Preparation of simian foamy virus type-1 vectors.

Foamy viruses (FVs) are nonpathogenic retroviruses that offer opportunities for efficient and safe gene transfer in various cell types from different species. These viruses have unique replication mechanisms that are distinct from other retroviruses, which may give an advantage to FV-mediated gene transfer. This protocol describes a method for simian foamy virus type-1 (SFV-1) vector preparation and concentration. A transient transfection of vector and packaging constructs allows generation of the SFV-1 vector with titers of 10(7)/mL. The vectors can be further concentrated by 100-200-fold without significant loss of vector titer.

The foamy virus genome remains unintegrated in the nuclei of G1/S phase-arrested cells, and integrase is critical for preintegration complex transport into the nucleus.

Foamy viruses are a member of the spumavirus subfamily of retroviruses with unique mechanisms of virus replication. Foamy virus replication is cell cycle dependent; however, the genome is found in the nuclei of cells arrested in the G(1)/S phase. Despite the presence of genome in the nuclei of growth-arrested cells, there is no viral gene expression, thus explaining its dependency on cell cycle. This report shows that the foamy virus genome remains unintegrated in G(1)/S phase-arrested cells. The foamy virus genome is detected by confocal microscopy in the nuclei of both dividing and growth-arrested cells. Alu PCR revealed foamy virus-specific DNA amplification from genomic DNA isolated in cycling cells at 24 h postinfection. In arrested cells no foamy virus DNA band was detected in cells harvested at 1 or 7 days after infection, and a very faint band that is significantly less than DNA amplified from cycling cells was observed at day 15. After these cells were arrested at the G(1)/S phase for 1, 7, or 15 days they were allowed to cycle, at which time foamy virus-specific DNA amplification was readily observed. Taken together, these results suggest that the foamy virus genome persists in nondividing cells without integrating. We have also established evidence for the first time that the foamy virus genome and Gag translocation into the nucleus are dependent on integrase in cycling cells, implicating the role of integrase in transport of the preintegration complex into the nucleus. Furthermore, despite the presence of a nuclear localization signal sequence in Gag, we observed no foamy virus Gag importation into the nucleus in the absence of integrase.

Accuracy estimation of foamy virus genome copying.

BACKGROUND: Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family. This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT). To investigate the accuracy of FV genome copying in vivo 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. RESULTS: We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to an in vivo error rate of approximately 4 x 10-4 per 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 bet 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 in vivo. 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 in vivo error rate of 1.1 x 10-5 per site per replication. In addition to the point mutations, we detected one 49 bp deletion within the analyzed 260000 nucleotides.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. CONCLUSION: 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.

Activity of TAR in inducible inhibition of HIV replication by foamy virus vector expressing siRNAs under the control of HIV LTR.

In this report we describe foamy virus vectors with conditional expression of short interfering RNAs (siRNAs) in HIV infected cells. Short hairpin RNAs (shRNAs) based on two targets in the 5' end of the untranslated region and one in the rev gene flanked with 5' and 3' microRNA 30 (miR30) sequences were synthesized and placed under the control of an HIV promoter for Tat-mediated expression. HIV permissive cells were transduced with foamy virus vectors containing each hybrid shRNA expression cassette and tested for their efficacy on the inhibition of HIV replication. Effective Tat dependent expression of the shRNAs, as well as GFP placed downstream each shRNA was evident. In addition the results show inhibition of HIV replication by greater than 98%. Interestingly, transduction of cells with a vector lacking an shRNA also revealed GFP expression in the presence of Tat with similar levels of inhibition of virus replication. When the TAR region was removed from this vector there was neither reduction in virus replication nor Tat-induced GFP expression. These results suggest that TAR in the vector, which Tat interacts to promote expression of the shRNA, is a potent inhibitor of virus replication. Previous studies with TAR regulated expression of antiviral genes ignore the contribution of TAR in the repression of virus replication. Interpretation of effective inhibition of HIV replication by antiviral genes located downstream of TAR while neglecting the efficacy of a potent repression by TAR is misleading.

Inhibition of simian immunodeficiency virus by foamy virus vectors expressing siRNAs.

