Progress 07/01/03 to 06/30/06
Outputs
OUTPUTS: Project activities involved the characterization of virus replicase interactions that are involved in virus replication and disease development. These studies were presented at the USM Virology symposia and the nantional meeting of the American Virology Society. In Addition, two graduate students, directly supported by this award, received training and mentoring. PARTICIPANTS: 1. James Culver: Principle Investigator 2. Sabrina Kramer: Graduate student responsible for the characterization of the replicase protein in vivo and its interactions with host proteins. 3. Menu Padmanabhan: Graduate student responsible for the characterization replicase interacting IAA protein. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Very little information exists regarding the structural organization of any RNA virus replicase complex. One goal of this award wass directed at determining the characteristics and importance of oligomer formation in viral replicase function. For this study the expression and purification of the C-terminal portion of the Tobacco mosaic virus (TMV) 126-kDa replicase protein in Escherichia coli was optimized to obtain soluble protein concentrations of >3mg/ml. Gel filtration chromatography, using a range of salt and pH conditions revealed that the TMV helicase domain (C-terminal portion of the replicase protein) produced a distinct set of higher-molecular weight complexes. Size filtration peaks representing the monomer, dimer, hexamer/dodecamer, and a higher order aggregate were identified. Electron microscopy studies of these fractions supported the presence of each aggregate. Interestingly, ATPase activity and RNA binding was detected mainly in monomer and dimer fractions and not in higher order fractions. This suggests that replicase enzymatic activity may reside within the monomer/dimer. These findings represent some of the first biophysical information related to the assembly products of a plant virus replicase protein. Another focus of this study involves the identification of virus replicase interactions that contribute to its localization and assembly. Efforts related to this objective have resulted in the identification of several host proteins that interact with the TMV viral replicase. Localization studies indicate that interaction with these host proteins can affect both replicase and host protein function. For example, replicase interaction with the transcription factor IAA26 correlates with symptom development. IAA26 functions in auxin regulation and plant development. Interaction with the TMV replicase alters the cellular localization of IAA26 and appears to promote its degradation. Presumably, this interaction disrupts IAA26 transcriptional control leading to the inappropriate expression of specific host genes. Our studies found that as much as 30% of the host genes that display transcriptional alterations in response to TMV contain auxin responsive promoter elements. As a result the appearance of specific viral induced developmental symptoms correlates with this interaction. Interestingly, we also determined that the TMV replicase protein similarly disrupts several additional IAA transcription factors and that related responses occur in a range of TMV hosts including tomato and tobacco. Thus, this interaction may account for a significant proportion of the disease response induced by this virus.
Publications
- Culver, J.N. and Padmanabhan, M.S. 2007. Virus-induced disease: altering host physiology one interaction at a time. Annu. Rev. Phytopathol. 45:221-243.
- Padmanabhan, M.S., Goregaoker, S.P., Golem, S., Shiferaw, H., and Culver, J.N. 2005. Interaction of the Tobacco mosaic virus replicase protein with the Aux/IAA proteins PAP1/IAA26 is associated with disease development. J. Virology, 79:2549-2558.
