Cell-cycle dependence of foamy virus vectors

Gillian S. Patton, Otto Erlwein{dagger} and Myra O. McClure

Jefferiss Research Trust Laboratories, Wright Fleming Institute, Division of Medicine, Imperial College London, London W2 1PG, UK

Correspondence
Myra O. McClure
m.mcclure{at}imperial.ac.uk


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Retroviruses differ in the extent to which they are dependent on host-cell proliferation for their replication, an aspect of their replication that impacts on their vector potential. Foamy viruses offer distinct advantages over other retroviruses for development as vectors for gene therapy. A vector derived from the prototypic foamy virus (PFV), formerly known as human foamy virus (HFV), transduced aphidicolin-arrested cells five- to tenfold more efficiently than one derived from murine leukemia virus (MLV), but several-fold less efficiently than a human immunodeficiency virus type 1 (HIV-1) vector. The same relative efficiency was found following transduction of cells that had been arrested by {gamma}-irradiation or with mitomycin C. Cells that were exposed to vector during aphidicolin arrest and were subsequently allowed to cycle were transduced significantly better by PFV than by MLV. Quiescent human CD34+ progenitor cells were transduced as efficiently by PFV as by HIV vectors (40–50 %) when transduction was assayed after the cells were allowed to cycle.

A figure showing the derivation of the constructs expressing the gag and pol genes is available as supplementary material in JGV Online.

{dagger}Present address: Institute for Biomedical Research, Georg-Speyer-Haus, Paul-Ehrlich-Str. 42–44, D-60596 Frankfurt/Main, Germany.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
We demonstrated previously that, like murine leukemia virus (MLV), wild-type foamy viruses do not productively infect G1/S- or G2-arrested cells and that mitosis is a prerequisite for virus protein expression (Bieniasz et al., 1995). It was demonstrated subsequently, however, that foamy virus-derived vectors transduced stationary-phase cells, albeit with low efficiency (Russell & Miller, 1996). Moreover, nuclear import of the foamy virus pre-integration complex (PIC) can take place in G1/S phase-arrested cells, possibly facilitated by the fact that the foamy virus Gag and Pol proteins, including the integrase, contain nuclear localization signals (NLSs) (Schliephake & Rethwilm, 1994; Saïb et al., 1997; Imrich et al., 2000), but the transduction efficiency remains low. After uncoating, the Gag protein, in association with the virus genome, targets the centrosome via the microtubule network and enters the nucleus, but no integration or virus gene expression takes place. Out of step with these studies, Mergia et al. (2001) demonstrated efficient transduction of arrested and stationary-phase cells by simian foamy virus type 1 (SFV-1)-based vectors, which the authors suggested was due to the high titre of vector and the cytomegalovirus (CMV) promoter, which is known to be active in arrested cells.

Hirata et al. (1996) showed that haematopoietic progenitor cells (HPCs), pre-stimulated into cycling by cytokines, were transduced similarly by PFV- and MLV-based vectors. However, more recent studies have shown that freshly isolated and hence quiescent (G0/G1) murine and human HPCs are transduced readily in vitro by both PFV (Vassilopoulos et al., 2001; Josephson et al., 2002) and SFV-1 (Zucali et al., 2002) vectors in the presence of cytokines. Also, in vivo studies in which HPCs were transduced ex vivo and reintroduced into NOD/SCID mouse models showed efficient transgene expression in engrafted cells (Vassilopoulos et al., 2001; Josephson et al., 2002, 2004; Zucali et al., 2002; Leurs et al., 2003).

The aim of this study was to compare the effect of growth arrest on the transduction of cell lines by MLV, PFV and HIV vectors at low titres and to assay the efficiency of these vectors on HPCs.

The MLV vectors were produced from the packaging cell lines FLY A4 LacZ 3 (Cosset et al., 1995) and FLY MFG NC (kindly provided by Professor Mary Collins). The resulting Moloney MLV (MoMLV) cores contain the LacZ and eGFP transgenes, respectively, driven by the MoMLV long terminal repeat (LTR) promoter, with the virions surrounded by the amphotrophic MLV envelope.

