Optimisation of expression and purification of the feline and primate foamy virus transmembrane envelope proteins using a 96 deep well screen

The production of recombinant transmembrane proteins is due to their biochemical properties often troublesome and time consuming. Here the prokaryotic expression and purification of the transmembrane envelope proteins of the feline and primate foamy viruses using a screening assay for optimisation of expression in 96 deep well plates is described. Testing simultaneously various bacterial strains, media, temperatures, inducer concentrations and different transformants, conditions for an about twentyfold increased production were quickly determined. These small scale test conditions could be easily scaled up, allowing purification of milligram amounts of recombinant protein. Proteins with a purity of about 95% were produced using a new purification protocol, they were characterised by gel filtration and circular dichroism and successfully applied in immunological assays screening for foamy virus infection and in immunisation studies. Compared to the previously described protocol (M. Mühle, A. Bleiholder, S. Kolb, J. Hübner, M. Löchelt, J. Denner, Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening, Virology 412 (2011) 333–340), proteins with similar characteristics but about thirtyfold increased yields were obtained. The screening and production method presented here can also be applied for the production of transmembrane envelope proteins of other retroviruses, including HIV-1

N-Terminally Myristoylated Feline Foamy Virus Gag Allows Env-Independent Budding of Sub-Viral Particles

Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation

Immunological properties of the transmembrane envelope protein of the feline foamy virus and its use for serological screening

The transmembrane envelope (TM) proteins of retroviruses are used as antigen in diagnostic immunoassays and they represent a conserved target for neutralizing antibodies. To analyze the situation in infections with the feline foamy virus (FFV), its recombinant TM protein was produced and used for ELISA and Western blot analyses. Screening sera from 404 German cats showed that 39% reacted against the TM protein, the same infection rate was determined using the Gag protein. Epitope mapping showed antibodies against the membrane proximal external region (MPER) of the TM protein in the sera from infected cats, but attempts to induce neutralizing antibodies by immunization with the recombinant TM protein failed. This is the first report demonstrating that the TM protein of the FFV is highly immunogenic and valuable for serological screening. Similar to HIV-1, but in contrast to different gammaretroviruses, immunization with the TM protein of FFV did not induce neutralizing antibodies

Foamy Virus Biology and Its Application for Vector Development

Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system

Molecular and functional interactions of cat APOBEC3 and feline foamy and immunodeficiency virus proteins: Different ways to counteract host-encoded restriction

AbstractDefined host-encoded feline APOBEC3 (feA3) cytidine deaminases efficiently restrict the replication and spread of exogenous retroviruses like Feline Immunodeficiency Virus (FIV) and Feline Foamy Virus (FFV) which developed different feA3 counter-acting strategies. Here we characterize the molecular interaction of FFV proteins with the diverse feA3 proteins. The FFV accessory protein Bet is the virus-encoded defense factor which is shown here to bind all feA3 proteins independent of whether they restrict FFV, a feature shared with FIV Vif that induces degradation of all feA3s including those that do not inactivate FIV. In contrast, only some feA3 proteins bind to FFV Gag, a pattern that in part reflects the restriction pattern detected. Additionally, one-domain feA3 proteins can homo- and hetero-dimerize in vitro, but a trans-dominant phenotype of any of the low-activity feA3 forms on FFV restriction by one of the highly-active feA3Z2 proteins was not detectable

Molecular dissection of the prototype foamy virus (PFV) RNA 5′-UTR identifies essential elements of a ribosomal shunt

