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ABSTRACT: Foamy Viruses (FVs) are unique among retroviruses in performing genome reverse transcription (RTr) late in replication resulting in an infectious DNA genome and also in their unusual Pol biosynthesis and encapsidation strategy. In addition, FVs display only very limited Gag and Pol processing by the viral protease (PR) during particle morphogenesis and disassembly, both thought to be crucial for viral infectivity. Here, we report the generation of functional prototype FV (PFV) particles from mature or partially processed viral capsid and enzymatic proteins with infectivity levels of up to 20% of wild type. Analysis of protein and nucleic acid composition, as well as infectivity, of virions generated from different Gag and Pol combinations (including both expression-optimized and authentic PFV ORFs) revealed that precursor processing of Gag, but not Pol, during particle assembly is essential for production of infectious virions. Surprisingly, when processed Gag (instead of Gag precursor) was provided together with PR-deficient Pol precursor during virus production, infectious, viral DNA-containing particles were obtained, even when using different vector or proviral expression systems. Although virion infectivity was reduced to 0.5-2% relative to respective parental constructs, this overturns the current dogma in the FV literature that viral PR activity is absolutely essential at some point during target cell entry. Furthermore, it demonstrates that viral PR-mediated Gag precursor processing during particle assembly initiates intra-particle RTr. Finally, it shows that RT and integrase are enzymatically active in the Pol precursor within the viral capsid, thus enabling productive host cell infection.
Journal of Virology 01/2013; · 5.40 Impact Factor
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ABSTRACT: Recently, insertion of immuno-modulatory or anti-apoptotic genes into corneal endothelial cells (HCECs) came into focus. Basic FGF-2 occurs in one secreted (low molecular weight, LMW, 18 kD) and four nuclear (high molecular weight, HMW, 22-34 kD) isoforms. HMW isoforms are known differentiation and survival factors, while LMW FGF-2 is a known mitogen. The effect of FGF-2 overexpression of each of the five known isoforms on HCEC cell survival after lentiviral gene transfer in different culture media was investigated.
Cells were transduced with lentiviral vectors encoding for each of the five FGF-2 isoforms. Transduction efficiency and expression of individual FGF-2 isoforms was assessed by marker gene transfer and western blotting. Primary HCECs were cultured and transduced in four different media previously described for HCEC cultivation or corneal organ cultivation. Cytotoxic effect of virus infection and a possible rescue effect of FGF-2 overexpression were determined by resazurin conversion assay.
Transduction with FGF-2 encoding lentiviral vectors resulted in overexpression of the respective isoform in all tested cell populations. Western blotting after total cell lysis proved nuclear localization of transgenic HMW isoforms. Overexpression of HMW FGF-2-especially 34 kD FGF-2-reduced lentiviral cytotoxicity, while overexpression of LMW FGF-2 aggravated viral cytotoxicity.
Cytotoxicity of lentiviral gene transfer in corneal endothelial cells may be reduced by using bicistronic vectors that encode for the target gene and the 34-kD isoform of human FGF-2. Such cotransduction of a survival factor may increase cell survival after gene transfer, thereby improving gene therapeutic approaches.
Investigative ophthalmology & visual science 05/2012; 53(6):3207-14. · 3.43 Impact Factor
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ABSTRACT: To evaluate retroviral vectors as a tool to transduce normal human corneal endothelial cells (HCECs) and to optimize transduction to increase gene transfer efficiency.
Enhanced green fluorescent protein (EGFP) encoding retroviral vectors based on HIV-1 or murine leukemia virus (MLV), pseudotyped with either vesicular stomatitis virus glycoprotein (VSV-G) or a modified foamy virus envelope protein (FV Env), and prototype foamy virus (PFV) were produced. Transduction was performed in four HCEC culture media that were previously described for specific cultivation of HCECs or organ culture of donor corneas, namely enriched HCEC growth medium F99(HCEC), its unsupplemented basal medium F99, MEM + 2% fetal calf serum (FCS) (MEM), and Human Endothelial-SFM (SFM). Transduction efficiency was evaluated by marker gene transfer assay, and cytotoxic effects of virus infection were evaluated by means of resazurin conversion assay.
