Integration of banana streak badnavirus into the Musa genome: molecular and cytogenetic evidence.
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Breeding and tissue culture of certain cultivars of bananas (Musa) have led to high levels of banana streak badnavirus (BSV) infection in progeny from symptomless parents. BSV DNA hybridized to genomic DNA of one such parent, Obino l'Ewai, suggesting integration of viral sequences. Sequencing of clones of Obino l'Ewai genomic DNA revealed an interface between BSV and Musa sequences and a complex BSV integrant. In situ hybridization revealed two different BSV sequence locations in Obino l'Ewai chromosomes and a complex arrangement of BSV and Musa sequences was shown by probing stretched DNA fibers. This is the first report of integrated sequences that possibly lead to a plant pararetrovirus episomal infection by a mechanism differing markedly from animal retroviral systems. (+info)
Evidence that badnavirus infection in Musa can originate from integrated pararetroviral sequences.
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When some virus- and disease-free Musa spp. (banana and plantain) are propagated by tissue culture, the resulting plants develop infections with banana streak badnavirus (BSV), a pararetrovirus. In sharp contrast to the virion DNA recovered from natural infections, the virion DNA from tissue culture-associated infections of different Musa spp. was highly similar if not identical. Although BSV does not employ integration during the infection cycle, BSV DNA was found to be integrated into the Musa genome. While one integration consisted of a partial BSV genome, a second contained more than one complete genome that was almost identical to BSV recovered from tissue culture-derived plants. The arrangement of this integrated BSV DNA suggests that it can yield an infectious episomal genome via homologous recombination. This report documents the first instance of integrated DNA of a nonintegrating virus giving rise to an episomal viral infection and identifies tissue culture as a possible trigger for the infection, raising the question of whether similar activatable viral sequences exist in the genomes of other plants and animals. (+info)
A short open reading frame terminating in front of a stable hairpin is the conserved feature in pregenomic RNA leaders of plant pararetroviruses.
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In plant pararetroviruses, pregenomic RNA (pgRNA) directs synthesis of circular double-stranded viral DNA and serves as a polycistronic mRNA. By computer-aided analysis, the 14 plant pararetroviruses sequenced so far were compared with respect to structural organization of their pgRNA 5'-leader. The results revealed that the pgRNA of all these viruses carries a long leader sequence containing several short ORFs and having the potential to form a large stem-loop structure; both features are known to be inhibitory for downstream translation. Formation of the structure brings the first long ORF into the close spatial vicinity of a 5'-proximal short ORF that terminates 5 to 10 nt upstream of the stable structural element. The first long ORF on the pgRNA is translated by a ribosome shunt mechanism discovered in cauliflower mosaic (CaMV) and rice tungro bacilliform viruses, representing the two major groups of plant pararetroviruses. Both the short ORF and the structure have been implicated in the shunt process for CaMV pgRNA translation. The conservation of these elements among all plant pararetroviruses suggests conservation of the ribosome shunt mechanism. For some of the less well-studied viruses, the localization of the conserved elements also allowed predictions of the pgRNA promoter region and the translation start site of the first long ORF. (+info)
Sequence changes in six variants of rice tungro bacilliform virus and their phylogenetic relationships.
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The DNA of three biological variants, G1, Ic and G2, which originated from the same greenhouse isolate of rice tungro bacilliform virus (RTBV) at the International Rice Research Institute (IRRI), was cloned and sequenced. Comparison of the sequences revealed small differences in genome sizes. The variants were between 95 and 99% identical at the nucleotide and amino acid levels. Alignment of the three genome sequences with those of three published RTBV sequences (Phi-1, Phi-2 and Phi-3) revealed numerous nucleotide substitutions and some insertions and deletions. The published RTBV sequences originated from the same greenhouse isolate at IRRI 20, 11 and 9 years ago. All open reading frames (ORFs) and known functional domains were conserved across the six variants. The cysteine-rich region of ORF3 showed the greatest variation. When the six DNA sequences from IRRI were compared with that of an isolate from Malaysia (Serdang), similar changes were observed in the cysteine-rich region in addition to other nucleotide substitutions and deletions across the genome. The aligned nucleotide sequences of the IRRI variants and Serdang were used to analyse phylogenetic relationships by the bootstrapped parsimony, distance and maximum-likelihood methods. The isolates clustered in three groups: Serdang alone; Ic and G1; and Phi-1, Phi-2, Phi-3 and G2. The distribution of phylogenetically informative residues in the IRRI sequences shared with the Serdang sequence and the differing tree topologies for segments of the genome suggested that recombination, as well as substitutions and insertions or deletions, has played a role in the evolution of RTBV variants. The significance and implications of these evolutionary forces are discussed in comparison with badnaviruses and caulimoviruses. (+info)
The rice tungro bacilliform virus gene II product interacts with the coat protein domain of the viral gene III polyprotein.
