[Role of Robotic Surgery in ENT]. / Bedeutung der transoralen robotischen Chirurgie in der HNO.

Since the beginning of the 21st century, surgical robots have been used in the ENT-environment. They primarily support surgeons in minimal invasive transoral operations, especially in multidisciplinary treatment concepts of head and neck tumors, but also in snoring surgery the robot provides a complement to the established transoral laser surgery. In the meantime there is a large number of data that deals with the importance of oncological results, function maintenance, economics and future perspectives.Operation areas of the current robot devices are still limited in the ENT-environment. As the number of cases are small, efforts are being made to connect centres on a national and international level. Thus, uniform training standards, targeted knowledge and data exchange as well as further development of systems would be managed better. The creation of small and agile ENT-specific equipment could expand the possibilities as a next step for the future and finally lead to a wide scale of ENT-surgical applications.

Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand.

The major birch pollen allergen Bet v 1 is the main elicitor of airborne type I allergies and belongs to the PR-10 family (pathogenesis-related proteins 10). Bet v 1 is the most extensively studied allergen, and is well characterized at a biochemical and immunological level; however, its physiological function remains elusive. In the present study, we identify Q3OS (quercetin-3-O-sophoroside) as the natural ligand of Bet v 1. We isolated Q3OS bound to Bet v 1 from mature birch pollen and confirmed its binding by reconstitution of the Bet v 1-Q3OS complex. Fluorescence and UV-visible spectroscopy experiments, as well as HSQC (heteronuclear single-quantum coherence) titration, and the comparison with model compounds, such as quercetin, indicated the specificity of Q3OS binding. Elucidation of the binding site by NMR combined with a computational model resulted in a more detailed understanding and shed light on the physiological function of Bet v 1. We postulate that the binding of Q3OS to Bet v 1 plays an important, but as yet unclear, role during the inflammation response and Bet v 1 recognition by IgE.

Structural requirements for enzymatic activities of foamy virus protease-reverse transcriptase.

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.

Foamy virus Gag p71-p68 cleavage is required for template switch of the reverse transcriptase.

In contrast to orthoretroviruses, processing of foamy viral p71 Gag is limited to a single cleavage site. Nevertheless, Gag maturation is essential for infectivity, but deletion of p3 results in a modest drop in infectivity. Here, we show that Gag processing of p71 to p68 and p3 is essential for full-length cDNA synthesis, while inactivation of Gag cleavage results in cDNAs containing only the RU5 region; cDNAs encompassing the U3 region were almost undetectable.

U1snRNP-mediated suppression of polyadenylation in conjunction with the RNA structure controls poly (A) site selection in foamy viruses.

BACKGROUND: During reverse transcription, retroviruses duplicate the long terminal repeats (LTRs). These identical LTRs carry both promoter regions and functional polyadenylation sites. To express full-length transcripts, retroviruses have to suppress polyadenylation in the 5'LTR and activate polyadenylation in the 3'LTR. Foamy viruses have a unique LTR structure with respect to the location of the major splice donor (MSD), which is located upstream of the polyadenylation signal. RESULTS: Here, we describe the mechanisms of foamy viruses regulating polyadenylation. We show that binding of the U1 small nuclear ribonucleoprotein (U1snRNP) to the MSD suppresses polyadenylation at the 5'LTR. In contrast, polyadenylation at the 3'LTR is achieved by adoption of a different RNA structure at the MSD region, which blocks U1snRNP binding and furthers RNA cleavage and subsequent polyadenylation. CONCLUSION: Recently, it was shown that U1snRNP is able to suppress the usage of intronic cryptic polyadenylation sites in the cellular genome. Foamy viruses take advantage of this surveillance mechanism to suppress premature polyadenylation at the 5'end of their RNA. At the 3'end, Foamy viruses use a secondary structure to presumably block access of U1snRNP and thereby activate polyadenylation at the end of the genome. Our data reveal a contribution of U1snRNP to cellular polyadenylation site selection and to the regulation of gene expression.

Delivery of the Brainshuttle™ amyloid-beta antibody fusion trontinemab to non-human primate brain and projected efficacious dose regimens in humans.

There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aß) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aß40 and Aß plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.

Insights into the structure and activity of prototype foamy virus RNase H.