PFV- and HIV-1-based vectors showed superior transduction efficiency compared with MLV-based vectors. Pseudotyping with a modified FV Env increased transduction efficiency compared with pseudotyping with VSV-G. In medium SFM, transduction efficiency of PFV, HIV-1-/FV Env, and MLV-based vectors was markedly reduced compared with the other culture media. When cells were cultured in F99-based media, cell viability was reduced by retroviral transduction compared with uninfected or mock infected controls, but remained unaffected when cells were cultured in SFM and was even increased when cells were cultured in MEM.
HIV-1-based vectors pseudotyped with FV Env can efficiently be used to transduce primary HCECs in vitro. However, transduction efficiency is dependent on culture conditions and impairs metabolic activity and viability of HCECs in vitro.
Investigative ophthalmology & visual science 07/2011; 52(9):6807-13. · 3.43 Impact Factor
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ABSTRACT: Foamy virus (FV) capsid proteins have few lysines. Basic residues are almost exclusively represented by arginines indicating positive selective pressure. To analyze the possible functions of this peculiarity, we mutated an infectious molecular clone of the prototypic FV (PFV) to harbor lysines in the Gag protein at arginine-specifying positions and analyzed various aspects of the FV replication cycle. The majority of mutants replicated equally as well in permanent cell cultures as the original wild-type (wt) virus and were genetically stable in gag upon 10 cell-free passages. With respect to the features of late reverse transcription, nucleic acid content, and infectiousness of the virion DNA genome, the majority of mutants behaved like the wt. Several mutants of PFV were ubiquitinated in Gag but unable to generate virus-like particles (VLPs) or to undergo pseudotyping by a heterologous envelope. Using primary cells, however, a replicative disadvantage of the majority of mutants was disclosed. This disadvantage was enhanced upon interferon (IFN) treatment. We found no evidence that the lysine-bearing gag mutants showed more restriction than the wt virus by tetherin (CD317) or Trim5α. A single lysine in PFV Gag was found to be nonessential for transient replication in permanent cell culture if replaced by an arginine residue. Upon replication in primary cells, even without IFN treatment, this mutant was severely impaired, indicating the importance of specifying at least this lysine residue in PFV Gag. The paucity of lysines in FV Gag proteins may be a consequence of preventing proteasomal Gag degradation.
Journal of Virology 02/2011; 85(8):3986-95. · 5.40 Impact Factor
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ABSTRACT: Many flavonoids, a major group of phenolic plant-derived secondary metabolites, are known to possess estrogen-like bioactivities. However, little is known about their estrogenic properties in the central nervous system due to the lack of suitable cellular models expressing sufficient amounts of functional estrogen receptor beta (ERbeta). To overcome this deficit, we have created a cellular model, which is serotonergic in origin, to study properties of estrogenic substances by stably transducing RN46A-B14 cells derived from raphe nuclei region of the rat brain with a lentiviral vector encoding a human ERbeta. We clearly showed that the transgenic human ERbeta is a spontaneously expressed and a functional receptor. We have further assessed the estrogenicity of three different isoflavones and four different naringenin-type flavanones in this cell line utilizing a luciferase reporter gene assay. Genistein (GEN), Daidzein (DAI), Equol (EQ), Naringenin (NAR) and 8-prenylnaringenin (8-PN) showed strong estrogenic activity in a concentration-dependent manner as compared to 7-(O-prenyl)naringenin-4'-acetate (7-O-PN) which was only slightly estrogenic and 6-(1,1-dimethylallyl)naringenin (6-DMAN) that neither showed estrogenic nor anti-estrogenic activity in our model. All observed effects could be antagonized by the anti-estrogen fulvestrant. Moreover, co-treatment of cells with 17beta-estradiol (E2) and either GEN or DAI showed a slight additive effect as compared to EQ. On the other hand, 8-PN in addition to 7-O-PN, but not NAR and 6-DMAN, were able to slightly antagonize the responses triggered by E2. Our newly established cellular model may prove to be a useful tool in explicating basic physiological properties of ERbeta in the brain and may help unravel molecular and cellular mechanisms involved in serotonergic mood regulation by estrogen or potential plant-derived secondary metabolites.