The HIV vector was made by transient transfection using the following constructs: either pH7G, which encodes eGFP under the control of the CMV promoter (Ikeda et al., 2003), or pH7nZ, which is the same as pH7G, but contains the LacZ transgene in place of eGFP, the HIV Gag-Pol construct, pSYNGP (Kotsopoulou et al., 2000) and the VSV-G envelope construct, pRV67 (Mitrophanous et al., 1999).

The pMD11 and pMD9 plasmids encode the PFV vector genome (Heinkelein et al., 2002). The pMD11 plasmid encodes LacZ and pMD9 encodes eGFP, both driven by the spleen focus-forming virus (SFFV) LTR promoter. pMD9 was adapted by exchanging the SFFV LTR for either a MoMLV LTR promoter [pMD9 (MoMLV)] or a CMV promoter [pMD9 (CMV)]. These vector constructs were co-transfected with plasmid pczHFVenv, which encodes the PFV envelope protein (Pietschmann et al., 1999), and the packaging construct pKOgp1, which is derived from pCgp1 (Fischer et al., 1998) and encodes the PFV Gag and Pol proteins under the control of the CMV promoter (see Supplementary Figure in JGV Online). Much of the U5 region and the leader sequence are absent, including the primer binding site and the RNA dimerization sequence (Erlwein et al., 1998; Heinkelein et al., 1998). The splice donor at position 51 (Muranyi & Flügel, 1991) is retained to facilitate Pol expression from its spliced mRNA. To do this, the gag sequences at positions 1274–2915 (numbers are according to the PFV infectious clone, HSRV-2; Rethwilm et al., 1990) were PCR-amplified by using high-fidelity Pwo polymerase (Roche). The forward primer sequence was 5'-TTTCCCTTGCTAAGGCCGCCGCCACCATGGCTTCAGGAAGTAATGTTGAAGAATATAAC-3' and included an optimized Kozak sequence (Kozak, 1986) upstream of the start codon of the gag ORF to optimize translation. The sequence of the reverse primer was 5'-TTTGAGGTTGGTAAGTACGGGGTCA-3'. The amplicon was cloned between the EcoNI site in R and the SwaI site in the gag ORF of pCgp1, thereby deleting nt 1087–1273 in the HSRV-2 leader sequence.

The PFV and HIV vectors were produced by transient transfection with polyethylenimine (PEI) (Sigma). Briefly, 293T cells were seeded at 4x106 cells per plate in 10 ml Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal calf serum (FCS), 1000 U penicillin ml–1, 100 µg streptomycin ml–1 and 2 mM L-glutamine (complete medium, CM) and incubated overnight at 37 °C. For the PFV vector, 5 µg pMD11, pMD9(MoMLV) or pMD9(CMV), 9 µg pKOGP and 3 µg pczHFVenv were co-transfected into cells and for the HIV vector, 8 µg pH7nZ or pH7G, 8 µg pSYNGP and 4 µg pRV67 were co-transfected. For each plate, plasmid DNA was diluted in 2·5 ml serum-free DMEM and added drop-wise with vortexing to 2·5 ml serum-free medium that contained 0·4 µM PEI. A PEI/DNA precipitate was formed after incubation at room temperature for 20 min and was added in full to fresh medium, which replaced the medium on the 293T cells prior to incubation for 4–5 h at 37 °C. The medium was changed to CM and the cells were incubated for a further 48 h at 32 °C. Vector supernatant was filtered through 0·45 µm filters (Millipore) and concentrated by ultracentrifugation at 50 000 g for 90 min at 4 °C. To produce MLV vector, FLY A4 LacZ 3 and FLY MFG NC cells were cultured to 70 % confluency. The medium was changed, the cells were incubated at 32 °C for 2 days and the supernatant was harvested and filtered. All vector stocks were titrated on D17 cells. Briefly, D17 cells were seeded at 3x104 cells per well and incubated at 37 °C overnight, then 250 µl vector (diluted serially tenfold to 10–5) was added. Transduced cells were incubated for 3 days at 37 °C and fixed in 4 % formaldehyde; {beta}-galactosidase expression was detected by the insoluble X-Gal assay (Bieniasz et al., 1995) and eGFP expression was detected by fluorescence microscopy.

