Foamy Virus Enzymes - Activity, Regulation and Resistance

Background: The foamy virus Pol protein is translated independently from Gag using a separate mRNA. Thus, in contrast to orthoretroviruses no Gag-Pol precursor protein is synthesized. Only the integrase domain is cleaved off from Pol resulting in a mature reverse transcriptase harboring the protease domain at the N-terminus (PR-RT). Although the homology between the PR-RTs from simian foamy virus from macaques (SFVmac) and the prototype foamy virus (PFV), probably originating from chimpanzee, exceeds 90%, several differences in the biophysical and biochemical properties of the two enzymes have been reported (i.e. SFVmac develops resistance to the nucleoside inhibitor azidothymidine (AZT) whereas PFV remains AZT sensitive even if the resistance mutations from SFVmac PR-RT are introduced into the PFV PR-RT gene). Moreover, contradictory data on the monomer/dimer status of the foamy virus protease have been published. Results: We set out to purify and directly compare the monomer/dimer status and the enzymatic behavior of the two wild type PR-RT enzymes from SFVmac and PFV in order to get a better understanding of the protein and enzyme functions. We determined kinetic parameters for the two enzymes, and we show that PFV PR-RT is also a monomeric protein. Conclusions: Our data show that the PR-RTs from SFV and PFV are monomeric proteins with similar biochemical and biophysical properties that are in some aspects comparable with MLV RT, but differ from those of HIV-1 RT. These differences might be due to the different conditions the viruses are confronted with in dividing and nondividing cells. Background Foamy viruses (FVs) belong to the family retroviridae, but differ in several aspects from orthoretrovirinae: (a) reverse transcription occurs before the virus leaves the host cell [1,2], (b) the pol-gene is expressed from a separate mRNA [3-5], and (c) the viral protease is not cleaved off from the Pol polyprotein. Only the integrase is removed from Pol [6,7]. Thus, the FV reverse transcriptase harbors a protease, polymerase and RNase H domain (PR-RT) (for review see [8,9]). Only recently, studies have focused on the biochemistry of the PR-RTs of FVs. Although the PR-RTs from simian foamy virus from macaques (SFVmac) and from the prototype foamy virus (PFV) exhibit more than 90% sequence homology at the protein level (79.5% identity; LALIGN, http://www.ch.embnet.org), some differences in their behavior have been reported. Bacterially expressed PFV PR-RT harbors many characteristics of orthoretroviral RTs; however, FV enzymes exhibit some peculiar features [10-16]. In comparison to human immunodeficiency virus type 1 (HIV-1) RT, PFV PR-RT appears to be a more processive polymerase [11]. This is probably due to differences in virus assembly. FV Pol packaging has been reported to require interactions of Pol with specific sequences in the RNA genome [17], and it has been suggested that there is a lower number of FV Pol molecules in the virus particle as compared to orthoretroviruses [11]. As a consequence, a highly processive polymerase is essential to enable synthesis of the complete double stranded genome. * Correspondence: birgitta.woehrl@uni-bayreuth.de Universität Bayreuth, Lehrstuhl für Struktur und Chemie der Biopolymere & Research, Center for Biomacromolecules, 95440 Bayreuth, Germany Hartl et al. Retrovirology 2010, 7:5 http://www.retrovirology. com/content/7/1/5 © 2010 Hartl et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. One antiretroviral drug that has been shown to inhibit FV replication is azidothymidine (AZT) [1,18,19]. In in vivo experiments SFVmac acquired high resistance to AZT by four mutations within the RT sequence [14,20]. PFV, however, did not develop resistance to AZT, and the introduction of the SFVmac mutations into the PFV RT gene did not result in viruses resistant to the nucleoside inhibitor [20]. Regarding the high amino acid homology of the two enzymes, this result was not to be expected. In SFVmac, the mechanism of resistance is due to the removal of already incorporated AZT-monophosphate (AZTMP) in the presence of ATP and thus resembles that of HIV-1 RT [14,21,22]. It has been shown previously that retroviral PRs are only active as homodimers. To create the active center, each subunit of the homodimer contributes catalytic residues located in the conserved motif DT/SG [23]. However, SFVmac PR-RT behaves as a monomer in solution, but nevertheless exhibits PR activity. Catalytic PR activity could only be observed at NaCl concentrations of 2-3 M [15], indicating that hydrophobic interactions might promote dimerization. Furthermore, by prevalent methods the separately expressed 12.6 kDa PR domain was also found to be monomeric but active [15]. Only further analyses using NMR paramagnetic relaxation enhancement proved that transient, lowly populated dimers are being formed (Hartl MJ, Schweimer K, Reger MH, Schwarzinger S, Bodem J, Rösch P, Wöhrl BM: Formation of transient dimers by a retroviral protease, submitted). Contradicting results were obtained by gel filtration analysis with a purified Cterminally extended 18 kDa PR domain of PFV, which indicated that PFV PR might be dimeric [6]. To clarify these issues and to shed more light on the properties of SFVmac and PFV PR-RT, we set out to purify both enzymes from bacterial lysates and directly compare their secondary structure, oligomerization state, and activities. Results and Discussion Protein purification Overexpression of PFV PR-RT in E. coli resulted in partial degradation by cellular proteases. Thus, we could not adopt the purification protocol established for SFVmac PR-RT [14]. Instead, we had to set up a new purification procedure for PFV PR-RT which includes Ni-affinity followed by hydrophobic interaction chromatography to remove the PR-RT degradation products. The yields were much lower than for SFVmac PR-RT. Nevertheless, pure soluble protein (> 95% purity, as judged from SDS-polyacrylamide gels) could be obtained. Biophysical properties To exclude that the purified PR-RTs are partially or completely unfolded, we analyzed the secondary structure of PFV and SFVmac PR-RT by circular dichroism (CD) spectroscopy. The shape of the CD spectra obtained for the two enzymes was highly similar, implying comparable ratios of a-helices and b-strands (Fig. 1A). In both cases, the curves showed a broad minimum between 205 nm and 222 nm, characteristic for a mixture of a-helical and b-strand structures, and high ellipticity near 200 nm. Thus, the spectra are indicative of predominantly folded proteins. Although the spectrum obtained for SFVmac PR-RT deviates slightly from that of PFV PR-RT, the calculated values (Table 1) confirm the accordance in the secondary structure contents of PFV and SFVmac PR-RT. However, crystal structure analyses will be necessary to obtain more information on the structural similarities and differences of the two enzymes. The three-dimensional structure will probably also shed more light on the differences between PFV and SFVmac PR-RT in developing AZT-resistance. Contradicting data have been published on the monomer/dimer status of FV PRs. PFV PR expressed separately was suggested to be dimeric [6], whereas we have shown by various analyses, like size exclusion chromatography and analytical ultracentrifugation that the full length PR-RT protein as well as the separate PR domain of SFVmac are monomeric, and only transient PR dimers are being formed [15] (Hartl MJ, Schweimer K, Reger MH, Schwarzinger S, Bodem J, Rösch P, Wöhrl BM: Formation of transient dimers by a retroviral protease, submitted). Previous results obtained by sucrose density gradient analyses with PR-RT purified from SFVmac particles also indicated that the protein is monomeric [24]. To clarify the monomer/dimer status of PFV PR-RT, we performed size exclusion chromatography (Fig. 1B). Our data revealed a single peak, which corresponded to a molecular mass of 85.4 kDa. This is in good agreement with the theoretical molecular mass of the monomeric PFV PR-RT of 86.5 kDa. Moreover, no dimer peak