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.

The solution structure of the prototype foamy virus RNase H domain indicates an important role of the basic loop in substrate binding.

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 ß-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.

The prototype foamy virus protease is active independently of the integrase domain.

BACKGROUND: Recently, contradictory results on foamy virus protease activity were published. While our own results indicated that protease activity is regulated by the viral RNA, others suggested that the integrase is involved in the regulation of the protease. RESULTS: To solve this discrepancy we performed additional experiments showing that the protease-reverse transcriptase (PR-RT) exhibits protease activity in vitro and in vivo, which is independent of the integrase domain. In contrast, Pol incorporation, and therefore PR activity in the viral context, is dependent on the integrase domain. To further analyse the regulation of the protease, we incorporated Pol in viruses by expressing a GagPol fusion protein, which supported near wild-type like infectivity. A GagPR-RT fusion, lacking the integrase domain, also resulted in wild-type like Gag processing, indicating that the integrase is dispensable for viral Gag maturation. Furthermore, we demonstrate with a trans-complementation assays that the PR in the context of the PR-RT protein supports in trans both, viral maturation and infectivity. CONCLUSION: We provide evidence that the FV integrase is required for Pol encapsidation and that the FV PR activity is integrase independent. We show that an active PR can be encapsidated in trans as a GagPR-RT fusion protein.

Regulation of foamy virus protease activity by viral RNA: a novel and unique mechanism among retroviruses.

Foamy viruses (FVs) synthesize the Pol precursor protein from a specific transcript. Thus, in contrast to what was found for orthoretroviruses, e.g., human immunodeficiency virus, no Gag-Pol precursor protein is synthesized. Foamy viral Pol consists of a protease (PR) domain, a reverse transcriptase domain, and an integrase domain and is processed into a mature protease-reverse transcriptase (PR-RT) fusion protein and the integrase. Protease activity has to be strictly regulated in order to avoid premature Gag and Pol processing before virus assembly. We have demonstrated recently that FV protease is an inactive monomer with a very weak dimerization tendency and postulated protease activation through dimerization. Here, we identify a specific protease-activating RNA motif (PARM) located in the pol region of viral RNA which stimulates PR activity in vitro and in vivo, revealing a novel and unique mechanism of retroviral protease activation. This mechanism is strikingly different to that of orthoretroviruses, where the protease can be activated even in the absence of viral RNA during the assembly of virus-like particles. Although it has been shown that the integrase domain is important for Pol uptake, activation of the foamy virus protease is integrase independent. We show that at least two foamy virus PR-RT molecules bind to the PARM and only RNAs containing the PARM result in significant activation of the protease. DNA harboring the PARM is not capable of protease activation. Structure determination of the PARM by selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) revealed a distinct RNA folding, important for protease activation and thus virus maturation.

Degradation and release of tannic acid from an injectable tissue regeneration bead matrix in vivo.

The development of multifunctional biomaterials as both tissue regeneration and drug delivery devices is currently a major focus in biomedical research. Tannic Acid (TA), a naturally occurring plant polyphenol, displays unique medicinal abilities as an antioxidant, an antibiotic, and as an anticancer agent. TA has applications in biomaterials acting as a crosslinker in polymer hydrogels improving thermal stability and mechanical properties. We have developed injectable cell seeded collagen beads crosslinked with TA for breast reconstruction and anticancer activity following lumpectomy. This study determined the longevity of the bead implants by establishing a degradation time line and TA release profile in vivo. Beads crosslinked with 0.1% TA and 1% TA were compared to observe the differences in TA concentration on degradation and release. We found collagen/TA beads degrade at similar rates in vivo, yet are resistant to complete degradation after 16 weeks. TA is released over time in vivo through diffusion and cellular activity. Changes in mechanical properties in collagen/TA beads before implantation to after 8 weeks in vivo also indicate loss of TA over a longer period of time. Elastic moduli decreased uniformly in both 0.1% and 1% TA beads. This study establishes that collagen/TA materials can act as a drug delivery system, rapidly releasing TA within the first week following implantation. However, the beads retain TA long term allowing them to resist degradation and remain in situ acting as a cell scaffold and tissue filler. This confirms its potential use as an anticancer and minimally invasive breast reconstructive device following lumpectomy.