The effect of cell-cycle arrest on transduction by three LacZ-expressing retrovirus vectors was assessed on HT1080, D17, HeLa and TE671 cells. Transduction was assayed as described for vector titration. Treatment with 5 µM aphidicolin, a reversible inhibitor of eukaryotic DNA synthesis, for 24 h prior to transduction caused cell-cycle arrest at G1/S in all cell lines, confirmed by flow cytometry using propidium iodide (Bieniasz et al., 1995). Cell viability remained >90 %, as assessed by Trypan blue exclusion. For each cell line and vector, the number of G1/S-arrested cells expressing LacZ was given as a percentage of the number that were observed in cycling cells.

Whilst the MLV vector failed to transduce any cells that were not dividing, the HIV and PFV vectors transduced different cells at quite different efficiencies: D17 (30 and 0·6 %, respectively), HeLa (21 and 1·4 %), HT1080 (70 and 0·3 %) and TE671 (32 and 10 %).

Transduction efficiency in HT1080 cycling cells was given a reference value of 100 %, to which the efficiency in arrested cells could be compared [Fig. 1a (A)]. Cells arrested in G1/S phase, exposed to LacZ-expressing vector for 4 h and maintained under similar conditions for another 48 h, poorly expressed the transgene following transduction by MLV and PFV vectors [Fig. 1a (B)]. When, on changing the medium, the cells were allowed to divide during the 4 h transduction and thereafter, transduction levels by PFV vector were comparable to those of HIV vector and threefold higher than those of MLV [Fig. 1a (C)]. When transduced cells were arrested during transduction before release (4 h), PFV vector transduction was slightly lower than that of HIV [Fig. 1a (D)]. When transduced cells were arrested for a further 24 h before release, the PFV vector again facilitated higher levels of transgene expression than the MLV vector, although this was reduced compared to the HIV vector [Fig. 1a (E)]. When cells in G1/S arrest were exposed to mitomycin C (at 10 µg ml–1 for 30 min pre-transduction) to prevent cycling through M phase following release from aphidicolin, vector titres were reduced to <0·05 % (MLV), 2 % (PFV) and 70 % (HIV) of the titres that were obtained in untreated cells [Fig. 1a (F)]. Cells arrested irreversibly in G2 by exposure to 9000 rad (=90 Gy) {gamma}-irradiation from a 137Cs source prior to seeding were transduced by PFV (1 %) and better by HIV (48 %), but not by MLV (<0·05 %) [Fig. 1a (G)].



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1. Cell-cycle dependency of transduction by MLV-, PFV- and HIV-based vectors. HT1080 cells were seeded on 48-well plates at a density of 1·5x104 per well and grown overnight under the conditions shown in Table 1. Transductions were performed in triplicate for each condition, 24 h after plating, using serial dilutions of MLV vector, PFV vector or HIV vector, all encoding LacZ. Vector was added to cells in a minimal volume for 4 h, the cells were washed twice with PBS and then fresh medium was added (cells showed 93 % viability post-arrest). Cells were fixed and stained for LacZ expression after 48 h. Transduction efficiencies were calculated (by using dilutions at which groups of cells arising from one transduced cell could be determined: 50–300 colonies per well) and then, assuming a value of 100 % for cycling cells, relative transduction levels of arrested to cycling cell populations were determined (mean±SEM, n=3). Transduction efficiencies are shown as columns in (a). Vector titre (x103) is shown above each column. A similar experiment, comparing MLV and PFV vector (both encoding eGFP driven by the MoMLV promoter), is shown in (b). A third experiment comparing MLV and PFV vectors, again encoding eGFP but driven by the CMV promoter, is shown in (c). Cell-cycle phase was determined at the time of infection via propidium iodide staining and flow-cytometry analysis.

 
One point to note about the above data is that different promoters directed LacZ expression in the three vectors. Arguably, the outcome may be different if expression under the control of the same promoter was compared. To address this, similar experiments were carried out by using vectors in which the eGFP transgene was expressed from the same promoter. The PFV vector with the transgene driven by the MoMLV LTR was compared with the MLV vector (Fig. 1b), and PFV vector with the transgene driven by the CMV promoter was compared with the HIV vector (Fig. 1c). There was little difference in the relative transduction efficiencies of the vectors, apart from where the cells were released from arrest 24 h after transduction [Fig. 1c (E)], when the transduction efficiency was fivefold higher than that of the SFFV-containing vector (Fig. 1a).

