Functional characterization of the alphavirus TF protein.

Alphavirus dogma has long dictated the production of a discrete set of structural proteins during infection of a cell: capsid, pE2, 6K, and E1. However, bioinformatic analyses of alphavirus genomes (A. E. Firth, B. Y. Chung, M. N. Fleeton, and J. F. Atkins, Virol. J. 5:108, 2008) suggested that a ribosomal frameshifting event occurs during translation of the alphavirus structural polyprotein. Specifically, a frameshift event is suggested to occur during translation of the 6K gene, yielding production of a novel protein, termed transframe (TF), comprised of a C-terminal extension of the 6K protein in the -1 open reading frame (ORF). Here, we validate the findings of Firth and colleagues with respect to the production of the TF protein and begin to characterize the function of TF. Using a mass spectrometry-based approach, we identified TF in purified preparations of both Sindbis and Chikungunya virus particles. We next constructed a panel of Sindbis virus mutants with mutations which alter the production, size, or sequence of TF. We demonstrate that TF is not absolutely required in culture, although disrupting TF production leads to a decrease in virus particle release in both mammalian and insect cells. In a mouse neuropathogenesis model, mortality was <15% in animals infected with the TF mutants, whereas mortality was 95% in animals infected with the wild-type virus. Using a variety of additional assays, we demonstrate that TF retains ion-channel activity analogous to that of 6K and that lack of production of TF does not affect genome replication, particle infectivity, or envelope protein transit to the cell surface. The TF protein therefore represents a previously uncharacterized factor important for alphavirus assembly.

Characterization of the first naturally thermostable terpene synthases and development of strategies to improve thermostability in this family of enzymes.

The terpenoid family of natural products is being targeted for heterologous microbial production as a cheaper and more reliable alternative to extraction from plants. The key enzyme responsible for diversification of terpene structure is the class-I terpene synthase (TS), and these often require engineering to improve properties such as thermostability, robustness and catalytic activity before they are suitable for industrial use. Improving thermostability typically relies on screening a large number of mutants, as there are no naturally thermostable TSs described upon which to base rational design decisions. We have characterized the first examples of natural TSs exhibiting thermostability, which catalyse the formation of the sesquiterpene τ-muurolol at temperatures up to 78 °C. We also report an enzyme with a kcat value of 0.95 s-1 at 65 °C, the highest kcat recorded for a bacterial sesquiterpene synthase. In turn, these thermostable enzymes were used as a model to inform the rational engineering of another TS, with the same specificity but low sequence identity to the model. The newly engineered variant displayed increased thermostability and turnover. Given the high structural homology of the class-I TS domain, this approach could be generally applicable to improving the properties of other enzymes in this class. DATABASE: Model data are available in the PMDB database under the accession number PM0080780.