The Journal of steroid biochemistry and molecular biology 06/2010; 120(4-5):208-17. · 2.66 Impact Factor
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Kristin Stirnnagel,
Daniel Lüftenegger,
Annett Stange,
Anka Swiersy,
Erik Müllers,
Juliane Reh, Nicole Stanke,
Arend Grosse,
Salvatore Chiantia,
Heiko Keller,
Petra Schwille,
Helmut Hanenberg,
Hanswalter Zentgraf,
Dirk Lindemann
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ABSTRACT: The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive.
In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction.
We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV--host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines resistant to Prototype Foamy Virus Env-mediated gene transfer. Interestingly, both cell lines still displayed FV Env-dependent attachment of fluorescent retroviral particles, implying a post-binding block potentially due to lack of putative FV entry cofactors. These cell lines might ultimately lead to the identification of the currently unknown ubiquitous cellular entry receptor(s) of FVs.
Retrovirology 01/2010; 7:45. · 6.47 Impact Factor
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ABSTRACT: Foamy viruses (FVs) are unconventional retroviruses with a replication strategy that is significantly different from orthoretroviruses and bears some homology to that of hepadnaviruses. Although some cellular proteins, such as APOBEC3, have been reported to block FVs, no restriction by Trim5alpha has been described to date. The sensitivity of three FV isolates of human-chimpanzee or prototypic (PFV), macaque (SFVmac), and feline (FFV) origin to a variety of primate Trim5alphas was therefore tested. PFV and SFVmac were restricted by Trim5alphas from most New World monkeys, but not from other primates, whereas FFV-based vectors were restricted by Trim5alphas from the great apes gorilla and orangutan. Trim5alphas from Old World monkeys did not restrict any FV isolate tested. Capuchin Trim5alpha was unique, as it restricted SFVmac and FFV but not PFV. Trim5alpha specificity for FVs was determined by the B30.2 domain, interestingly involving, in some instances, the same residues of the variable regions previously implicated as major determinants for human immunodeficiency virus type 1 restriction. FVs with chimeric Gags were made to map the viral determinants of sensitivity to restriction. The N-terminal half of the Gag molecule was found to contain the regions that control susceptibility. This region most likely corresponds to the capsid of conventional retroviruses. Due to their unique replication strategy, FVs should provide a valuable new system to examine the mechanism of retroviral restriction by Trim5alpha.
Journal of Virology 07/2008; 82(11):5429-39. · 5.40 Impact Factor
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ABSTRACT: Foamy virus (FV) particle egress is unique among retroviruses because of its essential requirement for Gag and Env coexpression for budding and particle release. The FV glycoprotein undergoes a highly unusual biosynthesis resulting in the generation of three particle-associated, mature subunits, leader peptide (LP), surface (SU), and transmembrane (TM), derived from a precursor protein by posttranslational proteolysis mediated by furin or furinlike proteases. Previously at least three LP products of different molecular weights were detected in purified FV particles. Here we demonstrate that the higher-molecular-weight forms gp28LP and gp38LP are ubiquitinated variants of the major gp18LP cleavage product, which has a type II membrane topology. Furthermore, we show that all five lysine residues located within the N-terminal 60-amino-acid cytoplasmic domain of gp18LP can potentially be ubiquitinated, however, there seems to be a preference for using the first three. Inactivation of ubiquitination sites individually resulted in no obvious phenotype. However, simultaneous inactivation of the first three or all five ubiquitination sites in gp18LP led to a massive increase in subviral particles released by these mutant glycoproteins that were readily detectable by electron microscopy analysis upon expression of the ubiquitination-deficient glycoprotein by itself or in a proviral context. Surprisingly, only the quintuple ubiquitination mutant showed a two- to threefold increase in single-cycle infectivity assays, whereas all other mutants displayed infectivities similar to that of the wild type. Taken together, these data suggest that the balance between viral and subviral particle release of FVs is regulated by ubiquitination of the glycoprotein LP.