Haematopoietic progenitor CD34+ cells were isolated from cord blood by magnetic microbead selection (Miltenyi Biotech). RetroNectin-coated 24-well plates were pre-incubated with 500 µl retrovirus vector supernatant at 3x106 ml–1 (titrated on D17 cells) for 2 h at 37 °C. CD34+ cells were added, to give a final concentration of 2x105 cells ml–1, to X-Vivo 10 medium (Biowhittaker) supplemented with the cytokines interleukin 3 (IL3; 20 ng ml–1), IL6 (20 ng ml–1), stem cell factor (SCF; 100 ng ml–1) and Flt-3 ligand (Flt-3; 100 ng ml–1) (Tebu). Cells and vector supernatant were centrifuged in the 24-well plate and incubated for 16 h at 37 °C, after which a further 500 µl vector, supplemented with cytokines, was added to the cells. Cells were harvested and assayed for {beta}-galactosidase activity 48 h after the second transduction. Mean vector transduction rates were: HIV, 48 %; PFV, 37 %; and MLV, 5–6 % (Fig. 2), reflecting differential dependence on host-cell division. Freshly isolated CD34+ cells are typically in the G0/G1 phase of the cell cycle and can be stimulated into division by cytokines. Our results are consistent with the premise that HIV vector delivery is largely independent of cellular mitosis, whereas MLV fails to transduce cells without prior cytokine stimulation (Uchida et al., 1998) and PFV can infect quiescent cells, but requires cell division for integration and transgene expression (Trobridge & Russell, 2004).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 2. Comparison of transduction of CD34+ cells by retrovirus vectors. A 24-well plate was seeded with 5x104 CD34+ cells per well in X-Vivo 10 medium containing the cytokines IL3, IL6, FLT-3 and SCF. Cells were transduced twice over the next 24 h with vectors at 3x106 ml–1, previously titrated on D17 cells.

 
Transduction efficiency by PFV vector was reduced uniformly in cells that were arrested at G1/S, in cells arrested irreversibly in G2 before infection and in cells infected during G1/S, but then allowed to pass through the cell cycle until arrest at M phase. In cells that were allowed to pass through mitosis after arrest, transduction was re-established more effectively for PFV than for MLV, reflecting the ease with which HIV, MLV and PFV access the nucleus of the non-dividing cell in order to integrate their genome into host DNA. For many viruses, it appears that after disassembly in the cytoplasm, NLS sequences within virus proteins are recognized by host-cell import factors that mediate nuclear targeting of PICs via the nuclear pore complex (Cullen, 2001). Several such sequences are present in HIV-1, most significantly in the virus integrase (IN) (Bukrinsky et al., 1993; Bouyac-Bertoia et al., 2001). No such NLS sequences have been found in C-type retroviruses such as MLV. Foamy virus Gag and Pol proteins, including IN, contain NLS sequences (Imrich et al., 2000) that, in combination, might account for the improved transduction ability of PFV over MLV in arrested cells. The lower efficiency in comparison with HIV could be due to the PFV NLS being partly occluded in the PIC. Saïb et al. (1997) showed that PFV generates 2-LTR circles in arrested cells. It is possible that there are further blocks to PFV integration after nuclear translocation of the DNA. As for wild-type PFV (Bieniasz et al., 1995), our vector results confirm that cells need to pass through M phase before integration can take place. Cells arrested in G2 by irradiation before transduction by PFV vector, and PFV-exposed cells allowed to pass through interphase, but halted before mitosis, show reduced levels of transgene expression compared with HIV.

Other factors besides NLS sequences could account for transduction differences in retrovirus vectors. Reverse transcription of PFV occurs mainly during PFV particle formation and the virions contain virus RNA and DNA (Yu et al., 1999); hence, the virions could be more stable. Our results demonstrate that the longer the time between vector entry and cell division, the lower the likelihood of successful integration. This is more marked for MLV than for PFV, suggesting that PFV is more stable intracellularly; it is possible that the PICs of foamy viruses are less susceptible to degradation by cellular nucleases than those of MLV.