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Rice tungro bacilliform virus (RTBV) is a plant pararetrovirus whose DNA genome contains four genes encoding three proteins and a large polyprotein. The function of most of the viral proteins is still unknown. To investigate the role of the gene II product (P2), we searched for interactions between this protein and other RTBV proteins. P2 was shown to interact with the coat protein (CP) domain of the viral gene III polyprotein (P3) both in the yeast two-hybrid system and in vitro. Domains involved in the P2-CP association have been identified and mapped on both proteins. To determine the importance of this interaction for viral multiplication, the infectivity of RTBV gene II mutants was investigated by agroinoculation of rice plants. The results showed that virus viability correlates with the ability of P2 to interact with the CP domain of P3. This study suggests that P2 could participate in RTBV capsid assembly. (+info)
Transcriptional activation of the rice tungro bacilliform virus gene is critically dependent on an activator element located immediately upstream of the TATA box.
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To investigate the transcriptional mechanisms of rice tungro bacilliform virus, we have systematically analyzed an activator element located immediately upstream of the TATA box in the rice tungro bacilliform virus promoter and its cognate trans-acting factors. Using electrophoretic mobility shift assays, we showed that rice nuclear proteins bind to the activator element, forming multiple specific DNA-protein complexes via protein-protein interactions. Copper-phenanthroline footprinting and DNA methylation interference analysis indicated that multiple DNA-protein complexes share a common binding site located between positions -60 to -39, and the proteins contact the activator element in the major groove. DNA UV cross-linking assays further showed that two nuclear proteins (36 and 33 kDa), found in rice cell suspension and shoot nuclear extracts, and one (27 kDa), present in root nuclear extracts, bind to this activator element. In protoplasts derived from a rice (Oryza sativa) suspension culture, the activator element is a prerequisite for promoter activity and its function is critically dependent on its position relative to the TATA box. Thus, transcriptional activation may function via interactions with the basal transcriptional machinery, and we propose that this activation is mediated by protein-protein interactions in a position-dependent mechanism. (+info)
The complexities of genome analysis, the Retroid agent perspective.
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MOTIVATION: The sequences of Retroid agents from a wide diversity of organisms constitute the largest set of complete genomes currently available for the study of genomic architecture and the transfer of information within and between organisms. These agents are ubiquitous in Eukaryotes, comprising 50-90% of the genomic information in some cases. RESULTS: Analyses conducted for over a decade illustrate that Retroid agents are engaged in a wide spectrum of molecular evolutionary events. A description of these complexities is presented as a three parameter conceptual framework that considers type, size, and mechanism of events that contribute to the evolution of genes, genomes, and organisms. The results of new data mining studies further illustrate the complexity of the network of relationships among and between Retroid agents and other organisms. A hidden Markov model construction strategy is presented that generates a multiple alignment more similar to those refined by human experts. CONTACT: [email protected]. edu (+info)
Sequence-specific and methylation-dependent and -independent binding of rice nuclear proteins to a rice tungro bacilliform virus vascular bundle expression element.
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Nuclear proteins from rice (Oryza sativa) were identified that bind specifically to a rice tungro bacilliform virus promoter region containing a vascular bundle expression element (VBE). One set of proteins of 29, 33, and 37 kDa, present in shoot and cell suspension extracts but hardly detectable in root extracts, bound to a site containing the sequence AGAAGGACCAGA within the VBE, which also contains two CpG and one CpNpG potential methylation motifs. Binding by these proteins was determined to be cytosine methylation-independent. However, a novel protein present in all analyzed extracts bound specifically to the methylated VBE. A region of at least 49 nucleotides overlapping the VBE and complete cytosine methylation of the three Cp(Np)G motifs was required for efficient binding of this methylated VBE-binding protein (MVBP). (+info)