Lentiviral Vectors Pseudotyped with Envelope Glycoproteins Derived from Gibbon Ape Leukemia Virus and Murine Leukemia Virus 10A1

Virology 273, 16–20 (2000) doi:10.1006/viro.2000.0394, available online at http://www.idealibrary.com on

RAPID COMMUNICATION Lentiviral Vectors Pseudotyped with Envelope Glycoproteins Derived from Gibbon Ape Leukemia Virus and Murine Leukemia Virus 10A1 J. Stitz,* C. J. Buchholz,* M. Engelsta¨dter,* W. Uckert,† U. Bloemer,‡ I. Schmitt,* and K. Cichutek* ,1 *Department of Medical Biotechnology, Paul-Ehrlich-Institut, 63225 Langen, Germany; †Max-Delbru¨ck-Zentrum fu¨r Molekulare Medizin, Berlin-Buch, Germany; and ‡Department of Neurosurgery, Medical School Hannover, 30625 Hannover, Germany Received March 22, 2000; accepted April 24, 2000 Lentiviral vectors pseudotyped with the envelope glycoproteins (Env) of amphotropic murine leukemia virus (MLV) and the G protein of vesicular stomatitis virus (VSV-G) have been successfully used in recent preclinical gene therapy studies. We report here the generation of infectious HIV-1-derived vector particles pseudotyped with the Env of the molecular clone 10A1 of MLV and with chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV. Formation of infectious HIV-1 (GaLV) pseudotype vectors was only possible with the substitution of the cytoplasmic tail of GaLV Env with that of MLV. The lentiviral vectors exhibited a host cell range identical with that of MLV(GaLV) and MLV(10A1) vectors, which are known to enter cells either via the GaLV-receptor Glvr-1 (Pit-1) or via the amphotropic receptor Ram-1 (Pit-2) in addition to Glvr-1, respectively. Thus, HIV-1(GaLV) and HIV-1(10A1) pseudotype vectors may be useful for efficient gene transfer into a variety of human tissues like primary human hematopoietic cells. © 2000 Academic Press Key Words: lentiviral vector; pseudotype vector; GaLV Env; 10A1 Env.

restricted to CD4 ⫹ cells, HIV-1 vectors are usually pseudotyped with VSV-G or the amphotropic MLV Env in order to broaden their target cell range (6). We have now successfully generated HIV-1-derived pseudotype vector particles harboring the 10A1 Env or a C-terminally modified GaLV Env. The modified GaLV Env encompassed the surface envelope glycoprotein (SU) of GaLV and a chimeric transmembrane (TM) protein derived from GaLV and from MLV-A. The resulting HIV-1(GaLV) and HIV1(10A1) vector particles have been shown to exhibit the cell tropism of GaLV and MLV-10A1, respectively. They may be useful for improving gene transfer efficiency into important target cells of gene therapy such as human HSCs and PBLs.

Murine leukemia virus (MLV)-derived vectors pseudotyped with the envelope glycoproteins (Env) of the molecular MLV clone 10A1 (MLV-10A1) or those of gibbon ape leukemia virus (GaLV) have been shown to allow efficient gene transfer into a variety of human cells including human CD34-positive cells and peripheral blood leukocytes (PBLs) (1, 2). Human CD34-positive cells, more specifically the hematopoietic stem cells (HSCs) contained within this cell population, and PBLs are less susceptible to transduction by amphotropic MLV (MLV-A) vectors, most likely due to the low cell surface expression of the amphotropic MLV receptor Ram-1 (3). MLV vector particles harboring the GaLV Env or the 10A1 Env have been shown to more efficiently transduce human CD34-positive cells and PBLs. One of the presumed reasons for the improved gene transfer efficiency is the usage of the GaLV receptor Glvr-1 by MLV(GaLV) and the usage of either Glvr-1 or Ram-1 by MLV(10A1) (4). However, MLV-derived vectors allow transduction of proliferating target cells only. In contrast, lentiviral vectors, e.g., those derived from HIV-1, have been used for efficient gene transfer into resting or slowly dividing human cells. In this respect lentiviral vectors are superior to oncoretroviral vectors (5). As the natural tropism of HIV-1 is