The prototype foamy virus (PFV) is a nonpathogenic retrovirus that shows promise as a vector for gene transfer. The PFV (pre)genomic RNA starts with a long complex leader that can be folded into an elongated hairpin, suggesting an alternative strategy to cap-dependent linear scanning for translation initiation of the downstream GAG open reading frame (ORF). We found that the PFV leader carries several short ORFs (sORFs), with the three 5′-proximal sORFs located upstream of a structural element. Scanning-inhibitory hairpin insertion analysis suggested a ribosomal shunt mechanism, whereby ribosomes start scanning at the leader 5′-end and initiate at the downstream ORF via bypass of the central leader regions, which are inhibitory for scanning. We show that the efficiency of shunting depends strongly on the stability of the structural element located downstream of either sORFs A/A′ or sORF B, and on the translation event at the corresponding 5′-proximal sORF. The PFV shunting strategy mirrors that of Cauliflower mosaic virus in plants; however, in mammals shunting can operate in the presence of a less stable structural element, although it is greatly improved by increasing the number of base pairings. At least one shunt configuration was found in primate FV (pre)genomic RNAs

Amdoparvoviruses in small mammals: expanding our understanding of parvovirus diversity, distribution, and pathology

Many new viruses have been discovered recently, thanks in part to the advent of next-generation sequencing technologies. Among the Parvoviridae, three novel members of the genus Amdoparvovirus have been described in the last 4 years, expanding this genus that had contained a single species since its discovery, Aleutian mink disease virus. The increasing number of molecular and epidemiological studies on these viruses around the world also highlights the growing interest in this genus. Some aspects of amdoparvoviruses have been well characterized, however, many other aspects still need to be elucidated and the most recent reviews on this topic are outdated. We provide here an up-to-date overview of what is known and what still needs to be investigated about these scientifically and clinically relevant animal viruses

The effect of three composite layering techniques evaluated for margin integrity in Class-II restorations using four adhesives, in vitro.