Journal of Virology 01/2006; 79(24):15074-83. · 5.40 Impact Factor
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ABSTRACT: The prototype foamy virus (PFV) glycoprotein, which is essential for PFV particle release, displays a highly unusual biosynthesis, resulting in posttranslational cleavage of the precursor protein into three particle-associated subunits, i.e., leader peptide (LP), surface (SU), and transmembrane (TM). Glycosidase digestion of metabolically labeled PFV particles revealed the presence of N-linked carbohydrates on all subunits. The differential sensitivity to specific glycosidases indicated that all oligosaccharides on LP and TM are of the high-mannose or hybrid type, whereas most of those attached to SU, which contribute to about 50% of its molecular weight, are of the complex type. Individual inactivation of all 15 potential N-glycosylation sites in PFV Env demonstrated that 14 are used, i.e., 1 out of 2 in LP, 10 in SU, and 3 in TM. Analysis of the individual altered glycoproteins revealed defects in intracellular processing, support of particle release, and infectivity for three mutants, having the evolutionarily conserved glycosylation sites N8 in SU or N13 and N15 in the cysteine-rich central "sheets-and-loops" region of TM inactivated. Examination of alternative mutants with mutations affecting glycosylation or surrounding sequences at these sites indicated that inhibition of glycosylation at N8 and N13 most likely is responsible for the observed replication defects, whereas for N15 surrounding sequences seem to contribute to a temperature-sensitive phenotype. Taken together these data demonstrate that PFV Env and in particular the SU subunit are heavily N glycosylated and suggest that although most carbohydrates are dispensable individually, some evolutionarily conserved sites are important for normal Env function of FV isolates from different species.
Journal of Virology 07/2005; 79(12):7664-72. · 5.40 Impact Factor
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ABSTRACT: Foamy viruses (FV) are unusual among retroviruses since they require both Gag and Env structural proteins for particle egress. Recently significant progress has been made towards the mechanistic understanding of the viral release process, in particular that of retroviruses, and the viral domains and cellular pathways involved. However little is currently known about domains of FV structural proteins and cellular proteins engaged in this process. By mutational analysis of sequence motifs in prototype FV (PFV) Gag, bearing homology to known late assembly (L) domains, a PSAP motif with L domain function that was functionally interchangeable by heterologous L domains was identified. In contrast the inactivation of a PPPI motif had no significant influence on PFV particle release, although mutant viral particles displayed reduced infectivity. Similarly mutation of an evolutionary conserved YXXL motif revealed no classical L-domain function but resulted in release of noninfectious viruslike particles. Biochemical and electron microscopy analysis demonstrated that these mutant particles incorporated all viral structural proteins but contained aberrantly capsid structures, suggesting a role in capsid assembly for this PFV Gag sequence motif. In line with the mutational analysis, overexpression of dominant negative (DN) mutants and wild-type TSG101 but not the DN mutant of AIP-1/ALIX reduced PFV particle release and infectivity. Furthermore, DN mutants of Vps4A, Vps4B, and CHMP3 inhibited PFV egress and infectivity. Taken together these results demonstrate that PFV, like other viruses, requires components of the vacuolar protein sorting (VPS) machinery for egress and enters the VPS pathway through interaction with TSG101.
Journal of Virology 06/2005; 79(9):5466-76. · 5.40 Impact Factor
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ABSTRACT: Analogous to cellular glycoproteins, viral envelope proteins contain N-terminal signal sequences responsible for targeting them to the secretory pathway. The prototype foamy virus (PFV) envelope (Env) shows a highly unusual biosynthesis. Its precursor protein has a type III membrane topology with both the N and C terminus located in the cytoplasm. Coexpression of FV glycoprotein and interaction of its leader peptide (LP) with the viral capsid is essential for viral particle budding and egress. Processing of PFV Env into the particle-associated LP, surface (SU), and transmembrane (TM) subunits occur posttranslationally during transport to the cell surface by yet-unidentified cellular proteases. Here we provide strong evidence that furin itself or a furin-like protease and not the signal peptidase complex is responsible for both processing events. N-terminal protein sequencing of the SU and TM subunits of purified PFV Env-immunoglobulin G immunoadhesin identified furin consensus sequences upstream of both cleavage sites. Mutagenesis analysis of two overlapping furin consensus sequences at the PFV LP/SU cleavage site in the wild-type protein confirmed the sequencing data and demonstrated utilization of only the first site. Fully processed SU was almost completely absent in viral particles of mutants having conserved arginine residues replaced by alanines in the first furin consensus sequence, but normal processing was observed upon mutation of the second motif. Although these mutants displayed a significant loss in infectivity as a result of reduced particle release, no correlation to processing inhibition was observed, since another mutant having normal LP/SU processing had a similar defect.
Journal of Virology 01/2005; 78(24):13865-70. · 5.40 Impact Factor