Results and Discussion. Generation of HIV-1 pseudotype vector particles using MLV-10A1 Env, but not wild- type GaLV Env. To investigate pseudotype formation of HIV-1 with GaLV and MLV-10A1, suitable env expression genes were constructed. The complete coding regions of the wild-type env genes from MLV-A, GaLV, and MLV-10A1 were inserted into the expression construct of plasmid pALF (7) comprising the FB29 Friend murine leukemia virus (FLV) promoter and the poly(A) signal sequence of the human phosphoglycerate kinase gene (Fig. 1A). Two days after transfection of 293T cells with all three constructs, Env expression was demonstrated by immunostaining of cells using a serum directed against gp71-SU of FLV (Fig. 2A, a–d). In order to generate HIV-1 vector particles, 293T cells were cotransfected with the

1 To whom correspondence and reprint requests should be addressed at Department of Medical Biotechnology, Paul-Ehrlich-Institut, Paul-Ehrlich-Strasse 51-59, 63225 Langen, Germany. Fax: ⫹49 6103 77 1255. E-mail: [email protected].

0042-6822/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

16

RAPID COMMUNICATION

17

FIG. 1. Schematic diagram of the env gene constructs. (A) All env genes described here were molecularly cloned into expression construct ALF (7) containing the LTR of FB29 strain of Friend MLV. A phleomycin-resistance gene (phleo) located downstream of the env gene was under the control of the LTR of Friend MLV C57. (B) Amino acid sequence alignment of the respective transmembrane regions (TMR) and C-tails of the transmembrane glycoproteins (TM) encoded by the env gene constructs. Sequences shaded in gray were derived from MLV-A; light gray and open boxes indicate regions derived from MLV-10A1 and GaLV, respectively.

packaging construct CMV⌬R8.2 encoding HIV-1 gag/pol, the packaging signal-positive transfer vector HR⬘CMVlacZ encoding ␤-Gal (8), and one of the respective envexpression constructs. MLV(MLV-A) vector particles were generated by transfecting the packaging construct HIT60 and the transfer vector construct HIT111 (9) and served as positive controls. As a negative control 293T cells were transfected with the respective packaging constructs and transfer vectors in the absence of an env gene construct. In order to assay transduction of cells, supernatants of the transfected cells harvested 2 days after transfection were incubated with human HT1080 fibrosarcoma cells (Ram-1⫹/Glvr-1⫹), canine D17 osteosarcoma cells (Ram-1⫹/Glvr-1⫹), and murine NIH-3T3 fibroblasts (Ram-1⫹/Glvr-1⫺) as described under Materials and Methods. Two days later, transduction was monitored by X-Gal staining of ␤-Gal-expressing cells. Gene transfer efficiency was determined as described previously (10). As shown in Table 1, infectious MLV pseudotype vector particles were generated with all three env gene constructs. As expected, they showed the target cell range of the viruses from which each respective Env was derived. No gene transfer was detectable following transfection of the relevant packaging and transfer constructs in the absence of any viral env gene

(data not shown). Similar to HIV-1 (MLV-A), efficient generation of infectious HIV-1(10A1) vector particles was detected. However, no HIV vector-mediated transduction was detected using the wild-type GaLV Env (GaLV wt Env). It was concluded either that GaLV wt Env incorporation was insufficient for the formation of infectious HIV-1 (GaLV wt) vector particles or that the resulting pseudotype vectors harboring GaLV wt Env were not infectious. Construction of chimeric env genes derived from GaLV and MLV-A. To allow formation of infectious retroviral vector particles the proteins used to form the vector envelope must be efficiently incorporated by the viral capsid particles. Once incorporated, they also must mediate binding of the resulting vector particles to a suitable cell surface receptor, fusion of the viral and cellular membrane, and entry into the cytoplasm. As we had observed that wild-type GaLV Env, although allowing the generation of infectious MLV pseudotype vectors, apparently did not efficiently mediate formation of infectious HIV-1 pseudotype vector particles, we assumed that this was caused by inefficient incorporation and/or low fusiogenicity. The C-tail of TM has been reported to influence Env incorporation of various retroviruses (11, 12). On the other hand, cellular and viral transmembrane proteins