Viral vectors available for gene therapy are either inefficient or suffer from safety concerns for human applications. Foamy viruses are non-pathogenic retroviruses that offer several unique opportunities for gene transfer in various cell types from different species. In this report, we describe the use of simian foamy virus type 1 (SFV-1) vector to examine the efficacy of therapeutic genes. Hairpin short-interfering RNA (siRNA) that targets the simian immunodeficiency virus (SIV) rev/env was placed under the control of the PolIII U6 snRNA promoter for expression and screened for silencing target genes using cognate target-reporter fusions. We have identified an effective siRNA (designated R2) which reduces the rev and env gene expression by 89% and 95%, respectively. Using the simian foamy virus type 1 (SFV-1) based vector, we delivered the PolIII expressed R2 siRNA into cultured cells and challenged with SIV. The results show that the R2 siRNA is a potent inhibitor of SIV replication as determined by p27 expression and reverse transcriptase assays. Vectors based on a non-pathogenic SFV-1 vector may provide a safe and efficient alternative to currently available vectors, and the SIV model will help devise protocols for effective anti-HIV gene therapy.

Transduction of umbilical cord blood CD34+ NOD/SCID-repopulating cells by simian foamy virus type 1 (SFV-1) vector.

Foamy viruses are nonpathogenic retroviruses that offer unique opportunities for gene transfer into various cell types including hematopoietic stem cells. We used a simian foamy virus type 1 vector (SFV-1) containing a LacZ reporter gene with a titer of 1-5 x 10(6) viral particles/ml that was free of replication-competent retrovirus to transduce human umbilical cord blood CD34+ cells. Transduced CD34+ cord blood cells were transplanted into NOD/SCID mice and plated in serum-free methylcellulose culture to determine the transduction efficiency of human hematopoietic progenitor cells. A transduction efficiency of about 20% was obtained. At 6-10 weeks posttransplantation, human hematopoietic cell engraftment and marking were determined. Marrow from transplanted mice demonstrated human cell engraftment by the presence of human (CD45+) cells containing both CD19+ lymphoid and CD33+ myeloid cells. Serial sampling of NOD/SCID bone marrow revealed the presence of 6.7-14.0% CD45+ cells at 6 weeks posttransplant as compared to 3.6-27.2% CD45+ cells at 9-10 weeks posttransplant. Human progenitors examined from NOD/SCID bone marrow cells 9 weeks posttransplant revealed from 7.4 to 25.9% of the colonies exhibiting X-gal staining. Our study demonstrates the ability of a simian foamy virus vector to transduce the SCID-repopulating cell and offers a promising new gene delivery system for use in hematopoietic stem cell gene therapy.

A minimal genome simian foamy virus type 1 vector system with efficient gene transfer.

Foamy viruses have several inherent features for the opportunity to develop efficient and versatile vectors for gene therapy. We have constructed a series of vectors and helper plasmids based on simian foamy virus type 1 (SFV-1) to establish the minimum vector genome required for efficient gene transduction. To characterize the efficiency of gene transduction by these vectors, the green fluorescent protein (GFP) coding sequence is linked to the human cytomegalovirus immediate gene promoter. Several deletion analyses of SFV-1 vectors revealed that the minimum genome with efficient GFP transduction contained the 5' untranslated region extending to the first 637 nucleotides of the gag gene, a 596 nucleotides of pol sequence from position 3137-3733, the 3' pol region at position 5200-5693, the 3' end polypurine tract, and the 3' LTR. An additional 1131 nucleotides can be removed from the 3' end LTR without affecting the efficiency of vector transduction. SFV-1 vector can therefore accommodate a minimum 8930 base-size heterologous DNA fragment. Furthermore, the efficiency of SFV-1 vector transduction was analyzed using different packaging plasmids. GFP transduction with packaging plasmid that contained the 5' R-U5 region of the LTR was compared with helper plasmids that had deletions in this region except for 22 nucleotides (positions 21-41), the first 61, 77, or 140 nucleotides of the R of the LTR. Transduction efficiencies were significantly reduced with the deletion mutations implicating that for optimum SFV-1 vector productions a packaging construct that includes the 5' R-U5 is required.