- Reverse Transkriptase (1) (remove)
- Foamy Virus Enzymes - Activity, Regulation and Resistance (2010)
- Foamy viruses or spumaretroviruses belong to the family of retroviridae but differ in several aspects from other retroviruses (orthoretroviruses). Viral particles contain DNA not RNA. The Pol protein, the precursor of the viral enzymes, is translated from a separate mRNA independently of the capsid and matrix proteins. The protease remains covalently bound to the reverse transcriptase, while in orthoretroviruses the protease is cleaved off autocatalytically. Thus, in mature spumaretroviruses a protease-reverse transcriptase protein (PR-RT) with three different catalytic activities is found: proteolysis, DNA polymerization and RNase H activity. In this work, the recombinant PR-RTs from the prototype foamy virus and a simian foamy virus isolate from macaques were purified and compared. The biophysical and enzymatic properties of the two enzymes were similar. However, their behavior towards the nucleoside inhibitor azidothymidine is different. This nucleoside analog inhibits the replication of foamy viruses by terminating polymerization. Prototype foamy virus was not able to develop resistance against azidothymidine, but we succeeded in the generation of an azidothymidine-resistant simian foamy virus. Up to four mutations within the reverse transcriptase were found to be necessary to confer high resistance against azidothymidine. To characterize the mechanism of resistance, the corresponding recombinant PR-RTs were investigated in vitro. The data reveal that the azidothymidine resistance is based on the excision of the incorporated inhibitor in the presence of ATP. Retroviral proteases are only active as homodimers. In this work, analysis of the PR-RT of prototype foamy virus and simian foamy virus isolated from macaques by analytical ultracentrifugation and size exclusion chromatography indicate, that foamy virus proteases are stable and inactive monomers in solution. The three-dimensional structure of the simian foamy virus protease domain was determined by nuclear magnetic resonance spectroscopy and revealed the typical folding of a monomer subunit of retroviral proteases. Furthermore, nuclear magnetic resonance analysis by paramagnetic relaxation enhancement suggested the formation of transient protease homodimers under native conditions. Finally, it is shown that polypurine rich sequences of the foamy virus RNA are able to activate protease activity. Chemical analysis of the secondary structure of these RNA sequences indicated a characteristic hairpin loop structure. Retardation and protein crosslinking experiments prove the formation of stable PR-RT dimers in the presence of the polypurine RNA sequences. Based on these in vitro data we propose a model for foamy virus assembly.