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1.
J Gen Virol ; 87(Pt 5): 1339-1347, 2006 May.
Article in English | MEDLINE | ID: mdl-16603537

ABSTRACT

Integration-site selection by retroviruses and retroviral vectors has gained increased scientific interest. Foamy viruses (FVs) constitute a unique subfamily (Spumavirinae) of the family Retroviridae, for which the integration pattern into the human genome has not yet been determined. To accomplish this, 293 cells were transduced with FV vectors and the integration sites into the cellular genome were determined by a high-throughput method based on inverse PCR. For comparison, a limited number of murine leukemia virus (MLV) and human immunodeficiency virus (HIV) integration sites were analysed in parallel. Altogether, 628 FV, 87 HIV and 141 MLV distinct integration sites were mapped to the human genome. The sequences were analysed for RefSeq genes, promoter regions, CpG islands and insertions into cellular oncogenes. Compared with the integration-site preferences of HIV, which strongly favours active genes, and MLV, which favours integration near transcription-start regions, our results indicate that FV integration has neither of these preferences. However, once integration has occurred into a transcribed region of the genome, FVs tend to target promoter-close regions, albeit with less preference than MLV. Furthermore, our study revealed a palindromic consensus sequence for integration, which was centred on the virus-specific, four-base-duplicated target site. In summary, it is shown that the integration pattern of FVs appears to be unique compared with those of other retroviral genera.


Subject(s)
Spumavirus/physiology , Cell Line , Genome/genetics , Humans , Kidney , Molecular Sequence Data , Promoter Regions, Genetic/genetics , Virus Integration
2.
J Virol ; 79(19): 12464-76, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16160174

ABSTRACT

Among the Retroviridae, foamy viruses (FVs) exhibit an unusual way of particle assembly and a highly specific incorporation of envelope protein into progeny virions. We have analyzed deletions and point mutants of the prototypic FV gag gene for capsid assembly and egress, envelope protein incorporation, infectivity, and ultrastructure. Deletions introduced at the 3' end of gag revealed the first 297 amino acids (aa) to be sufficient for specific Env incorporation and export of particulate material. Deletions introduced at the 5' end showed the region between aa 6 and 200 to be dispensable for virus capsid assembly but required for the incorporation of Env and particle egress. Point mutations were introduced in the 5' region of gag to target residues conserved among FVs from different species. Alanine substitutions of residues in a region between aa 40 and 60 resulted in severe alterations in particle morphology. Furthermore, at position 50, this region harbors the conserved arginine that is presumably at the center of a signal sequence directing FV Gag proteins to a cytoplasmic assembly site.


Subject(s)
Gene Products, gag/genetics , Gene Products, gag/physiology , Spumavirus/genetics , Spumavirus/physiology , Virus Assembly , Amino Acid Sequence , Amino Acid Substitution , Cell Line , Gene Products, gag/chemistry , Humans , Microscopy, Electron , Molecular Sequence Data , Point Mutation , Protein Structure, Tertiary , Sequence Deletion , Spumavirus/pathogenicity , Spumavirus/ultrastructure , Virion/ultrastructure
3.
Cancer Gene Ther ; 12(12): 947-53, 2005 Dec.
Article in English | MEDLINE | ID: mdl-15905857

ABSTRACT

A replication competent foamy virus derived retroviral vector expressing suicide genes has been constructed and characterized in vitro. Here we used vectors expressing the purine nucleoside phosphorylase (FOV-7/pnp), the nitroreductase (FOV-7/ntr), or the thymidine kinase (FOV-7/tk) suicide gene in an in vivo athymic (nude) mice/human glioblastoma tumor model. Gliomas were induced by subcutanous injection of U87 tumor cells. The virus vector was injected when the tumor became visible. Mice with vector virus-injected tumors were treated with the respective prodrug. The treatment resulted in significant inhibition of tumor growth. Surprisingly, in mice with vector virus-injected tumors without prodrug treatment a similar suppression of tumor growth was observed. In 65% (pnp vector), 75% (ntr vector) and 37% (tk vector) of these mice the tumors stopped growing or vanished and the animals remained tumor free for the 25 weeks of the experiment, whereas all mice of the control groups had to be killed because of the tumor growth. In control experiments, the suppression of tumor growth could also be observed when wild-type foamy virus was injected instead of the suicide gene-transducing vectors. Similar results were obtained using the nude mice/G59 human glioblastoma tumor model. In conclusion, the experiments demonstrate an oncolytic activity of foamy virus replication in a nude-mice glioblastoma xenograft tumor model. The analysis of vector virus DNA by PCR revealed that the vector persisted in different organs of the animals irrespective of the use of a prodrug or the elimination of a tumor.