18

RAPID COMMUNICATION

FIG. 2. Expression of the envelope proteins in transfected 293T cells. (A) Using an antiserum directed against FLV gp71-SU immunostaining was performed as described under Materials and Methods: (a) mock; (b) MLV-A Env; (c) MLV-10A1 Env; (d) GaLV wt Env; (e) GaLV CM Env; (f) GaLV TM Env. (B) Detection of the indicated Env on the cell surface by FACS using the anti-FLV gp71-SU serum and secondary antibodies conjugated with phycoerythrin.

have been shown to be efficiently incorporated by HIV-1 virions independent of any C-terminal modifications (13, 14). Moreover, the C-terminal domain following the transmembrane-spanning region (C-tail) of retroviral TM has been shown to trigger the fusion capacity of Env. For example, the C-tail of MLV TM is processed by the viral protease during virion maturation. Cleavage of a small peptide (R-peptide) of 16 amino acids from the TM CTABLE 1 Infectivity of Vector Particles Pseudotyped with Retroviral Envelope Proteins Target cells a Vector particle

HT1080

D17

3T3

MLV(MLV-A) MLV(10A1) MLV(GaLV wt)

1.0 ⫻ 10 5 8.2 ⫻ 10 4 2.5 ⫻ 10 5

1.2 ⫻ 10 5 1.0 ⫻ 10 4 2.8 ⫻ 10 5

1.3 ⫻ 10 5 4.5 ⫻ 10 4 ⬍10 1

HIV(MLV-A) HIV(10A1) HIV(GaLV wt)

2.7 ⫻ 10 4 5.0 ⫻ 10 4 ⬍10 1

2.3 ⫻ 10 3 9.0 ⫻ 10 3 ⬍10 1

1.0 ⫻ 10 3 6.0 ⫻ 10 2 ⬍10 1

a

MLV and HIV-1 pseudotype vectors were titrated on the target cells indicated. Titers are reported as lacZ i.u./ml. Data shown resulted from one representative experiment of three.

terminus results in vastly enhanced fusiogenicity of the Env (15). MLV-A Env efficiently confers infectivity to HIV-1 pseudotype vectors and has been demonstrated to be processed by the HIV-1 protease (16). We therefore tested whether a substitution of the C-terminal amino acids of GaLV TM by those of MLV TM would allow the formation of infectious HIV-1 vector particles. First, we constructed chimeric env genes comprising the gp71-SU coding region of GaLV and the N-terminal region of the GaLV TM except for the C-terminal amino acids and/or the transmembrane region (TMR), which were replaced by those of the MLV TM. The chimeric env gene coding regions were generated by PCR and fusion-PCR, respectively, and inserted into the env expression construct ALF. As shown in Fig. 1B, in Env-variant GaLV TM the TMR and the C-terminal amino acids following the TMR were replaced by those of the MLV TM. The env gene product GaLV CM encompassed only the C-tail of the MLV TM, whereas all other GaLV TM coding regions were retained. After transfection of constructs pALF-GaLV TM and pALF-GaLV CM into 293T cells, Env expression was demonstrated by immunostaining as described above (Fig. 2A, e and f). To demonstrate the functional expression and presence on the cell surface of the packaging cells, the transfected cells were stained with anti-FLV gp71-SU antibodies and phycoerythrin-labeled second-

RAPID COMMUNICATION TABLE 2 Infectivity of Vector Particles Pseudotyped with Chimeric Envelope Proteins Derived from MLV and GaLV Target cells a Vector particle

HT1080

D17

3T3

MLV(MLV-A) MLV(GaLV wt) MLV(GaLV CM) MLV(GaLV TM)

