Accuracy estimation of foamy virus genome copying

<p>Abstract</p> <p>Background</p> <p>Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the <it>in vitro </it>error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying <it>in vivo </it>we analyzed the occurrence of mutations in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient vector system. Furthermore, the frequency of FV recombination by template switching (TS) and the cross-packaging ability of different FV strains were analyzed.</p> <p>Results</p> <p>We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an <it>in vivo </it>error rate of approximately 4 × 10^-4per site per replication cycle. Surprisingly, all mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for the majority of mutations detected in our experimental system. In line with this, we detected a late but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed 170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-interfering foamy viral <it>bet </it>gene and observed a significant 50% drop in G to A mutations, indicating that APOBEC activity indeed contributes substantially to the foamy viral replication error rate <it>in vivo</it>. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that residual APOBEC activity accounted for most of the observed mutations. If we subtract these APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic <it>in vivo </it>error rate of 1.1 × 10^-5per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.</p> <p>Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a template switching (TS) event within a 1 kilobase (kb) region. This corresponds to a 98% probability that FVs undergo at least one additional TS event per replication cycle. We also show that a given FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than the homologous vector.</p> <p>Conclusion</p> <p>Our results indicate that the copying of the FV genome is more accurate than previously thought. On the other hand recombination among FV genomes appears to be a frequent event.</p

AZT-resistant foamy virus

AbstractAzidothymidine (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

Targeting deregulated AMPK/mTORC1 pathways improves muscle function in myotonic dystrophy type I

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 (HSALR mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in HSALR muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in HSALR mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in HSALR mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease

Generation of an improved foamy virus vector by dissection of cis-acting sequences

Im Vergleich zu anderen Retroviren, zeichnen sich Foamyviren durch eine Reihe von Eigenschaften aus, die sie besonders attraktiv für die Vektorentwicklung und somatische Gentherapie machen. Foamyviren exprimieren ihr Pol Prekursorprotein unabhängig von Gag, d.h. von ihrer eigenen gespleisten mRNA. Zwar ist der genaue Pol-Verpackungsmechanismus von Foamyviren noch nicht vollständig aufgeklärt, frühere Studien zeigten jedoch, dass die prägenomische RNA essentiell für die Pol-Enkapsidierung ist. Zwei Pol-Verpackungssequenzen (PES) wurden identifiziert, welche sich in den cis-aktiven Sequenzen (CAS) der prägenomischen RNA befinden (Heinkelein et al., 1998; Peters et al., 2005). In dieser Arbeit wurde untersucht, ob die PESI und PESII Sequenzen alleine ausreichend für die Pol-Verpackung sind. Zusätzich wurde der Einfluss von verschiedenen Teilen der ca. 2000 nt langen CASII Sequenz auf den Vektortransfer ohne Verlust der Pol-Enkapsidierung untersucht. Es konnte gezeigt werden, dass PESI und PESII alleine nicht ausreichend für die Pol-Verpackung ins foamyvirale Partikel sind. Die Verkürzung des CASII Elements zeigte keinen Effekt auf die Pol-Verpackung und den Vektortransfer. Das Einfügen eines zusätzlichen zentralen Polypurintraktes führte jedoch zur signifikanten Erhöhung der Transduktionseffizienz von FV Vektoren. Diese Ergebnisse führten zur Entwicklung eines neuen foamyviralen Vektors (pTW01), der ca. 850nt kürzer ist als die früher etablierten FV Vektoren, aber immer noch die gleiche Transduktionseffizienz auf Fibroblasten und humanen Stammzellen zeigt. Dieser Vektor mit einer höheren Verpackungskapazität und Sicherheit, eignet sich hervorragend für den Einsatz in gentherapeutischen Studien. Zusätzlich konnte gezeigt werden, dass eine heterologe Verpackung zwischen zwei unterschiedlichen Foamyviren (PFV und SFVmac) zu einem geringen Prozentsatz stattfindet. Als erster Schritt in der Entwickung eines neues Systems für eine einfache und kostengünstige Vektorvirusproduktion wurde gezeigt, dass die Expression der foamyviralen Gag, Pol und Env Proteine in Saccharomyces cerevisiae stattfinden kann.Foamy viruses harbor some unique features which make them, compared to other retroviruses, especially attractive for vector development and somatic gene therapy. Foamy viruses express their Pol precursor protein independently of Gag, i.e. from their own spliced mRNA. While the exact mechanism by which Pol is incorporated into the foamy virus particle is still unknown, previous studies have shown that pregenomic RNA is essential for Pol incorporation. Two cis-active sequences (CAS) were identified, within which two essential Pol encapsidation sequences (PES) were mapped (Heinkelein et al., 1998; Peters et al., 2005). Using the prototype foamy virus (PFV) as a model, this work investigated whether the previously identified PESI and PESII sequences in an FV vector are alone sufficient for Pol encapsidation. Additionly, the influence of various parts of the 2000 bp CASII sequence on vector transfer efficiency without the loss of Pol encapsidation was studied. The obtained results indicate that the PESI and PESII alone are not sufficient for Pol incorporation into a foamy virus particle. The truncation of the CASII element has no effect on Pol incorporation and vector transfer. However, if an additional central poly purine tract is generated into a foamy virus vector, it significantly increases the FV vector transduction rate. These results led to a generation of an improved foamy virus vector (pTW01), about 850 bp shorter than the previously established vectors, yet still as effective in transducing fibroblasts and primary human cells. These data add to the packaging limit of the PFV vectors for gene therapy, as well as to the safety of these vectors. In addition to these findings, it was shown that the cross-packaging between two different foamy viruses (PFV and SFVmac) takes place very rarely. Finally, as a first step in finding a new low-cost possibility to produce large amounts of vector viruses, it was shown that an expression of the three foamy virus proteins: Gag, Pol and Env can take place in Saccharomyces cerevisiae

RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5′ long terminal repeat and one at the 3′ end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins

Genetic characterization and improved genotyping of the dysferlin-deficient mouse strain Dysf (tm1Kcam)

BACKGROUND: Mouse models of dysferlinopathies are valuable tools with which to investigate the pathomechanisms underlying these diseases and to test novel therapeutic strategies. One such mouse model is the Dysf (tm1Kcam) strain, which was generated using a targeting vector to replace a 12-kb region of the dysferlin gene and which features a progressive muscular dystrophy. A prerequisite for successful animal studies using genetic mouse models is an accurate genotyping protocol. Unfortunately, the lack of robustness of currently available genotyping protocols for the Dysf (tm1Kcam) mouse has prevented efficient colony management. Initial attempts to improve the genotyping protocol based on the published genomic structure failed. These difficulties led us to analyze the targeted locus of the dysferlin gene of the Dysf (tm1Kcam) mouse in greater detail. METHODS: In this study we resequenced and analyzed the targeted locus of the Dysf (tm1Kcam) mouse and developed a novel PCR protocol for genotyping. RESULTS: We found that instead of a deletion, the dysferlin locus in the Dysf (tm1Kcam) mouse carries a targeted insertion. This genetic characterization enabled us to establish a reliable method for genotyping of the Dysf (tm1Kcam) mouse, and thus has made efficient colony management possible. CONCLUSION: Our work will make the Dysf (tm1Kcam) mouse model more attractive for animal studies of dysferlinopathies

Interlaboratory Analysis of Isavuconazole Plasma Concentration Assays Among European Laboratories

Background: Under certain circumstances, clinicians treating patients with isavuconazole for invasive aspergillosis or mucormycosis may use therapeutic drug monitoring. However, the accuracy and reproducibility of the various assays used by different laboratories for the quantification of isavuconazole plasma concentrations have yet to be determined. Methods: Human plasma samples spiked with known concentrations of isavuconazole were provided to 27 European laboratories that took part in a round-robin test (an interlaboratory test performed independently at least 2 times; 2 rounds performed in the current study). Assay methods included liquid chromatographytandem mass spectrometry (LC-MS/MS), LC with ultraviolet detection (LC-UV), LC with fluorescence detection (LC-FL), and bioassay. The accuracy and reproducibility compared with the known concentrations for each sample in each round were compared overall, between assays, and between laboratories. Results: Twenty-seven laboratories participated in the study (LCMS/MS, n = 15; LC-UV; n = 9; LC-FL, n = 1; bioassay, n = 2). In round 1, for nominal concentrations of 1000, 1700, 2500, and 4000 ng/mL, the mean (SD) determined concentrations were 1007 (183), 1710 (323), 2528 (540), and 3898 (842) ng/mL, respectively. In round 2, for nominal concentrations of 1200, 1800, 2400, and 4000 ng/mL, the mean (SD) determined concentrations were 1411 (303), 2111 (409), 2789 (511), and 4723 (798) ng/mL, respectively. Over both rounds, determined concentrations were consistently within 15% of the nominal concentrations for 10 laboratories (LCMS/MS, n = 4; LC-UV, n = 5; bioassay, n = 1) and consistently exceeded the upper 15% margin for 7 laboratories (LC-MS/MS and LC-UV, n = 3 each; LC-FL, n = 1). Conclusions: Alignment of methodologies among laboratories may be warranted to improve the accuracy and reproducibility of therapeutic drug measurements