Titelblatt und Inhaltsverzeichnis Einleitung Literaturübersicht Fragestellung Material und Methode Ergebnisse Diskussion Schlussfolgerung Zusammenfassung Summary Literaturverzeichnis AnhangDas Ziel der vorliegenden In-vitro-Studie war, den Einfluss von drei verschiedenen Schichttechniken in Kombination mit vier Adhäsivsystemen auf die marginale Integrität zervikal dentinbegrenzter Klasse-II- Kompositrestaurationen zu beurteilen. In 96 menschliche extrahierte Prämolaren wurde eine standardisierte Klasse-II- Kavität mit einer Dentinstufe präpariert (H: 6 mm, B: 4 mm, T: 1,5-2 mm). Folgende vier Adhäsivsysteme der vierten, fünften, sechsten und siebten Generation wurden verwendet: OptiBond FL (OPT), Excite (EXC), Clearfil SE Bond (CSE) und Adper Prompt-L-Pop (PLP). Die Applikation des Komposits Filtek Z250 erfolgte in einer von drei Schichttechniken: Horizontal-, Diagonal- und Zentripetal-Schichttechnik (Schalentechnik), wobei jeweils 4 Inkremente in die Kavität eingebracht wurden. Die Kompositfüllungen wurden unter direkter Sicht ausgearbeitet und die Zähne anschließend für 21 Tage in Wasser gelagert. Vor (T1) und nach (T2) einer thermischen Wechselbelastung (5° - 55°C, 2000 Zyklen) und nach einer mechanischen Belastung (T3) im Münchener Kausimulator (50 N, 125.000 Zyklen) wurden Replika angefertigt. Diese wurden mit Epoxidharz ausgegossen und mit Gold besputtert, so dass eine Beurteilung der Randqualität nach definierten Kriterien bei 200facher Vergrößerung im Rasterelektronenmikroskop (REM) mittels quantitativer Randanalyse erfolgen konnte. Zur statistischen Auswertung wurden der KRUSKAL-WALLIS-Test mit BONFERRONI-Adjustierung und der WILCOXON-Test durchgeführt (p< 0,05). Hinsichtlich der zur Füllungslegung angewandten Schichttechniken wurden keine statistisch signifikanten Unterschiede (p>0,05) der Randqualität im Dentin festgestellt. Infolgedessen wurden die Daten der verschiedenen Schichttechniken für das jeweilige Adhäsivsystem gepoolt. Bezüglich der verwendeten Adhäsivsysteme - einschließlich der verschiedenen Auswertungsbereiche zervikaler horizontaler Rand und zervikale Kavitätenecke - konnten jedoch nach T1, T2 und T3 signifikante Unterschiede (p<0,05) der Randqualität ermittelt werden. Zu den drei Evaluationszeitpunkten (T1/T2/T3) lagen die Medianwerte des prozentualen Anteils an kontinuierlichen Rändern ( Note 1 ) im Bereich des horizontal verlaufenden Füllungsrands bei: OPT (100 %/ 100 %/ 87,5 %), CSE (100 %/ 100 %/ 86,9 %), EXC (81,2 %/ 63,3 %/ 17,8 %) und PLP (82,3 %/ 64,7 %/ 27,7 %). Aufgrund der Ergebnisse konnte für die getesteten Adhäsivsysteme folgende Rangordnung erstellt werden: OPT = CSE > EXC = PLP. Zusammenfassend kann man feststellen, dass keine der drei Schichttechniken die Randqualität für das jeweils angewandte Adhäsivsystem signifikant beeinflusst. Für das Etch&amp;Rinse-Drei-Schritt-System; und für das selbstkonditionierende Zwei-Schritt-System wurden signifikant bessere Randqualitäten ermittelt, als für das Etch&amp;Rinse-Zwei-Schritt-System; und das selbstkonditionierende All-in-one -Adhäsiv.Objective: To evaluate the influence of three different layering techniques on gingival margin integrity of Class-II composite resin restorations with cervical margins in dentin using four adhesive systems, in vitro. Methods: 96 extracted human premolars were prepared for standardized Class-II restorations (6 mm high with cervical margins in dentin, 4 mm wide and 1.5-2 mm deep). Four adhesive systems, OptiBond FL (OPT), Excite (EXC), Clearfil SE Bond (CSE) and Adper Prompt L-Pop (PLP), so-called 4th, 5th, 6th and 7th generation adhesives respectively, were used.The restorative Filtek Z250 was placed by three different layering techniques: 4 horizontal layers, 4 diagonal layers and a centripetal technique where the initial composite layer is applied against the matrix band followed by 3 diagonal layers. Gingival margins were evaluated after 21 days of water storage (TM1), then after thermocycling of 2000 cycles 5° to 55°C (TM2), and finally after an additional mechanical loading of 125,000 cycles with 50 N (TM3). Quantitative SEM margin analysis was performed at a magnification of 200X using replicas, produced at each stage, to quantify gap formation, irregularities and the continuity of margins. The statistical evaluation was performed by using the KRUSKAL-WALLIS-test with a BONFERRONI-adjustment and the WILCOXON-test (p<0.05). Results: No statistically significant differences (p>0.05) were found for the results of the three different layering techniques at any time of evaluation. Therefore the data for the different layering techniques could be pooled for the respective adhesive system. Regarding the used adhesive systems including different areas of analysis horizontal cervical margin and cervical cavity angle significant differences (p<0.05) of the margin quality at TM1, TM2 and TM3 were found. The results for the three evaluations (TM1/TM2/TM3) showed the following median values of continuous margins in % of the entire horizontal cervical margin lengths in dentin: OPT (100 %/ 100 %/ 87,5 %), CSE (100 %/ 100 %/ 86,9 %), EXC (81,2 %/ 63,3 %/ 17,8 %) and PLP (82,3 %/ 64,7 %/ 27,7 %). From the results for the adhesive systems the following ranking was evaluated: OPT = CSE > EXC = PLP. Conclusion: There is no effect on the integrity of cervical margins of Class- II restorations by the three different layering techniques, however, the three-step etch&amp;rinse; and the two-step self-etching adhesive systems show significantly better results than the two-step etch&amp;rinse; and the tested all-in-one adhesive