Progress 08/01/03 to 07/31/05
Outputs
This project led to the identification of yeast genes that (i) affect tombusvirus replication and (ii) interact with the tombusvirus replicase proteins. Yeast genome-wide screen reveals host genes affecting tombusvirus replication. Viruses are devastating pathogens of humans, animals and plants. To further our understanding of how viruses utilize the resources of infected cells, we systematically tested the yeast single-gene knockout library for the effect of each host gene on replication of Tomato bushy stunt virus (TBSV), a positive-strand RNA virus of plants. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the TBSV replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins and other compounds and in protein targeting/transport. Comparison with published genome-wide screens revealed that replication of TBSV and Brome mosaic virus, which belongs to a different
supergroup among plus-strand RNA viruses, are affected by vastly different yeast genes. Moreover, a set of yeast genes, involved in vacuolar targeting of proteins and vesicle-mediated transport, both affected replication of the TBSV replicon and enhanced the cytotoxicity of the Parkinson's disease-related alpha-synuclein when this protein was expressed in yeast. In addition, a set of host genes involved in ubiquitin-dependent protein catabolism affected both TBSV replication and the cytotoxicity of a mutant huntingtin protein, a candidate agent in Huntington's disease. This suggests that virus infection and disease-causing proteins might use or alter similar host pathways and may indicate connections between chronic diseases and prior virus infection. Identification of host proteins within the Cucumber necrosis virus (CNV) replicase complex. Two-step affinity-based purification was used to obtain highly pure tombusvirus replicase preparations. Briefly, the enriched membrane fraction
from yeast co-expressing CNV replication proteins (p33 and p92) and the replicon RNA was solubilized using nonionic detergent, followed first by Nickel-affinity purification and, second, by immuno-affinity purification based on the FLAG-tag. Two-dimensional (2D)-PAGE analysis revealed that the obtained CNV replicase preparation contained functional replicase consisting of p33, p92, viral RNA and two additional abundant host proteins, which are not present in the control preparations obtained using the same two-step method from yeast co-expressing the replicon RNA, p33 and p92 without the affinity tags. After isolation of the co-purified viral- and host proteins from 2D-PAGE, MALDI-TOF analysis was employed to determine their identities: this analysis revealed that the CNV replicase, in addition to p33 and p92, contains glyceraldehyde-3-phosphate dehydrogenase (GPD2 or Tdh2p in Saccharomyces cerevisiae) and Ssa2p, a member of the 70kDa heat shock protein family. Because these host
proteins were abundant in five separately obtained CNV replicase preparations, but were not detected in the control preparations, it is highly likely that they have specific roles in CNV replicase formation/function.
Impacts
Plant viruses are important pathogens of crop plants in the USA. Viruses use host factors to infect plants and for their replication. In this project, host factors involved in Tombusvirus replication were identified. Understanding host factor involvement in viral replication should lead to improved control of virus infections.
Publications
- Panavas, T., Serviene, E., Brasher, J. and Nagy, P.D. 2005. Yeast genome-wide screen reveals dissimilar set of host genes affecting replication of RNA viruses. Proc Natl Acad Sci U S A. (PNAS) 102:7326-31.
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Progress 01/01/04 to 12/31/04
Outputs
This project led to the identification of yeast genes that (i), affect tombusvirus replication and (ii), interact with the tombusvirus replicase proteins. Yeast genome-wide screen reveals host genes affecting tombusvirus replication. To further our understanding of how viruses utilize the resources of infected cells, the yeast single-gene knockout library was systematically tested for the effect of each host gene on replication of tombusviruses. The genome-wide screen identified 96 host genes whose absence either reduced or increased the accumulation of the Tomato bushy stunt virus replicon. The identified genes are involved in the metabolism of nucleic acids, lipids, proteins and other compounds as well as in protein targeting/transport. Identification of host proteins within the Cucumber necrosis virus (CNV) replicase complex. Two-step affinity-based purification was used to obtain highly pure tombusvirus replicase preparations. Briefly, the enriched membrane
fraction from yeast co-expressing CNV replication proteins (p33 and p92) and the replicon RNA was solubilized using nonionic detergent, followed first by Nickel-affinity purification and second by immuno-affinity purification based on the FLAG-tag. Two-dimensional (2D)-PAGE analysis revealed that the obtained CNV replicase preparation contained functional replicase consisting of p33, p92, viral RNA and two additional abundant host proteins, which are not present in the control preparations obtained using the same two-step method from yeast co-expressing the replicon RNA, p33 and p92 without the affinity tags. After isolation of the co-purified viral- and host proteins from 2D-PAGE, MALDI-TOF analysis was employed to determine their identities: this analysis revealed that the CNV replicase, in addition to p33 and p92, contains glyceraldehyde-3-phosphate dehydrogenase (GPD2 or Tdh2p in Saccharomyces cerevisiae) and Ssa2p, a member of the 70kDa heat shock protein family. Because these
host proteins were abundant in five separately obtained CNV replicase preparations, but were not detected in the control preparation, it is highly likely that they have specific roles in CNV replicase formation/function.
Impacts
Development of yeast as a model host makes Tombusviruses as one of the premier systems to study RNA virus replication and recombination. Plant viruses are important pathogens of crop plants in the USA. Viruses use host factors to infect plants and for their replication. In this project, host factors involved in Tombusvirus replication were identified. Understanding host factor involvement in viral replication should lead to improved control of virus infections.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
The PI and associates have developed yeast, an excellent model host for genetic and biochemical studies, as an efficient Tomato bushy stunt virus RNA replication system by co-expressing the replicase proteins and a defective interfering (DI) RNA template. Replication of DI RNA in yeast cells was confirmed by (i) detection of minus-strand DI RNA intermediates only when p33 and p92 were co-expressed; (ii) reduction of DI RNA levels when RIII(-) replication enhancer was deleted; (iii) the presence of active tombusvirus replicase in transformed yeast. We also observed DI RNA recombinants in yeast.
Impacts
Development of yeast as a model host makes Tombusviruses as one of the premier systems to study RNA virus replication and recombination.
Publications
- Panavas, T., and Nagy, P. D. 2003. Yeast as a model host to study replication and recombination of defective interfering RNA of Tomato bushy stunt virus. Virology 314: 315-325.
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