Viruses are serious pathogens of animals and plants. To cause disease, viruses must carry out many defined tasks. Since virus genomes are small, while the number of tasks they need to accomplish are great, it is common for viral proteins to be multifunctional. How multifunctional viral proteins integrate their activities is unknown, but it is likely that protein self-association is required. If self-association of a viral protein is important for infection then it should be possible to prevent disease by impairing that interaction. This could be accomplished by identifying the domain responsible for self-association and employing it as a dominant negative inhibitor. Here, we examine the multifunctional gene VI product (P6) of Cauliflower mosaic virus (CaMV). Inclusion body (IB) formation is one of the many functions of P6. CaMV lBs are thought to be sites of virus genome replication, protein synthesis, and virion assembly. Therefore, by studying P6, we obtain information on lBs as well as on the workings of a multifunctional protein. We have found that P6 self-associates and have identified a conserved region (ID6) important for this interaction. The work described here analyzes ID6 and examines its role in virus infection.
The Specific Aims of this proposal are: 1. To Characterize The P6-P6 Interaction Domain, ID6. 2. To Elucidate The Role Of ID6 In P6 Self-Association. 3. To Determine The Role Of ID6 In Viral Infection. 4. To Test If ID6 Can Be Employed As An Inhibitor Of P6 Self-Association.To address Specific Aim 1, we will perform mutagenesis on ID6 and test these mutants for their ability to bind to P6. This will be accomplished using site-directed mutagenesis of conserved residues in ID6. We will also generate random mutations in ID6 by chemical mutagenesis. The mutants will then be tested for their ability to bind P6 by yeast two-hybrid analysis.
For Specific Aim 2, we will generate mutations in the full length P6 protein equivalent to those that prevent ID6 binding to the gene VI product. These mutant proteins will then be examined for their ability to self-associate and bind to the CaMV movement protein using the yeast two-hybrid system. We will then test these mutants for their translational transactivation activity. To accomplish Specific Aim 3, we will generate the mutations defined above in gene VI of a CaMV genome. These mutant viruses will then be examined for their propagation, symptom formation, distribution, and lB formation. To accomplish Specific Aim 4, we will use a modified form of the yeast two-hybrid system to test if ID6 interferes with P6 self-association. By performing this work, we will better understand the activities of a multifunctional viral protein and provide useful training to undergraduate students.
| Lutz, Lindy; Okenka, Genevieve; Schoelz, James et al. (2015) Mutations within A 35 amino acid region of P6 influence self-association, inclusion body formation, and Caulimovirus infectivity. Virology 476:26-36 |
| Lutz, Lindy; Raikhy, Gaurav; Leisner, Scott M (2012) Cauliflower mosaic virus major inclusion body protein interacts with the aphid transmission factor, the virion-associated protein, and gene VII product. Virus Res 170:150-3 |
| Hapiak, Michael; Li, Yongzhong; Agama, Keli et al. (2008) Cauliflower mosaic virus gene VI product N-terminus contains regions involved in resistance-breakage, self-association and interactions with movement protein. Virus Res 138:119-29 |
