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Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae . The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

RNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg(2+) needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data, in silico docking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In contrast to orthoretroviruses, the foamy virus protease is only active as a protease-reverse transcriptase fusion protein and requires viral RNA for activation. Maturation of foamy viral proteins seems to be restricted to a single cleavage site in Gag and Pol. We provide evidence that unprocessed Gag is required for optimal infectivity, which is unique among retroviruses. Analyses of the cleavage site sequences of the Gag and Pol cleavage sites revealed a high similarity compared to those of Lentiviruses. We show that positions P2׳ and P2 are invariant and that Gag and Pol cleavage sites are processed with similar efficiencies. The RNase H domain is essential for protease activity, but can functionally be substituted by RNase H domains of other retroviruses. Thus, the RNase H domain might be involved in the stabilization of the protease dimer, while the RT domain is essential for RNA dependent protease activation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Reverse transcriptases (RTs) are pivotal in the life cycle of retroviruses and convert the genomic viral RNA into double-stranded DNA. The RT polymerase domain is subdivided into fingers, palm, thumb, and the connection subdomain, which links the polymerase to the C-terminal RNase H domain. In contrast to orthoretroviruses, mature RT of foamy viruses harbors the protease (PR) domain at its N-terminus (PR-RT). Therefore and due to low homology to other RTs, it is difficult to define the boundaries and functions of the (sub)domains. We introduced N- and C-terminal deletions into simian foamy virus PR-RT to investigate the impact of the truncations on the catalytic activities. Both, the RNase H domain and the connection subdomain contribute substantially to polymerase integrity and stability as well as to polymerase activity and substrate binding. The 42 amino acids long region C-terminal of the PR is important for polymerase stability and activity. PR activation via binding of PR-RT to viral RNA requires the presence of the full length PR-RT including the RNase H domain. In vitro, the cleavage efficiencies of FV PR for the Gag and Pol cleavage site are comparable, even though in virus particles only the Pol site is cleaved to completion suggesting that additional factors control PR activity and that virus maturation needs to be strictly regulated., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: The ribonuclease H (RNase H) domains of retroviral reverse transcriptases play an essential role in the replication cycle of retroviruses. During reverse transcription of the viral genomic RNA, an RNA/DNA hybrid is created whose RNA strand needs to be hydrolyzed by the RNase H to enable synthesis of the second DNA strand by the DNA polymerase function of the reverse transcriptase. Here, we report the solution structure of the separately purified RNase H domain from prototype foamy virus (PFV) revealing the so-called C-helix and the adjacent basic loop, which both were suggested to be important in substrate binding and activity., Results: The solution structure of PFV RNase H shows that it contains a mixed five-stranded β-sheet, which is sandwiched by four α-helices (A-D), including the C-helix, on one side and one α-helix (helix E) on the opposite side. NMR titration experiments demonstrate that upon substrate addition signal changes can be detected predominantly in the basic loop as well as in the C-helix. All these regions are oriented towards the bound substrate. In addition, signal intensities corresponding to residues in the B-helix and the active site decrease, while only minor or no changes of the overall structure of the RNase H are detectable upon substrate binding. Dynamic studies confirm the monomeric state of the RNase H domain. Structure comparisons with HIV-1 RNase H, which lacks the basic protrusion, indicate that the basic loop is relevant for substrate interaction, while the C-helix appears to fulfill mainly structural functions, i.e. positioning the basic loop in the correct orientation for substrate binding., Conclusions: The structural data of PFV RNase H demonstrate the importance of the basic loop, which contains four positively charged lysines, in substrate binding and the function of the C-helix in positioning of the loop. In the dimeric full length HIV-1 RT, the function of the basic loop is carried out by a different loop, which also harbors basic residues, derived from the connection domain of the p66 subunit. Our results suggest that RNases H which are also active as separate domains might need a functional basic loop for proper substrate binding.

Background: RNase H is an endonuclease that hydrolyzes the RNA strand in RNA/DNA hybrids. Retroviral reverse transcriptases harbor a C-terminal RNase H domain whose activity is essential for viral replication. The RNase H degrades the viral genomic RNA after the first DNA strand is synthesized. Here, we report the biophysical and enzymatic properties of the RNase H domain of prototype foamy virus (PFV) as an independently purified protein. Sequence comparisons with other retroviral RNases H indicated that PFV RNase H harbors a basic protrusion, including a basic loop and the so-called C-helix, which was suggested to be important for activity and substrate binding and is absent in the RNase H domain of human immunodeficiency virus. So far, no structure of a retroviral RNase H containing a C-helix is available., Results: RNase H activity assays demonstrate that the PFV RNase H domain is active, although its activity is about 200-fold reduced as compared to the full length protease-reverse transcriptase enzyme. Fluorescence equilibrium titrations with an RNA/DNA substrate revealed a KD for the RNase H domain in the low micromolar range which is about 4000-fold higher than that of the full-length protease-reverse transcriptase enzyme. Analysis of the RNase H cleavage pattern using a [32P]-labeled substrate indicates that the independent RNase H domain cleaves the substrate non-specifically. The purified RNase H domain exhibits a well defined three-dimensional structure in solution which is stabilized in the presence of Mg2+ ions., Conclusions: Our data demonstrate that the independent PFV RNase H domain is structured and active. The presence of the C-helix in PFV RNase H could be confirmed by assigning the protein backbone and calculating the chemical shift index using NMR spectroscopy.

Foamy virus (FV) replication, while related to that of orthoretroviruses, differs at a number of steps. Several of these differences involve the reverse transcriptase (RT). There appear to be fewer RTs present in FV than in orthoretroviruses; we previously proposed that the polymerase of FV RT was more active than orthoretroviral RTs to compensate for the numerical difference. Here we present further characterization of the RT of FV. The polymerase activity of FV RT was greater than that of human immunodeficiency virus type 1 RT in a variety of assays. We also examined the RNase H activity of FV RT, and we propose that FV RT has a basic loop in the RNase H domain. Although the sequence of the basic loop of FV RT is different from the basic loop of either Moloney leukemia virus RNase H or Escherichia coli RNase H, the FV RT basic loop appears to have a similar function.

Foamy viruses form a separate group of retroviruses encoding a pol protein with at least four domains based on comparative sequence alignments. The polymerase and ribonuclease H domains of the human foamy virus (HFV) pol gene were expressed in Escherichia coli either individually or in combination. The histidine-tagged HFV fusion proteins were subsequently purified to near homogeneity by affinity Ni2+ chelate column chromatography. The polymerase and RNase H activities were characterized by performing conventional DNA polymerase and ribonuclease H assays and in situ gel assays. Six purified recombinant HFV proteins were enzymatically active either individually as DNA polymerase and ribonuclease H or as combined domains. The HFV enzymatic activities were characterized with respect to cation preferences and pH optima. Western blots with antibodies against the RNase H domain, in situ reverse transcriptase (RT), and RNase H gel assays showed that in HFV-infected cells pol proteins of 120 and 80 kDa were detectable. A novel activity band of 60 kDa was found in situ RT gel assays. Recombinant RNase H protein additionally purified by fast performance liquid chromatography was capable of removing the primer for minus-strand DNA synthesis when labeled tRNA(Lys1,2) model substrates were used. Specific cleavages occurred at the phosphodiester bonds one to three nucleotides 5' of the RNA-DNA junction. The results revealed biochemical properties of the HFV pol gene products that define functional domains of the HFV pol gene that are distinct but comparable to other retroviruses.

Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.