Subject(s)
Genes, Transgenic, Suicide/genetics , Genetic Therapy , Genetic Vectors/genetics , Neoplasms/genetics , Neoplasms/therapy , Spumavirus/genetics , Animals , Cell Line, Tumor , Cell Proliferation , Cricetinae , DNA, Viral/genetics , Humans , Mice , Mice, Nude , Neoplasms/pathology , Survival Rate , Virus Replication , Xenograft Model Antitumor Assays
4.
J Virol ; 79(11): 7005-13, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15890940

ABSTRACT

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.


Subject(s)
Gene Products, pol/genetics , Gene Products, pol/physiology , RNA, Viral/genetics , RNA, Viral/metabolism , Spumavirus/genetics , Spumavirus/physiology , Cell Line , Chromosome Mapping , Genes, Viral , Humans , Spumavirus/growth & development , Viral Proteins/genetics , Viral Proteins/physiology , Virion/genetics , Virion/growth & development , Virion/physiology , Virus Assembly/genetics , Virus Assembly/physiology
5.
J Virol ; 79(9): 5466-76, 2005 May.
Article in English | MEDLINE | ID: mdl-15827161

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.


Subject(s)
Gene Products, gag/genetics , Spumavirus/genetics , Amino Acid Motifs/genetics , Amino Acid Sequence , Cell Line , Cell Line, Tumor , Consensus Sequence , Gene Products, gag/metabolism , Humans , Molecular Sequence Data , Sequence Alignment , Spumavirus/pathogenicity , Spumavirus/ultrastructure , Virus Replication
6.
Genesis ; 39(2): 94-9, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15170694

ABSTRACT

Here we describe the efficient generation of eGFP-transgenic rats using a lentiviral approach. Analysis of the founder generation demonstrated that 46% of the offspring had stably integrated the provirus into the genome and of those 92% expressed eGFP in all blood-derived leukocytes. In contrast to their offspring, all founder rats were mosaic with regard to eGFP-expression, suggesting delayed viral transduction after injection. The expression level of eGFP in the F1 generation is influenced by and segregates with the site of proviral integration. Interestingly, a single copy of the transgene is sufficient for reliable detection by flow cytometry, irrespective of the leukocyte subtype analyzed. Adoptive transfer of purified CD4(+) T-lymphocytes from transgenic rats and subsequent reisolation from various organs further demonstrated that expression of the lentiviral transgene is maintained in a foreign host and therefore allows for efficient tracking of transferred cells. Taken together, lentivirally generated eGFP-transgenic rats are a powerful tool for various applications in immunology and presumably also many other fields.


Subject(s)
Animals, Genetically Modified/metabolism , Green Fluorescent Proteins/metabolism , Lentivirus/genetics , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/metabolism , Female , Flow Cytometry , Gene Transfer Techniques , Genetic Vectors , Leukocytes/metabolism , Male , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Transduction, Genetic , Transgenes/physiology
7.
J Virol ; 78(5): 2472-7, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14963145

ABSTRACT

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5' end of the U3 region in the 3' LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.


Subject(s)
Spumavirus/physiology , Virus Integration , Animals , Base Sequence , Cell Line , Cricetinae , Genetic Vectors/genetics , Humans , Mutation/genetics , Proviruses/genetics , Proviruses/physiology , Recombination, Genetic/genetics , Spumavirus/genetics , Terminal Repeat Sequences/genetics , Virus Integration/genetics , Virus Replication
8.
J Virol ; 77(21): 11324-31, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14557618

ABSTRACT

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.