Formation of transient dimers by a retroviral protease.

Retroviral proteases have been shown previously to be only active as homodimers. They are essential to form the separate and active proteins from the viral precursors. Spumaretroviruses produce separate precursors for Gag and Pol, rather than a Gag and a Gag-Pol precursor. Nevertheless, processing of Pol into a PR (protease)-RT (reverse transcriptase) and integrase is essential in order to obtain infectious viral particles. We showed recently that the PR-RT from a simian foamy virus, as well as the separate PRshort (protease) domain, exhibit proteolytic activities, although only monomeric forms could be detected. In the present study, we demonstrate that PRshort and PR-RT can be inhibited by the putative dimerization inhibitor cholic acid. Various other inhibitors, including darunavir and tipranavir, known to prevent HIV-1 PR dimerization in cells, had no effect on foamy virus protease in vitro. 1H-15N HSQC (heteronuclear single quantum coherence) NMR analysis of PRshort indicates that cholic acid binds in the proposed PRshort dimerization interface and appears to impair formation of the correct dimer. NMR analysis by paramagnetic relaxation enhancement resulted in elevated transverse relaxation rates of those amino acids predicted to participate in dimer formation. Our results suggest transient PRshort homodimers are formed under native conditions but are only present as a minor transient species, which is not detectable by traditional methods.

Docetaxel, cisplatin and 5-FU compared with docetaxel, cisplatin and cetuximab as induction chemotherapy in advanced squamous cell carcinoma of the head and neck: Results of a randomised phase II AGMT trial.

PURPOSE: Induction chemotherapy (ICT) with cisplatin (P), 5-FU (F) and taxanes (T) is a therapeutical option in patients suffering from locally advanced or unresectable stage III or IV squamous cell carcinoma of the head and neck (SCCHN). The role of ICT is controversial, and toxicity and/or delay of radiotherapy (RT) may reduce the potential benefit of this treatment regimen. Here, we report the results of a randomised phase II trial comparing TPF with TP + cetuximab (C). PATIENTS AND METHODS: In this trial, 100 patients with locally advanced stage III or IV SCCHN were included in the analysis. Patients were randomly assigned to either TPF-ICT (N = 49) or TPC-ICT (N = 51), both followed by RT + C. The primary end-point of the study was overall response rate (ORR) three months after RT + C was finished. RESULTS: On an intention-to-treat basis, the ORR (complete remission + partial remission) was 74.5% in the TPC arm compared with 63.3% in the TPF arm (p = 0.109). OS was similar in both arms 400 days after treatment was initiated (86.1% [95% confidence interval {CI}, 73.0-93.1%] in the TPC arm and 78.5% [95% CI, 63.7-87.8%] in the TPF arm). TPC resulted in slightly less serious adverse events and in less haematological, but more skin toxicities. Two patients randomised in the TPC arm died during ICT and RT. Four patients in the TPF arm died after completion of RT. No delay from the end of ICT to RT + C was observed. A total of 83.1% of patients (80% in the TPC arm; 86% in the TPF arm) received RT without dose reduction and/or modification. CONCLUSION: TPC-containing ICT for patients with locally advanced SCCHN was found to be an effective and tolerable one-day regimen. Further prospective evidence from larger trials is warranted.

Biophysical and enzymatic properties of the simian and prototype foamy virus reverse transcriptases.

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 non-dividing cells.

AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP.

Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K(M)-values for polymerization but no change in K(D)-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.

Ligand Recognition of the Major Birch Pollen Allergen Bet v 1 is Isoform Dependent.

Each spring millions of patients suffer from allergies when birch pollen is released into the air. In most cases, the major pollen allergen Bet v 1 is the elicitor of the allergy symptoms. Bet v 1 comes in a variety of isoforms that share virtually identical conformations, but their relative concentrations are plant-specific. Glycosylated flavonoids, such as quercetin-3-O-sophoroside, are the physiological ligands of Bet v 1, and here we found that three isoforms differing in their allergenic potential also show an individual, highly specific binding behaviour for the different ligands. This specificity is driven by the sugar moieties of the ligands rather than the flavonols. While the influence of the ligands on the allergenicity of the Bet v 1 isoforms may be limited, the isoform and ligand mixtures add up to a complex and thus individual fingerprint of the pollen. We suggest that this mixture is not only acting as an effective chemical sunscreen for pollen DNA, but may also play an important role in recognition processes during pollination.