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Progress 10/01/04 to 09/30/05
Outputs
Very little information exists regarding the structural organization of any RNA virus replicase complex. One goal of this award is directed at determining the characteristics and importance of oligomer formation in viral replicase function. For this study the expression and purification of the C-terminal portion of the Tobacco mosaic virus (TMV) 126-kDa replicase protein in Escherichia coli was optimized to obtain soluble protein concentrations of >3mg/ml. Gel filtration chromatography, using a range of salt and pH conditions revealed that the TMV helicase domain (C-terminal portion of the replicase protein) produced a distinct set of higher-molecular weight complexes. Size filtration peaks representing the monomer, dimer, hexamer/dodecamer, and a higher order aggregate were identified. Electron microscopy studies of these fractions supported the presence of each aggregate. Interestingly, ATPase activity and RNA binding was detected mainly in monomer and dimer
fractions and not in higher order fractions. This suggests that replicase enzymatic activity may reside within the monomer/dimer. These findings represent some of the first biophysical information related to the assembly products of a plant virus replicase protein. Another focus of this study involves the identification of virus replicase interactions that contribute to its localization and assembly. Efforts related to this objective have resulted in the identification of several host proteins that interact with the TMV viral replicase. Localization studies indicate that interaction with these host proteins can affect both replicase and host protein function. For example, replicase interaction with the transcription factor IAA26 correlates with symptom development. IAA26 functions in auxin regulation and plant development. Interaction with the TMV replicase alters the cellular localization of IAA26 and appears to promote its degradation. Presumably, this interaction disrupts IAA26
transcriptional control leading to the inappropriate expression of specific host genes. Our studies found that as much as 30% of the host genes that display transcriptional alterations in response to TMV contain auxin responsive promoter elements. As a result the appearance of specific viral induced developmental symptoms correlates with this interaction. Interestingly, we also determined that the TMV replicase protein similarly disrupts several additional IAA transcription factors and that related responses occur in a range of TMV hosts including tomato and tobacco. Thus, this interaction may account for a significant proportion of the disease response induced by this virus
Impacts
Genome replication is one of the most fundamental processes in biology. For positive-stranded RNA viruses, an essential aspect of this process is provided by viral encoded replicase genes. In general, these replicase genes encode a number of functional domains (e.g. methyltransferase, helicase, and RNA-dependent RNA-polymerase) that participate in sequence-specific RNA synthesis, virus transport, cross-protection and disease development. Work in this project will result in better understandings of how virus replicase complexes assemble, function and interact with their host to induce disease responses.
Publications
- Padmanabhan, M.S., Goregaoker, S.P., Golem, S., Shiferaw, H., and Culver, J.N. 2005. Interaction of the Tobacco mosaic virus replicase protein with the Aux/IAA proteins PAP1/IAA26 is associated with disease development. J. Virology, 79:2549-2558.
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Progress 10/01/03 to 09/30/04
Outputs
Very little information exists regarding the structural organization of any RNA virus replicase complex. One goal of this award is directed at determining the characteristics and importance of oligomer formation in viral replicase function. For this study the expression and purification of the C-terminal portion of the Tobacco mosaic virus (TMV) 126-kDa replicase protein in Escherichia coli was optimized to obtain soluble protein concentrations of >3mg/ml. Gel filtration chromatography, using a range of salt and pH conditions revealed that the TMV helicase domain (C-terminal portion of the replicase protein) produced a distinct set of higher-molecular weight complexes. Size filtration peaks representing the monomer, dimer, hexamer/dodecamer, and a higher order aggregate were identified. Electron microscopy studies of these fractions supported the presence of each aggregate. These findings represent some of the first biophysical information related to the assembly
products of a plant virus replicase protein. Another focus of this study involves the identification of virus replicase interactions that contribute to its localization and assembly. Efforts related to this objective have resulted in the identification of several host proteins that interact with the TMV viral replicase. Localization studies indicate that interaction with these host proteins can affect both replicase and host protein function. For example, replicase interaction with the transcription factor IAA26 correlates with symptom development. IAA26 functions in auxin regulation and plant development. Interaction with the TMV replicase appears to promote IAA26 degradation, disrupting its role in plant development. As a result the appearance of specific viral induced developmental symptoms correlates with this interaction. Other interactions are currently being examined for their effect on replicase localization and assembly.
Impacts
Genome replication is one of the most fundamental processes in biology. For positive-stranded RNA viruses, an essential aspect of this process is provided by viral encoded replicase genes. In general, these replicase genes encode a number of functional domains (e.g. methyltransferase, helicase, and RNA-dependent RNA-polymerase) that participate in sequence-specific RNA synthesis, virus transport, cross-protection and disease development. Work in this project will result in better understandings of how virus replicase complexes assemble and function, providing insight into the design of novel anti-viral strategies.
Publications
- Padmanabhan, M.S., Goregaoker, S.P., Golem, S., Shiferaw, H., and Culver, J.N. 2004. Interaction of the Tobacco mosaic virus replicase protein with the Aux/IAA proteins PAP1/IAA26 is associated with disease development. J. Virology, 79:2549-2558.
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