Progress 01/01/04 to 12/31/04
Outputs
The baculovirus AcMNPV encodes its RNA polymerase. We determined the functional regions of the largest subunit, late expression factor-8, and a functional domain of another subunit, late expression factor-9. We are currently defining protein-protein and protein-DNA interactions between these subunits and their target promoter regions.
Impacts
Baculoviruses are the leading viral insect pest control agents presently being developed. This research program aims to improve the efficacy of baculoviruses as bioinsecticides by studying the genes that control DNA replication and transcription at late times. These genes drive the expression of foreign genes that improve the ability of the virus to destroy pest species. Overall, this study will aid in developing a safe food supply and improve human health.
Publications
- No publications reported this period
|
Progress 12/01/00 to 11/30/04
Outputs
This project focused on two main objectives: (1) functional characterization of the baculovirus AcMNPV RNA polymerase that is composed of four subunits designated LEF-8, LEF-9, LEF-4, and P47; and (2) testing the ability of late gene expression factors (lefs) from related and more divergent viruses in complementing late gene expression in cells permissive for AcMNPV infection. Objective 1: We carried out detailed mutagenesis of lef-8 and found that regions throughout the gene were required for function. We are finalizing experiments to define protein-protein and protein-DNA interactions between these subunits and their target promoter regions. Objective 2: We have finalized studies involving the functionality of lefs encoded by BmNPV and SeMNPV in SF-21 cells. We found that the BmNPV lefs, p143, ie-2, and p35, were not able to substitute for the corresponding AcMNPV in a functional assay. We focused on the inability of BmNPV p143 to substitute for the AcMNPV p143 and
found that the BmNPV P143 was not localizing to the nucleus of cells. This was due to its inability to interact with LEF-3, the factor that shuttles it to the nucleus of cells or due to its inability to fold correctly in the cells used in this study. Only the SeMNPV lef-5 was able to substitute for the AcMNPV lef-5.
Impacts
Baculoviruses are the leading viral insect pest control agents presently being developed. This research program aims to improve the efficacy of baculoviruses as bioinsecticides by studying the genes that control DNA replication and transcription at late times. These genes drive the expression of foreign genes that improve the ability of the virus to destroy pest species. Overall, this study will aid in developing a safe food supply and improve human health.
Publications
- No publications reported this period
|
Progress 01/01/03 to 12/31/03
Outputs
Work that defined the functional domains of the baculovirus AcMNPV late expression factor-8 gene, the largest of four subunits that make up the virus-encoded RNA polymerase required for the specific transcription of late-stage genes, has been completed. The work highlighted the important contributions to late gene expression of regions throughout the protein and from an RNA polymerase conserved motif that may serve as part of the catalytic site. We have also mapped a functional domain in another viral RNA polymerase subunit, late expression factor-9, that may also contribute to catalysis. We are currently defining protein-protein and protein-DNA interactions.
Impacts
Baculoviruses are the leading viral insect pest control agents presently being developed. This research program aims to improve the efficacy of baculoviruses as bioinsecticides by studying the genes that control DNA replication and transcription at late times. These genes drive the expression of foreign genes that improve the ability of the virus to destroy pest species. Overall, this study will aid in developing a safe food supply and improve human health.
Publications
- Titterington, J. S., T. K. Nun, and A. L. Passarelli. 2003. Functional dissection of the baculovirus late expression factor-8: Sequence requirements for late gene promoter activation. J. Gen. Virol. 84:1817-1826.
|
Progress 01/01/02 to 12/31/02
Outputs
We have completed the mutagenesis of the baculovirus AcMNPV late expression factor-8 gene, the largest subunit of the virus-encoded RNA polymerase required for the specific transcription of late-stage genes. We found that regions throughout this subunit were important for function, highlighting the complex and numerous interactions that occur during transcription of viral genes. We are currently defining these interactions in vivo and in vitro. We are in the process of determining the factors necessary for productive baculovirus infections in permissive and non-permissive hosts by comparing gene activities of potential host range genes from baculoviruses with different host ranges. The role of these factors in genetic recombination events is also being evaluated. To this end, we developed an assay that allowed the identification of viral genes necessary for homologous recombination. The ability of these viruses to infect insect hosts may be closely linked to their
ability to replicate and repair their genomes using both viral and host enzymes.
Impacts
Industry and agriculture would benefit from baculoviruses with a wider host range to effectively control pests. In order to accomplish this successfully, we are identifying and characterizing baculovirus host range genes. Recombination in baculoviruses will help understand genetic heterogeneity and speciation that are important in the assessment of bioinsecticides. Overall, this study will aid in developing a safe food supply and improve human health.
Publications
- Crouch, E. A. and A. L. Passarelli. 2002. Genetic requirements for homologous recombination in Autographa californica nucleopolyhedrovirus. J. Virol. 76:9323-9334.
|
Progress 01/01/01 to 12/31/01
Outputs
We have almost completed mutagenesis of the baculovirus AcMNPV late expression factor-8 (lef-8) gene that encodes the largest subunit of the virus encoded DNA-directed RNA polymerase required to transcribe late and very late viral genes, in order to map domains required for function. We found that a 13-amino acid domain with striking homology to the catalytic center of enzyme subunits in prokaryotic and eukaryotic RNA polymerases is required for optimal viral late gene expression. In addition, we found that regions of 150 nucleotides throughout the lef-8 gene are also required for function. We are presently defining these regions in more detail by performing site-directed mutagenesis. Also, we are currently defining the host specific requirement for late gene promoter stimulation mediated by lef genes of different baculoviruses in cells permissive or nonpermissive for viral infection.
Impacts
The AcMNPV RNA polymerase is the simplest multimeric eukaryotic RNA polymerase known and the only DNA-directed RNA polymerase encoded by a nuclear replicating DNA-containing virus. This four-subunit polymerase has no overall homology to more complex enzymes yet achieves the same task. Thus, structure-function studies of LEF-8 will yield information about viral transcription mechanisms that can be extrapolated to other systems and shed light into the evolutionary origin of this polymerase.
Publications
- No publications reported this period
|
|