3.1 ⫻ 10 5 2.4 ⫻ 10 5 5.6 ⫻ 10 4 1.3 ⫻ 10 5

3.0 ⫻ 10 5 2.9 ⫻ 10 5 5.3 ⫻ 10 4 9.3 ⫻ 10 4

3.1 ⫻ 10 5 ⬍10 1 ⬍10 1 ⬍10 1

HIV(MLV-A) HIV(GaLV wt) HIV(GaLV CM) HIV(GaLV TM)

8.3 ⫻ 10 4 ⬍10 1 9.3 ⫻ 10 3 1.6 ⫻ 10 4

4.4 ⫻ 10 3 ⬍10 1 7.3 ⫻ 10 3 1.1 ⫻ 10 4

1.5 ⫻ 10 3 ⬍10 1 ⬍10 1 ⬍10 1

a MLV and HIV-1 pseudotype vectors were titrated on the target cells indicated. Titers are reported as lacZ i.u./ml. Data shown resulted from one representative experiment of three.

ary antibodies and subsequently subjected to fluorescence activated cell sorting (FACS). Cells transfected with either the MLV-A or the GaLV wt env genes served as positive controls, whereas mock-transfected cells were used as negative controls. As shown in Fig. 2B, all Env variants were expressed on the surface of the transfected 293T cells. Chimeric Env variants GaLV TM and GaLV CM allow efficient formation of infectious HIV-1 pseudotype vectors exhibiting the target cell range of GaLV. To investigate the potential of the chimeric Env variants GaLV TM and GaLV CM to confer infectivity to HIV-1 and MLV cotransfection of 293T cells, vector harvesting and transduction of human HT1080, canine D17, and murine NIH-3T3 cells were performed as described above. As shown in Table 2, both chimeric envelopes allowed the formation of infectious MLV vector particles and were able to mediate infectivity in HT1080 and D17 cells (Glvr-1⫹), but not NIH-3T3 cells (Glvr-1⫺), as expected for envelopes harboring the GaLV gp71-SU. The vector titers measured were approximately twofold (GaLV TM) and fourfold (GaLV CM) lower than those measured after transfection of GaLV wt Env in conjunction with the relevant packaging and transfer constructs. More importantly, and in contrast to the GaLV wt Env, only GaLV CM and GaLV TM mediated formation of infectious HIV-1 pseudotype vector particles. HIV-1(GaLV TM) vector particles seemed to be slightly more efficiently generated, as titers of about 1.0 ⫻ 10 4 infectious units per milliliter (i.u./ml) were obtained. However, both pseudotype vector particles exhibited the host cell range of MLV(GaLV wt) pseudotype vector particles, indicating the usage of Glvr-1 for cell entry as expected. In addition, and in contrast to MLVbased vectors, HIV-1-derived vectors pseudotyped with Env of MLV-A, GaLV TM, GaLV CM, and MLV-10A1 were able to efficiently transduce growth-arrested HT1080 cultured in the presence of the DNA-polymerase inhibitor