It is clear that foamy viruses allow infection of non-dividing cells at a level intermediate between onco- and lentivirus vectors, but the integration efficiency remains significantly lower than that of lentiviruses unless cells are allowed to pass through mitosis. The data presented in this paper confirm our own findings on wild-type foamy viruses, as well as those published recently by Trobridge & Russell (2004), but are contrary to the findings of Mergia et al. (2001), possibly for reasons alluded to above. Hence, our data are likely to end the controversy that surrounds the question of whether PFV and vectors derived from the virus are likely to be effective in gene therapy targeted to end-differentiated cells.


View this table:
[in this window]
[in a new window]
 
Table 1. Cell-cycle conditions prior to and during vector transduction

 

   ACKNOWLEDGEMENTS
 
We thank Oxford BioMedica (Oxford Science Park, Oxford, UK) for providing the HIV constructs pH7G, pH7nZ, pSYNGP and pRV67, and Professor M. K. L. Collins (Dept of Immunology and Molecular Pathology, Windeyer Institute, University College London, London W1T 4JF, UK) for the FLY packaging cell lines. This work was funded by the Jefferiss Trust.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Bieniasz, P. D., Weiss, R. A. & McClure, M. O. (1995). Cell cycle dependence of foamy retrovirus infection. J Virol 69, 7295–7299.[Abstract]

Bouyac-Bertoia, M., Dvorin, J. D., Fouchier, R. A. M., Jenkins, Y., Meyer, B. E., Wu, L. I., Emerman, M. & Malim, M. H. (2001). HIV-1 infection requires a functional integrase NLS. Mol Cell 7, 1025–1035.[CrossRef][Medline]

Bukrinsky, M. I., Haggerty, S., Dempsey, M. P. & 7 other authors (1993). A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666–669.[CrossRef][Medline]

Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R. A. & Collins, M. K. L. (1995). High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 69, 7430–7436.[Abstract]

Cullen, B. R. (2001). Journey to the center of the cell. Cell 105, 697–700.[CrossRef][Medline]

Erlwein, O., Bieniasz, P. D. & McClure, M. O. (1998). Sequences in pol are required for transfer of human foamy virus-based vectors. J Virol 72, 5510–5516.[Abstract/Free Full Text]

Fischer, N., Heinkelein, M., Lindemann, D., Enssle, J., Baum, C., Werder, E., Zentgraf, H., Müller, J. G. & Rethwilm, A. (1998). Foamy virus particle formation. J Virol 72, 1610–1615.[Abstract/Free Full Text]

Heinkelein, M., Schmidt, M., Fischer, N., Moebes, A., Lindemann, D., Enssle, J. & Rethwilm, A. (1998). Characterization of a cis-acting sequence in the pol region required to transfer human foamy virus vectors. J Virol 72, 6307–6314.[Abstract/Free Full Text]

Heinkelein, M., Dressler, M., Jármy, G., Rammling, M., Imrich, H., Thurow, J., Lindemann, D. & Rethwilm, A. (2002). Improved primate foamy virus vectors and packaging constructs. J Virol 76, 3774–3783.[Abstract/Free Full Text]

Hirata, R. K., Miller, A. D., Andrews, R. G. & Russell, D. W. (1996). Transduction of hematopoietic cells by foamy virus vectors. Blood 88, 3654–3661.[Abstract/Free Full Text]

Ikeda, Y., Takeuchi, Y., Martin, F., Cosset, F. L., Mitrophanous, K. & Collins, M. (2003). Continuous high-titer HIV-1 vector production. Nat Biotechnol 21, 569–572.[CrossRef][Medline]

Imrich, H., Heinkelein, M., Herchenröder, O. & Rethwilm, A. (2000). Primate foamy virus Pol proteins are imported into the nucleus. J Gen Virol 81, 2941–2947.[Abstract/Free Full Text]

Josephson, N. C., Vassilopoulos, G., Trobridge, G. D., Priestley, G. V., Wood, B. L., Papayannopoulou, T. & Russell, D. W. (2002). Transduction of human NOD/SCID-repopulating cells with both lymphoid and myeloid potential by foamy virus vectors. Proc Natl Acad Sci U S A 99, 8295–8300.[Abstract/Free Full Text]

