Timothy C. Hall's Lab

Virus Group: Strategies in Replication and Pathogenesis

Research Outline

Plant viruses are pathogens of major crops. Because major groups of viruses contain animal, insect and plant pathogens, basic studies on plant viruses provide important insight to general strategies for virus replication and pathogenesis. Our studies have focused on brome mosaic virus (BMV), a pathogen of grasses and cereals. BMV has a total genome of 8 kb, divided among three single-stranded, (+)-sense RNAs. RNA-1 (3234 nt) encodes a 109,208 kD protein that contributes helicase, methylation and other properties that, together with the core replicase encoded by RNA-2 (2865 nt) and currently unidentified host factors, are essential to virus replication. The other genomic component, RNA-3 (2117 nt), encodes a protein thought to participate in cell-to cell movement and the capsid protein that is expressed from subgenomic RNA-4 (876 nt).

We found that the 3' end of each RNA component of BMV has a tRNA-like structure that is capable of accepting tyrosine with similar efficacy to that of tRNA^Tyr (Hall et al., 1972). This structure is multifunctional, serving also as the recognition site for (-) strand replicase and nucleotidyl transferase (Bujarski et al., 1986). We published many studies characterizing the promoters for (-) strand and subgenomic (+) strand synthesis and developed a novel hypothesis concerning (+) strand promoter activity which invokes the participation of host pol III factors in replication, based on the similarity of viral sequence motifs to internal control regions (ICRs) 1 and 2 of tRNA gene promoters (see reviews by (Duggal et al., 1994; Rao et al., 1994)). We initiated studies on RNA-protein binding and a search for host factors involved in the viral replication complex (Duggal and Hall, 1995).

We have used the natural plant host barley (Hordeum vulgare) for many studies in vivo that employed infectious transcripts of cDNA clones containing a wide range of mutations introduced by site-directed mutagenesis and other approaches. We have also used Chenopodium hybridum, a local lesion host, in novel approaches for understanding RNA recombination events that are intrinsic to viral evolution and pathogenesis (Rao and Hall, 1993).

Recent work was directed towards elucidating the mechanism involved in gene-specific silencing of RNA-2 replication (Iyer and Hall, 2000). We made transgenic Nicotiana benthamiana plants that express the p2 protein encoded by BMV RNA-2. Protoplasts from leaves of these plants support replication of RNAs -1 and -3. However, when these protoplasts are transfected with virion RNA that contains RNA-2 as well as RNAs-1 and -3, no replication of RNA-2 occurs. By inoculating protoplasts with RNAs -2 and -3, we have shown that there is no preferential degradation of RNA-2 in the transgenic (p2) protoplasts and, hence, induction of an RNA-2-specific nuclease is unlikely to be involved. We have also inserted segments of RNA-2 (that collectively cover the entire genome) into RNA-3; the fact that no silencing of the chimeric RNA-3 was detected further suggests that the silencing seen in this system is protein rather than RNA-mediated. It is possible that competitive interaction of excess p2 present in the transgenic protoplasts with the replicase complex displaces nascent RNA-2 during replication.

Publications

(Click here for a complete listing of our lab publications)

Bujarski, J.J., Ahlquist, P., Hall, T.C., Dreher, T.W. and Kaesberg, P. 1986. Modulation of replication, aminoacylation, and adenylation in vitro and infectivity in vivo of BMV RNAs containing deletions within the multifunctional 3' end. EMBO J. 5: 1769-1774.

Duggal, R. and Hall, T.C. 1995. Interaction of host proteins with the plus-strand promoter of brome mosaic virus RNA-2. Virology 214: 638-641.

Duggal, R., Lahser, F.C., Rao, A.L.N. and Hall, T.C. 1994. Cis-acting sequences in the replication of plant viruses with (+)-sense RNA genomes. In: R.J. Cook (ed) Ann. Rev. Phytopathol., pp. 287-309. Annual Reviews, Inc., Palo Alto.

Hall, T.C., Shih, D.S. and Kaesberg, P. 1972. Enzyme-mediated binding of tyrosine to brome-mosaic-virus ribonucleic acid. Biochem. J. 129: 969-976.

Iyer, L.M. and Hall, T.C. 2000. Virus recovery is induced in brome mosaic virus p2 transgenic plants showing synchronous complementation and RNA-2-specific silencing. Mol. Plant Microbe Interact. 13: 247-258.

Rao, A.L. and Hall, T.C. 1993. Recombination and polymerase error facilitate restoration of infectivity in brome mosaic virus. J Virol 67: 969-79.

Rao, A.L.N., Duggal, R., Lahser, F.C. and Hall, T.C. 1994. Analysis of RNA replication in plant viruses. In: K.W. Adolph (Eds.) Methods in Molecular Genetics: Molecular Virology, Academic Press, Inc., Orlando, FL., pp. 216-236.