Biomedical subjects

Axel Rethwilm

Publications and source records attributed to Axel Rethwilm.

27 records · Page 2Linked to original sources

PubMed2003

Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells.

The use of recombinant vectors based on wild-type viruses that are absent in humans and are not associated with any disease in their natural animal hosts or in accidentally infected humans would add an additional level of safety for human somatic gene therapy approaches. These criteria are fulfilled by foamy viruses (FVs), a family of complex retroviruses whose members are widely found among mammals and are apathogenic in all hosts. Here, we show by comparison of identically designed vector constructs that recombinant retroviral vectors based on FVs were as efficient as lentiviral vectors in transducing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice repopulating human CD34(+) cord blood (CB) cells. The FV vector was able to achieve gene transfer levels up to 84% of engrafted human cells in a short overnight transduction protocol. In contrast, without prestimulation of the target cells, a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector pseudotyped with gibbon ape leukemia virus envelope (GALV Env) was nearly as inefficient as murine leukemia virus (MLV)-based oncoretroviral vectors in transducing NOD/SCID repopulating cells. The same HIV vector pseudotyped with the vesicular stomatitis virus glycoprotein G (VSV-G) achieved high marking efficiency. Clonality analysis of bone marrow samples showed oligoclonal hematopoiesis with single to multiple insertions per cell, both for FV and HIV vectors. These data demonstrate that vectors based on FVs warrant further investigation and development for medical use.

Animals↗

PubMed2003

Evaluation of four commercially available Epstein-Barr virus enzyme immunoassays with an immunofluorescence assay as the reference method.

Four commercially available enzyme immunoassays (EIAs) (Novitec, Biotest, Virotech, and DiaSorin) were evaluated, with an indirect immunofluorescence assay as the reference method, for Epstein-Barr virus (EBV) VCA (viral capsid antigen) immunoglobulin G (IgG), VCA IgM, or EBNA (EBV nuclear antigen) IgG at three different locations (Homburg, Stuttgart, and Dresden). Serum samples from 66 immunocompetent patients with infectious mononucleosis, 73 patients without prior EBV infection, and 96 patients with past EBV infections and 29 serum samples with possible cross-reactions to other herpesviruses were included. In addition, 25 samples from an extensively pretested panel that is commercially available (Boston Biomedica) were tested. Each sample was tested at only one location. The four EIAs varied considerably in performance. When analyzing for EBV diagnosis, the Novitec assay performed the best, with 4.9% discrepant diagnoses, followed by the Biotest, Virotech, and DiaSorin assays, with 6.8, 11.7, and 14.0% discrepant diagnoses, respectively. On the basis of single-parameter analysis, the Novitec assay also showed the lowest number of discrepant results, with 3.5%, compared with the Virotech, Biotest, and DiaSorin assays, which produced 5.4, 6.4, and 8.6% discrepant results, respectively. VCA assays using affinity-purified native antigens performed better than assays with recombinant or synthetic antigens. The synthetic EBNA-1s showed the lowest concordance with the reference compared to recombinant p72. Commercially available EBV EIAs differed considerably in performance; however, some proved to be reliable and convenient alternatives to the indirect immunofluorescence assay for routine diagnostics. Native antigens, rather than synthetic peptides, are favored for EBV serology testing.

Antigens, Viral↗

PubMed2003

Replication-competent hybrids between murine leukemia virus and foamy virus.

Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae, orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope (env) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers.

Amino Acid Sequence↗

PubMed2003

Feline foamy virus genome and replication strategy.

Crucial aspects of the foamy virus (FV) replication strategy have so far only been investigated for the prototypic FV (PFV) isolate, which is supposed to be derived from nonhuman primates. To study whether the unusual features of this replication pathway also apply to more-distantly related FVs, we constructed feline FV (FFV) infectious molecular clones and vectors. It is shown by quantitative RNA and DNA PCR analysis that FFV virions contain more RNA than DNA. Full-length linear DNA was found in extracellular FFV by Southern blot analysis. Similar to PFV, azidothymidine inhibition experiments and the transfection of nucleic acids extracted from extracellular FFV indicated that DNA is the functional relevant FFV genome. Unlike PFV, no evidence was found indicating that FFV recycles its DNA into the nucleus.

Animals↗

PubMed2003

Retrotransposition and cell-to-cell transfer of foamy viruses.

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenröder, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.

Cell Fusion↗

PubMed2003

Foamy virus envelope glycoprotein-mediated entry involves a pH-dependent fusion process.

In general, enveloped viruses use two different entry strategies and are classified accordingly into pH-dependent and pH-independent viruses. Different members of the retrovirus family use one or the other strategy. Little is known about the uptake of foamy viruses (FV), a special group of retroviruses, into the target cells. In this study, we examined the pH dependence of FV entry by analyzing FV envelope glycoprotein (Env)-mediated infection of target cells with murine leukemia virus or FV vector pseudotypes in the presence of various lysosomotropic agents. Similar to vesicular stomatitis virus glycoprotein G (VSV-G)-mediated uptake, FV Env-mediated entry was inhibited by various lysosomotropic agents, suggesting a pH-dependent endocytic pathway. However, in contrast to its effect on VSV-G pseudotypes, chloroquine failed to reduce the infectivity of FV Env pseudotypes, implying that the pathway is different from that of VSV-G. Glycoproteins of various other FV species showed inhibition profiles similar to that of the prototype FV (PFV) Env. Analysis of the pH dependence of the FV Env-mediated fusion process in a cell-to-cell fusion assay revealed an induction of syncytium formation by a short exposure to acidic pH, peaking around pH 5.5. Interestingly, of all FV Env species analyzed, only the PFV Env had a significant fusion activity at neutral pH. Taken together, these data suggest a pH-dependent endocytic pathway for infection of target cells by FV.

Ammonium Chloride↗

PubMed2002

Pregenomic RNA is required for efficient incorporation of pol polyprotein into foamy virus capsids.

The foamy virus (FV) Pol polyprotein is translated independently of Gag from a spliced mRNA. This method of expression raises the question of how Pol is associated with the viral particle. Using a transient FV vector transfection system, it is shown that pregenomic RNA is required for efficient virion incorporation of functionally active Pol and that protein-protein interactions of Pol with Gag are not sufficient to complete particle assembly.

Capsid↗

PubMed2002

Improved primate foamy virus vectors and packaging constructs.

Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5' splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5' SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5' SD or an unrelated intron sequence was substituted for the FV 5' untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.

5' Untranslated Regions↗

PubMed2000

Primate foamy virus Pol proteins are imported into the nucleus.

Mouse monoclonal antibodies (MAbs) that specifically detect the 127 kDa Pol precursor and the 85 kDa reverse transcriptase/RNase H (RT/RN) or pr127 and the 40 kDa integrase (IN) in immunoblot and immunofluorescence assays (IFA) were used to investigate the subcellular localization of primate foamy virus (PFV) proteins. IFA of cells infected with PFV using the anti-Pol MAbs and rabbit anti-capsid (Gag) serum revealed that both the Gag and Pol proteins are transported into the nucleus. Transfection of cells with eukaryotic expression constructs for pr127(Pol), p85(RT/RN) and p40(IN) served to show Gag-independent subcellular localization of Pol proteins. Interestingly, not only the Pol precursor and IN molecules were found to be localized to the nucleus, but also the RT/RN subdomain. It is therefore suggested that PFV cores bear at least three separate nuclear localization signals, one in Gag and two in Pol. The latter appear to be localized to the two Pol subdomains.

3T3 Cells↗