Enlarging the toolbox for allergen epitope definition with an allergen-type model protein.

BACKGROUND: Birch pollen-allergic subjects produce polyclonal cross-reactive IgE antibodies that mediate pollen-associated food allergies. The major allergen Bet v 1 and its homologs in plant foods bind IgE in their native protein conformation. Information on location, number and clinical relevance of IgE epitopes is limited. We addressed the use of an allergen-related protein model to identify amino acids critical for IgE binding of PR-10 allergens. METHOD: Norcoclaurine synthase (NCS) from meadow rue is structurally homologous to Bet v 1 but does not bind Bet v 1-reactive IgE. NCS was used as the template for epitope grafting. NCS variants were tested with sera from 70 birch pollen allergic subjects and with monoclonal antibody BV16 reported to compete with IgE binding to Bet v 1. RESULTS: We generated an NCS variant (Δ29NCSN57/I58E/D60N/V63P/D68K) harboring an IgE epitope of Bet v 1. Bet v 1-type protein folding of the NCS variant was evaluated by 1H-15N-HSQC NMR spectroscopy. BV16 bound the NCS variant and 71% (50/70 sera) of our study population showed significant IgE binding. We observed IgE and BV16 cross-reactivity to the epitope presented by the NCS variant in a subgroup of Bet v 1-related allergens. Moreover BV16 blocked IgE binding to the NCS variant. Antibody cross-reactivity depended on a defined orientation of amino acids within the Bet v 1-type conformation. CONCLUSION: Our system allows the evaluation of patient-specific epitope profiles and will facilitate both the identification of clinically relevant epitopes as biomarkers and the monitoring of therapeutic outcomes to improve diagnosis, prognosis, and therapy of allergies caused by PR-10 proteins.

Fast mapping of biomolecular interfaces by Random Spin Labeling (RSL).

Random spin labeling (RSL) is a method for rapid mapping of biomolecular interaction surfaces using an interaction partner with SL and an interaction partner enriched in (13)C or (15)N nuclei for paramagnetic relaxation enhanced NMR-based detection. The SL reaction is conducted in a manner resulting in a heterogeneous reaction product consisting of different populations of the protein carrying a varying number of spin labels at different positions. Preparation of the paramagnetic probe is complete within a few hours and hence much faster than site selective SL. RSL is applicable to tightly interacting systems but shows its particular strength when applied to systems involving weak or transient contacts.

The solution structure of the simian foamy virus protease reveals a monomeric protein.

In contrast to orthoretroviruses, foamy viruses (FVs) express their Pol polyprotein from a separate pol-specific transcript. Only the integrase domain is cleaved off, leading to a protease-reverse transcriptase (PR-RT) protein. We purified the separate PR domain (PRshort) of simian FV from macaques by expressing the recombinant gene in Escherichia coli. Sedimentation analyses and size exclusion chromatography indicate that PRshort is a stable monomer in solution. This allowed us to determine the structure of the PRshort monomer using 1426 experimental restraints derived from NMR spectroscopy. The superposition of 20 conformers resulted in a backbone atom rmsd of 0.55 A for residues Gln8-Leu93. Although the overall folds are similar, the macaque simian FV PRshort reveals significant differences in the dimerization interface relative to other retroviral PRs, such as HIV-1 (human immunodeficiency virus type 1) PR, which appear to be rather stable dimers. Especially the flap region and the N- and C-termini of PRshort are highly flexible. Neglecting these regions, the backbone atom rmsd drops to 0.32 A, highlighting the good definition of the central part of the protein. To exclude that the monomeric state of PRshort is due to cleaving off the RT, we purified the complete PR-RT and performed size exclusion chromatography. Our data show that PR-RT is also monomeric. We thus conclude adoption of a monomeric state of PR-RT to be a regulatory mechanism to inhibit PR activity before virus assembly in order to reduce packaging problems. Dimerization might therefore be triggered by additional viral or cellular factors.

AZT-resistant foamy virus.

Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.