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Sequence and structural elements promoting positive-strand RNA synthesis in brome mosaic virus
Abstract
Sequences at the 5' termini of the genomic RNAs of brome mosaic virus (BMV) and other (+)-stranded RNA viruses have been shown to resemble the internal control regions (ICRs 1 and 2, or A and B boxes) of tRNA genes. In order to examine the role of these sequences in viral replication, mutations were introduced initially into the ICR2-like sequence of a BMV RNA-2 deletion mutant, pRNA-2 M/S (parasitic RNA), that does not encode a functional viral protein but replicates in the presence of genomic RNAs-1 and -2. The accumulation of (+) strand progeny in barley protoplasts from pRNAs bearing single and double base substitutions was 70 to 91% lower than that of wild type (wt), while addition of single bases at position 8 reduced replication by 80%. These results demonstrated that the wt composition of this motif is essential for proper RNA replication. Substitutions introduced at sequences surrounding the ICR2 motif yielded levels of pRNA replication that differed, depending on the maintenance of a putative 5' stem -loop structure in the (+) strand of the viral genome. The requirement for this structure provided the basis for a new model describing how these sequence and structural elements act in the production of virus (+) strand RNA. Deletion of the 5' ICR2 motif or substitutions at both the G1, and G2, positions of this motif prevented the replication of genomic RNA-2. However, the protein p2a, translated from such non-replicating mutants, supported the replication of the co-inoculated RNAs-1 and -3. Several RNA-2 mutants, which were greatly debilitated in replication as assayed in protoplasts, supported both local lesion and systemic infections on plants. Deletion of the 5' ICR2-like sequence in RNA-3 had little effect on its replication, but removing an ICR2 motif in the intercistronic region reduced (+) strand replication by 80%. RNA-3 mutants bearing deletions of ICR2-like sequences were impaired with respect to supporting infections of intact plants. Several mutants of RNA-2, bearing internal deletions, failed to replicate when co-inoculated with RNAs-1 and -2. However, the inoculum RNA corresponding to these deletion mutants was extremely stable. One of these mutants, NRI RNA Nc/S, interfered with the accumulation of the co-inoculated BMV RNAs. At an NRI:genomic RNA inoculum molar ratio of 1:1, NRI RNA Nc/S reduced the accumulation of all helper virus RNAs by 55%. The ability of NRI RNAs to interfere with virus replication suggests that their expression in transgenic plants may provide a new approach for generating plants resistant to virus infection.
Description
Typescript (photocopy).Subject
Major biology1992 Dissertation P746
Plant viruses
Genetics
Mosaic diseases
RNA viruses
Reproduction
RNA
Synthesis
Collections
Citation
Pogue, Gregory Paul (1992). Sequence and structural elements promoting positive-strand RNA synthesis in brome mosaic virus. Texas A&M University. Texas A&M University. Libraries. Available electronically from https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1281134.
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