19

aphidicolin [(20 ␮g/ml), data not shown]. Thus, substitution of the C-terminal domain of GaLV TM by that of MLV TM was shown to be sufficient to overcome the inability of the GaLV wt Env to mediate formation of infectious HIV-1 pseudotype vector particles. Additional substitution of the GaLV TM TMR by that of MLV led to a slight additional increase of vector titers. Whether this is due to more efficient incorporation of the chimeric GaLV-derived Env into HI virions and/or processing of the respective TM protein that may have resulted in enhanced fusiogenicity of Env remains unclear and is currently under investigation. However, the ability to target HIV-1 vector particles to the cell surface receptor Glvr-1 by pseudotyping with Env of MLV-10A1 and Env variants GaLV CM or GaLV TM offers the opportunity to further enhance gene transfer into resting or slowly dividing human cells. In particular, human PBLs and HSCs are attractive targets for lentiviral gene transfer in future gene therapy scenarios, e.g., for the treatment of immune dysfunctions such as severe combined immunodeficiency, T-cell lymphomas, or AIDS. Materials and Methods. Cloning of retroviral env genes. Plasmid pHIT456 (9) encoding the MLV-A env gene [molecular clone 4070A (18)] was digested with XbaI and ClaI. The Env encoding gene fragment was isolated and inserted into the backbone of plasmid pALF-PERV A (kindly donated by Y. Takeuchi, Chester Beatty Laboratories, London, UK), thus replacing the porcine endogenous retrovirus (PERV) env gene coding region that is followed by the C-tail encoding sequence of MLV. The env gene coding region of MLV-10A1 (GenBank Accession No. M33470) was amplified by PCR using DNA of plasmid pcDNA3.1 10A1 env as a template and the oligonucleotide primers 10A1envXba⫹, 5⬘-GCTCTAGAATGGAAGGTCCAGCGTTCTCA-3⬘, and 10A1envXba⫺, 5⬘-GCTCTAGATCATGGCTCGTACTCTATAGG-3⬘. The resulting DNA fragment was also digested with XbaI and ClaI and inserted into the backbone fragment of plasmid pALF-PERV A as described above. For the construction of the GaLV wt env expression gene, we used plasmid pMOV-GaLV (17) as the template for PCR and the oligonucleotide primers GaLVenvXba⫹, 5⬘-GCTCTAGAATGGTATTGCTGCCTGGGTCC-3⬘, and GaLVwtCla⫺, 5⬘CCATCGATTTAAAGGTTACCTTCGTTCTC-3⬘. GaLV CM env was constructed using the primer GaLVwtXba⫹ and the primer GaLVTMR/Cla⫺-, 5⬘-GACCAAATCGATTGATGATGCATGGCCC-3⬘. To generate the env gene variant GaLV TM, two fragments were amplified using (i) pMOV-GaLV and the primers GaLVenvXba⫹ and FUTMR⫺, 5⬘-GGTGGTAAACCAGGGGAGGTATTGAACCATCC-3⬘, and (ii) pHIT456 and the primers FUTMR⫹, 5⬘-GGATGGTTCAATACCTCCCCCTGGTTTACCACC-3⬘ and MLV-AXba⫺, 5⬘-GCTCTAGATCATGGCTCGTACTCTATGGG3⬘. The resulting env gene fragments were then

20

RAPID COMMUNICATION

assembled by fusion-PCR as described previously (19). Cells, transfections, and infections. HT1080 human fibrosarcoma (ATCC CCL121) and D17 canine osteosarcoma cells (ATCC CCL 183) were purchased from the American Tissue Culture Collection. These cells as well as murine NIH-3T3 fibroblasts and human kidney 293T cells were cultured in high glucose (4.5 g/liter) DMEM (Gibco, Eggenstein, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom, Berlin, Germany). Transfection of 293T cells was performed using a total amount of 3 ␮g plasmid DNA (1 ␮g of each plasmid encoding one of the vector components) and the transfection reagent Lipofectamin Plus (Gibco) according to the manufacturer’s instructions. Contaminating packaging cells were removed from the vector particle containing supernatants by passage through a 0.45-␮m filter. The supernatants were then used in various dilutions for the transduction of 1 ⫻ 10 5 target cells seeded in 12-well dishes (Nunc, Wiesbaden, Germany) 24 h prior to infection. Polybrene (10 ␮g/ml; Sigma, Deisenhofen, Germany) was added to the target cells during incubation with the vector particles. After 2 h target cells were washed with PBS and expanded for another 2 days before being subjected to X-Gal staining as described previously (10). Immunostaining and analysis by FACS. Two days posttransfection Env-expressing cells were subjected to immunostaining as described elsewhere (10) using a goat anti-FLV gp71-SU serum (kind donation of H. Schwarz, Max-Planck-Institut, Tu¨bingen, Germany) diluted 1:400 in PBS/1% BSA and 1:1000 diluted peroxidase-coupled protein G (Sigma). For FACS analysis 5 ⫻ 10 5 transfected cells were washed with PBA (PBS with 2% FCS and 0.1% sodium azide) and incubated with the anti-gp71-SU serum dilution for 1 h at 4°C. Cells were then washed twice with PBA and incubated at 4°C for 45 min with 1:40 diluted phyocoerythrin-labeled secondary antibodies directed against goat IgG (Sigma). After being washed twice with PBA, cells were subjected to FACS analysis (FACScan, Becton Dickinson, San Jose, CA). ACKNOWLEDGMENTS This work was supported by Grant 01 KV9919 (01KV9550) of the BMBF and Grant 328-135000/03 of the BMG to K.C. We are indebted to S. Kingsman and A. Kingsman for the donation of the pHIT-plasmid panel, to Y. Takeuchi for the donation of the construct pALF-PERV A, and to H. Schwarz for providing the anti-FLV gp71 serum. We thank R. Wenig and M. Selbert for excellent technical assistance and E. Flory for helpful discussions.