Subject(s)
Genome, Viral , Spumavirus/physiology , Virus Replication , Animals , Cats , Cell Line , Cricetinae , DNA, Viral/genetics , DNA, Viral/metabolism , Humans , Mice , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Viral/genetics , RNA, Viral/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Spumavirus/genetics , Spumavirus/pathogenicity , Transfection , Virion/metabolism , Virion/pathogenicity
9.
J Virol ; 77(21): 11855-8, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14557671

ABSTRACT

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.


Subject(s)
Gene Products, env/genetics , Genome, Viral , Retroelements/genetics , Spumavirus/genetics , Spumavirus/physiology , Cell Fusion , Cell Line , Genetic Markers , Genetic Vectors , HeLa Cells/virology , Humans , Mutation , Transfection
10.
Hum Gene Ther ; 14(6): 509-19, 2003 Apr 10.
Article in English | MEDLINE | ID: mdl-12718762

ABSTRACT

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.


Subject(s)
Antigens, CD34/analysis , Genetic Vectors , Hematopoietic Stem Cell Transplantation , Retroviridae/genetics , Spumavirus/genetics , Transduction, Genetic , Animals , Cell Line , Fetal Blood/cytology , Green Fluorescent Proteins , HIV-1/genetics , Hematopoietic Stem Cells/chemistry , Humans , Lentivirus/genetics , Leukemia Virus, Murine/genetics , Luminescent Proteins/genetics , Mice , Mice, Inbred NOD , Mice, SCID
11.
J Virol ; 76(19): 10069-73, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12208988

ABSTRACT

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.


Subject(s)
Capsid/metabolism , Gene Products, pol/metabolism , RNA, Viral/physiology , Spumavirus/physiology , Virus Assembly , Virion/physiology
13.
J Virol ; 76(8): 3774-83, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11907217

ABSTRACT

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.


Subject(s)
Genetic Vectors , Primates , Spumavirus/genetics , Virus Assembly , 5' Untranslated Regions/genetics , Animals , Cell Line , Gene Products, gag/genetics , Gene Products, gag/metabolism , Gene Products, pol/genetics , Gene Products, pol/metabolism , Humans , Plasmids/genetics , RNA, Viral/metabolism
14.
J Gen Virol ; 81(Pt 12): 2941-2947, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11086125

ABSTRACT

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.


Subject(s)
Cell Nucleus/metabolism , Gene Products, pol/metabolism , Primates/virology , Spumavirus , 3T3 Cells , Active Transport, Cell Nucleus , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Blotting, Western , Cell Line , Cell Nucleus/virology , Cricetinae , Fluorescent Antibody Technique , Gene Products, gag/genetics , Gene Products, gag/immunology , Gene Products, gag/metabolism , Gene Products, pol/genetics , Gene Products, pol/immunology , Integrases/genetics , Integrases/immunology , Integrases/metabolism , Mice , Mice, Inbred C57BL , Nuclear Localization Signals , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/immunology , RNA-Directed DNA Polymerase/metabolism , Ribonuclease H/genetics , Ribonuclease H/immunology , Ribonuclease H/metabolism , Spumavirus/enzymology , Spumavirus/genetics , Spumavirus/metabolism , Transfection
15.
Pathol Oncol Res ; 3(1): 62-67, 1997.
Article in English | MEDLINE | ID: mdl-11173628

ABSTRACT

Infection with the human immunodeficiency virus (HIV) causes gradual depletion of CD4+ T helper lymphocytes and destruction of the lymphoid tissue, which ultimately leads to a fatal defect of the cellular immune system. Paramount to the understanding of the pathogenesis of HIV infection is to elucidate the mechanism which underlies the loss of T helper cells. Various ideas have been proposed in order to explain this issue. Several hypotheses have focused on the role of the envelope glycoprotein in this process. This review summarizes the data obtained and concepts proposed regarding the involvement of the HIV glycoprotein in the pathology of CD4+ T cell depletion.

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