RNA viruses are among the most important pathogens of humans, animals and plants. In several, replication involves a tRNA (e.g., retroviruses) or a complex folded 3' tRNA-like structure (e.g., brome mosaic virus, BMV). The maintenance in the RNA genome of properties such as aminoacylation implies the operation of a positive selection pressure, e.g., a controlling role in infection. Our overall objective is to identify how this function contributes to infective processes. A 3' 128 nucleotide (nt) fragment, common to all four BMV RNAs functions in vitro as a substrate for aminoacylation. Recombinant DNA techniques will be used to construct novel BMV RNA sequences with specified nucleotide substitutions, deletions or insertions. Their functionality will be tested in vitro using aminoacyl-tRNA synthetase and our unique BMV RNA template-specific and -dependent replicase. Such experiments, impossible by classical genetics, also permit evaluation of models proposed for secondary and tertiary structures of 3' sequences of viral RNAs. We have placed a cDNA clone of the 3'-terminal 200 nt of BMV RNAs under the control of an SP6 phage promoter and used SP6 RNA polymerase to transcribe microgram quantities of RNA from this DNA template. The cDNA clones was engineered to contain a Tth111 I site, and transcription of cDNA templates cleaved at this site yielded RNAs accurately terminating in -CCA. These transcripts are functional in aminoacylation and replication. Altered RNAs will be constructed by oligodeoxynucleotide-directed site-specific mutations in the cDNA clone and by other cloning techniques. Subsequently, full-length BMV RNA transcripts containing native and altered 3' sequences will be tested for infectivity in protoplasts. We have devised a hybrid-arrested replication assay to localize regions within BMV RNA intrinsic to replication events. We propose to further define regions of native and modified viral RNAs involved in initiation of replication and in binding of replicase (and synthetase) by photocrosslinking and other techniques. We also intend to expand our characterization of replicase products to provide sensitive detection of de novo (+) strand synthesis during replication in vitro. Together with proposed approaches for improvements in the purity and activity of our replicase, the results of these studies are expected to yield an understanding of replication processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI022354-05
Application #
3133331
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1984-09-01
Project End
1990-05-31
Budget Start
1988-06-01
Budget End
1989-05-31
Support Year
5
Fiscal Year
1988
Total Cost
Indirect Cost
Name
Texas A&M University
Department
Type
Schools of Arts and Sciences
DUNS #
City
College Station
State
TX
Country
United States
Zip Code
77845
Marsh, L E; Huntley, C C; Pogue, G P et al. (1991) Regulation of (+):(-)-strand asymmetry in replication of brome mosaic virus RNA. Virology 182:76-83
Pogue, G P; Marsh, L E; Hall, T C (1990) Point mutations in the ICR2 motif of brome mosaic virus RNAs debilitate (+)-strand replication. Virology 178:152-60
Rao, A L; Sullivan, B P; Hall, T C (1990) Use of Chenopodium hybridum facilitates isolation of brome mosaic virus RNA recombinants. J Gen Virol 71 ( Pt 6):1403-7
Rao, A L; Huntley, C C; Marsh, L E et al. (1990) Analysis of RNA stability and (-) strand content in viral infections using biotinylated RNA probes. J Virol Methods 30:239-50
Rao, A L; Hall, T C (1990) Requirement for a viral trans-acting factor encoded by brome mosaic virus RNA-2 provides strong selection in vivo for functional recombinants. J Virol 64:2437-41
Rao, A L; Dreher, T W; Marsh, L E et al. (1989) Telomeric function of the tRNA-like structure of brome mosaic virus RNA. Proc Natl Acad Sci U S A 86:5335-9
Dreher, T W; Rao, A L; Hall, T C (1989) Replication in vivo of mutant brome mosaic virus RNAs defective in aminoacylation. J Mol Biol 206:425-38
Dreher, T W; Hall, T C (1988) Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation. J Mol Biol 201:41-55
Dreher, T W; Hall, T C (1988) Mutational analysis of the sequence and structural requirements in brome mosaic virus RNA for minus strand promoter activity. J Mol Biol 201:31-40
Marsh, L E; Dreher, T W; Hall, T C (1988) Mutational analysis of the core and modulator sequences of the BMV RNA3 subgenomic promoter. Nucleic Acids Res 16:981-95

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