base Sequence↗
PubMed1996

RNA-mediated virus resistance in transgenic plants.

In recent years the concept of pathogen-derived resistance (PDR) has been successfully exploited for conferring resistance against viruses in many crop plants. Starting with coat protein-mediated resistance, the range has been broadened to the use of other viral genes as a source of PDR. However, in the course of the efforts, often no clear correlation could be made between expression levels of the transgenes and observed virus resistance levels. Several reports mentioned high resistance levels using genes incapable of producing protein, but in these cases, even plants accumulating high amounts of transgene RNA were not most resistant. To accommodate these unexplained observations, a resistance mechanism involving specific breakdown of viral RNAs has been proposed. Recent progress towards understanding the RNA-mediated resistance mechanism and similarities with the co-suppression phenomenon will be discussed.

Gene Expression↗
PubMed1996

Distinct functional domains in the cowpea mosaic virus movement protein.

Cell-to-cell movement of cowpea mosaic virus particles in plants takes place with the help of tubules that penetrate presumably modified plasmodesmata. These tubules, which are built up by the virus-encoded 48-kDa movement protein (MP), are also formed on single protoplast cells. To determine whether the MP contains different functional domains, the effect of mutations in its coding region was studied. Mutations between amino acids 1 and 313 led to complete abolishment of the tubule-forming capacity, while a deletion in the C-terminal region resulted in tubules that could not take up virus particles. From these observations, it is concluded that the MP contains at least two distinct domains, one that is involved in tubule formation and that spans amino acids 1 and 313 and a second that is probably involved in the incorporation of virus particles in the tubule and that is located in the C terminus between amino acids 314 and 331.

Comovirus↗
PubMed1994

Expression and subcellular location of the NSM protein of tomato spotted wilt virus (TSWV), a putative viral movement protein.

The 33.6-kDa nonstructural (NSM) protein gene, located on the ambisense M RNA segment of tomato spotted wilt virus (TSWV), was cloned and expressed using the Escherichia coli pET-11t expression system. The protein thus produced was purified and used for the production of a polyclonal antiserum. Western immunoblot analyses of TSWV-infected Nicotiana rustica plants showed NSM synthesis only during a short period early in systemic infection. Although NSM was found associated with cytoplasmic nucleocapsid preparations, it was absent from purified virus particles. Analyses of subcellular fractions from young, systemically infected leaves showed the presence of NSM in fractions enriched for cell walls and cytoplasmic membranes, respectively. Furthermore, immunogold labeling of tissue sections of TSWV-infected N. rustica plants showed that this protein was found associated with nucleocapsid aggregates in the cytoplasm and in close association with plasmodesmata. The data obtained provide evidence that NSM represents the viral movement protein of TSWV, involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures.

Animals↗
PubMed1994

Adaptation of positive-strand RNA viruses to plants.

The vast majority of positive-strand RNA viruses (more than 500 species) are adapted to infection of plant hosts. Genome sequence comparisons of these plant RNA viruses have revealed that most of them are genetically related to animal cell-infecting counterparts; this led to the concept of "superfamilies". Comparison of genetic maps of representative plant and animal viruses belonging to the same superfamily (e.g. cowpea mosaic virus [CPMV] versus picornaviruses and tobacco mosaic virus versus alphaviruses) have revealed genes in the plant viral genomes that appear to be essential adaptations needed for successful invasion and spread through their plant hosts. The best studied example represents the "movement protein" gene that is actively involved in cell-to-cell spread of plant viruses, thereby playing a key role in virulence and pathogenesis. In this paper the host adaptations of a number of plant viruses will be discussed, with special emphasis on the cell-to-cell movement mechanism of comovirus CPMV.

Adaptation, Physiological↗
PubMed1993

Detection of the L protein of tomato spotted wilt virus.

The 5'-terminal and 3'-terminal parts of the single open reading frame (ORF) in the L RNA of tomato spotted wilt virus (TSWV) were expressed using a prokaryotic expression system. Using antibodies raised against the translational products obtained a 330-kDa protein could be specifically detected in preparations of purified virions and in nucleocapsid preparations from TSWV-infected leaf tissue. The results obtained indicate that the L protein of TSWV, though much larger than that of the animal-infecting bunyaviruses, is present in virus particles in an unprocessed, intact form.

Base Sequence↗
PubMed1993

Taxonomic relationships between distinct potato virus Y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions.

Detailed comparisons were made of the sequences of the coat protein (CP) cistrons and 3'-nontranslated regions (3'-NTR) of 21 (geographically) distinct isolates of potato virus Y (PVY) and a virus isolate initially described as pepper mottle virus (PepMoV). Multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host range, symptomatology and serology. Virus isolates belonging to the same subgroup shared a number of characteristic CP amino acid and 3'-NTR nucleotide residues indicating that, by using sequences from the 3'-terminal region of the potyvirus genome, a distinction could be made between different isolates of one virus species as well as between different virus species. RNA secondary structure analysis of the 3'-NTR of twelve PVY isolates revealed four major stem-loop structures of which, surprisingly, the loop sequences gave a similar clustering of isolates as resulting from the overall comparisons of CP and 3'-NTR sequences. This implies a biological significance of these structural elements.

