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Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

ABSTRACT: Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 production, as well as RNA1 and RNA2 replication. Two cis elements controlling RNA3 production were found. A proximal subgenomic control element was located just upstream of the RNA3 start site (nucleotides [nt] 2282 to 2777). A short distal element also controlling RNA3 production (distal subgenomic control element) was identified 1.5 kb upstream, at nt 1229 to 1239. Base pairing between these distal and proximal elements was shown to be essential for RNA3 production by covariation analysis and in vivo selection of RNA3-expressing replicons from plasmid libraries containing random sequences in the distal element. Two distinct RNA1 replication elements (RE) were mapped within the 3' quarter of RNA1: the intRE (nt 2322 to 2501) and the 3'RE (nt 2735 to 3011). The 3'RE significantly overlaps the RNA3 region in RNA1, and this information was applied to produce improved RNA3-based vectors for foreign-gene expression. In addition, replication of an RNA2 derivative was dependent on RNA1 templates capable of forming the long-distance interaction that controls RNA3 production.

SUBMITTER: Lindenbach BD

PROVIDER: S-EPMC136111 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

Lindenbach Brett D BD Sgro Jean-Yves JY Ahlquist Paul P

Journal of virology 20020401 8

Replication of flock house virus (FHV) RNA1 and production of subgenomic RNA3 in the yeast Saccharomyces cerevisiae provide a useful tool for the dissection of FHV molecular biology and host-encoded functions involved in RNA replication. The replication template activity of RNA1 can be separated from its coding potential by supplying the RNA1-encoded replication factor protein A in trans. We constructed a trans-replication system in yeast to examine cis-acting elements in RNA1 that control RNA3 ...[more]

PMID: 11907230

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Project description:The larger segment (RNA 1) of the bipartite, positive-sense RNA genome of the nodavirus flock house virus encodes the viral RNA-dependent RNA polymerase. Two nonstructural viral proteins are made during the self-directed replication of this RNA: protein A (110 kDa), the translation product of RNA 1 itself, and protein B (11 kDa), the translation product of a subgenomic RNA (RNA 3) that is produced from RNA 1 during replication. To examine the roles of these proteins in RNA replication, specialized T7 transcription plasmids that contained wild-type or mutant copies of flock house virus RNA 1 cDNA were constructed and used in cells infected with the vaccinia virus-T7 RNA polymerase recombinant to make full-length transcripts that directed their own replication. Sequences in the primary transcripts that extended beyond the ends of the authentic RNA 1 sequence inhibited self-directed RNA replication, but plasmids that were constructed to minimize these terminal extensions produced primary transcripts that replicated as abundantly as authentic RNA 1. Truncation or mutation of the open reading frame for protein A eliminated self-directed replication, although the mutant RNA 1 remained a competent template for replication by wild-type protein A supplied in trans. These results showed that protein A was essential for RNA replication and that the process was not inseparably coupled to complete translation of the template. In contrast, protein B could be eliminated without inhibiting replication by mutations that disrupted the second of the two overlapping open reading frames on RNA 3. Furthermore, a mutant of RNA 1 in which the first nucleotide of the RNA 3 region was changed from G to U replicated at levels as high as those of the wild type without making either RNA 3 or protein B. However, diminishing replication levels were observed during subsequent replicative passages of RNA from both the mutants that could not make protein B. Roles for this protein that could account for the subtle phenotype of these mutants are discussed.

Dataset Information

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

Publications

Long-distance base pairing in flock house virus RNA1 regulates subgenomic RNA3 synthesis and RNA2 replication.

Similar Datasets

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