Josephson, N. C., Trobridge, G. & Russell, D. W. (2004). Transduction of long-term and mobilized peripheral blood-derived NOD/SCID repopulating cells by foamy virus vectors. Hum Gene Ther 15, 87–92.[CrossRef][Medline]

Kotsopoulou, E., Kim, V. N., Kingsman, A. J., Kingsman, S. M. & Mitrophanous, K. A. (2000). A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene. J Virol 74, 4839–4852.[Abstract/Free Full Text]

Kozak, M. (1986). Bifunctional messenger RNAs in eukaryotes. Cell 47, 481–483.[Medline]

Leurs, C., Jansen, M., Pollok, K. E. & 8 other authors (2003). Comparison of three retroviral vector systems for transduction of nonobese diabetic/severe combined immunodeficiency mice repopulating human CD34+ cord blood cells. Hum Gene Ther 14, 509–519.[CrossRef][Medline]

Mergia, A., Chari, S., Kolson, D. L., Goodenow, M. M. & Ciccarone, T. (2001). The efficiency of simian foamy virus vector type-1 (SFV-1) in nondividing cells and in human PBLs. Virology 280, 243–252.[CrossRef][Medline]

Mitrophanous, K. A., Yoon, S., Rohll, J. B., Patil, D., Wilkes, F. J., Kim, V. N., Kingsman, S. M., Kingsman, A. J. & Mazarakis, N. D. (1999). Stable gene transfer to the nervous system using a non-primate lentiviral vector. Gene Ther 6, 1808–1818.[CrossRef][Medline]

Muranyi, W. & Flügel, R. M. (1991). Analysis of splicing patterns of human spumaretrovirus by polymerase chain reaction reveals complex RNA structures. J Virol 65, 727–735.[Medline]

Pietschmann, T., Heinkelein, M., Heldmann, M., Zentgraf, H., Rethwilm, A. & Lindemann, D. (1999). Foamy virus capsids require the cognate envelope protein for particle export. J Virol 73, 2613–2621.[Abstract/Free Full Text]

Rethwilm, A., Baunach, G., Netzer, K.-O., Maurer, B., Borisch, B. & ter Meulen, V. (1990). Infectious DNA of the human spumaretrovirus. Nucleic Acids Res 18, 733–738.[Abstract]

Russell, D. W. & Miller, A. D. (1996). Foamy virus vectors. J Virol 70, 217–222.[Abstract]

Saïb, A., Puvion-Dutilleul, F., Schmid, M., Périès, J. & de Thé, H. (1997). Nuclear targeting of incoming human foamy virus Gag proteins involves a centriolar step. J Virol 71, 1155–1161.[Abstract]

Schliephake, A. W. & Rethwilm, A. (1994). Nuclear localization of foamy virus Gag precursor protein. J Virol 68, 4946–4954.[Abstract]

Trobridge, G. & Russell, D. W. (2004). Cell cycle requirements for transduction by foamy virus vectors compared to those of oncovirus and lentivirus vectors. J Virol 78, 2327–2335.[Abstract/Free Full Text]

Uchida, N., Sutton, R. E., Friera, A. M., He, D., Reitsma, M. J., Chang, W. C., Veres, G., Scollay, R. & Weissman, I. L. (1998). HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc Natl Acad Sci U S A 95, 11939–11944.[Abstract/Free Full Text]

Vassilopoulos, G., Trobridge, G., Josephson, N. C. & Russell, D. W. (2001). Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors. Blood 98, 604–609.[Abstract/Free Full Text]

Yu, S. F., Sullivan, M. D. & Linial, M. L. (1999). Evidence that the human foamy virus genome is DNA. J Virol 73, 1565–1572.[Abstract/Free Full Text]

Zucali, J. R., Ciccarone, T., Kelley, V., Park, J., Johnson, C. M. & Mergia, A. (2002). Transduction of umbilical cord blood CD34+ NOD/SCID-repopulating cells by simian foamy virus type 1 (SFV-1) vector. Virology 302, 229–235.[CrossRef][Medline]

Received 21 April 2004; accepted 1 July 2004.