Amino Acid Sequence↗
PubMed1993

Complex formation determines the activity of ribozymes directed against potato virus YN genomic RNA sequences.

A ribozyme was synthesized against a conserved region in the RNA-dependent RNA-polymerase encoding cistron of the important plant pathogen potato virus Y (PVY). This ribozyme was shown to cleave PVY-specific RNA-transcripts efficiently in vitro, with up to 95% of the substrate RNA being cleaved within 2 h incubation at 37 degrees C. A second ribozyme, designed with much shorter viral complementary arms in an attempt to optimize the efficiency of the cleavage reaction, surprisingly failed to cleave the substrates previously cleaved by the longer ribozyme. A much shorter PVY specific RNA-transcript of only 37 nucleotides (nt), however, was cleaved by this short ribozyme proving its ribozymic activity and indicating that the cleavage activity of the ribozyme is, in part, determined by the substrate involved. Analysis of cleavage reactions on non-denaturing polyacrylamide (PAA) gels indicated that incorrect basepairing, interfering with correct formation of the hammerhead structure, was likely to be responsible for the absence of detectable cleavage of the larger substrates by the short ribozyme.

Base Sequence↗
PubMed1993

Sequence data to settle the taxonomic position of bean common mosaic virus and blackeye cowpea mosaic virus isolates.

The nucleotide sequences of the coat protein genes and 3' non-translated regions (3'-NTRs) of three isolates of bean common mosaic virus (NL1, NL3 and NY15) and one isolate of blackeye cowpea mosaic virus (W) were determined. Comparison of these sequences revealed that the coat proteins of NL1, NY15 and W were identical in size (287 amino acids) and exhibited an overall sequence similarity (94 to 97%), and 84 to 98% in their N-terminal regions. Furthermore, their 3'-NTRs were very similar in length [253 to 256 nucleotides (nt)] and sequence (93 to 96% similarity). In contrast, the coat protein of NL3 had only 261 amino acids and showed 87 to 89% similarity with NL1, NY15 and W whereas its N-terminal region revealed only 46 to 61% similarity. The 3'-NTR of NL3 also displayed appreciable differences, both in length (240 nt) and sequence (56 to 63% similarity). These results, in combination with earlier serological findings, justify the conclusion that NL1, NY15 and W should be considered strains of the same virus, i.e. bean common mosaic virus, and that NL3 is a strain of a different potyvirus for which the name 'bean black root virus' is proposed.

Amino Acid Sequence↗
PubMed1993

RNA sequence of potato virus X strain HB.

The genomic RNA of the potato virus X (PVX) strain HB, isolated in Bolivia and able to overcome all known resistance genes, has been cloned and sequenced. The PVXHB RNA sequence is 6432 nucleotides long and contains, similarly to the RNAs of other PVX strains, five open reading frames encoding proteins of M(r)s 165.1K, 24.5K, 12.4K, 7.6K and 25.1K (coat protein), respectively. Multiple amino acid sequence alignments of the coat proteins of four PVX strains identified eight amino acid residues unique for PVXHB. Structural prediction comparisons of the coat proteins of PVXHB and of the other strains suggest a general structural similarity. However, two of the eight amino acid residues unique for strain HB gave rise to a change in the predicted coat protein structure, suggesting a possible involvement in the resistance-breaking activity of PVXHB.

Amino Acid Sequence↗
PubMed1993

Multiplication of tomato spotted wilt virus in its insect vector, Frankliniella occidentalis.

The accumulation of two proteins, the nucleocapsid (N) protein and a non-structural (NSs) protein both encoded by the S RNA of tomato spotted wilt virus (TSWV), was followed in larvae during development and in adults of Frankliniella occidentalis after ingesting the virus for short periods on infected plants. The amounts of both proteins increased, as shown by ELISA and Western blot analysis, within 2 days above the levels ingested, indicating multiplication of TSWV in these insects. Accumulation of these proteins and of virus particles was further confirmed by in situ immunolabelling of the salivary glands and other tissues of adult thrips. The accumulation of large amounts of N and NSs protein, the occurrence of several vesicles with virus particles in the salivary glands and the massive numbers of virus particles in the salivary gland ducts demonstrate that the salivary glands are a major site of TSWV replication. The occurrence of virus particles in the salivary vesicles is indicative of the involvement of the Golgi apparatus in the maturation of the virus particles and its transport to the salivary ducts.

Animals↗