REFERENCES 1. von Kalle, C., Kiem, H. P., Goehle, S., Darovsky, B., Torok-Strob, B., Strob, R., and Schuening, F. G. (1994). Increased gene transfer into human hematopoietic progenitor cells by extended in vitro exposure to a pseudotyped retroviral vector. Blood 84, 2890–2897. 2. Uckert, W., Becker, C., Gladow, M., Klein, D., Kammertoono, T., Pedersen, L., and Blankenstein, T. (2000). Efficient gene transfer into primary human CD8 ⫹ T lymphocytes by MaLV-10A1 retrovirus pseudotype. Hum. Gene Ther. 11, 1005–1014.

3. Richardson, C., and Bank, A. (1996). Developmental-stage-specific expression and regulation of an amphotropic retroviral receptor in hematopoietic cells. Mol. Cell. Biol. 16, 4240–4247. 4. Miller, D. G., and Miller, A. D. (1994). A family of retroviruses that utilize related phosphate transporters for cell entry. J. Virol. 68, 8270–8276. 5. Uchida, N., Sutton, R. E., Friera, A. M., He, D., Reitsman, M. J., Chang, W. C., Veres, G., Scollay, R., and 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. USA 95, 11939–11944. 6. Mochizuki, H., Schwartz, J. P., Tanaka, K., Brady, R. O., and Reiser, J. (1998). High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. J. Virol. 72, 8873–8883. 7. Cosset, F.-L., Takeuchi, Y., Battini, J. L., Weiss, R. A., and Collins, M. K. (1995). High. titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69, 7430–7436. 8. Naldini, L., Blomer, U., Gage, F. H., Trono, D., and Verma, I. M. (1996). Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proc. Natl. Acad. Sci. USA 93, 11382–11388. 9. Soneoka, Y., Cannon, P. M., Ramsdale, E. E., Griffiths, J. C., Romano, G., Kingsman, S. M., and Kingsman, A. J. (1995). A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acids Res. 23, 628–633. 10. Schnierle, B. S., Stitz, J., Bosch, V., Nocken, F., Merget-Millitzer, H., Engelstadter, M., Kurth, R., Groner, B., and Cichutek, K. (1997). Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells. Proc. Natl. Acad. Sci. USA 94, 8640–8645. 11. Bugelski, P. J., Maleeff, B. E., Klinker, A. M., Ventre, J., and Hart, T. K. (1995). Ultrastructural evidence of an interaction between Env and Gag proteins during assembly of HIV type 1. AIDS Res. Hum. Retroviruses 11, 55–64. 12. Sakalian, M., and Hunter, E. (1998). Molecular events in the assembly of retrovirus particles. Adv. Exp. Med. Biol. 440, 329–339. 13. Wilk, T., Pfeiffer, T., and Bosch, V. (1992). Retained in vitro infectivity and cytopathogenicity of HIV-1 despite truncation of the Cterminal tail of the env gene product. Virology 189, 167–177. 14. Henriksson, P., Pfeiffer, T., Zentgraf, H., Alke, A., and Bosch, V. (1999). Incorporation of wild. type and C-terminally truncated human epidermal growth factor receptor into human immunodeficiency virus-like particles: Insight into the processes governing glycoprotein incorporation into retroviral particles. J. Virol. 73, 9294–9302. 15. Rein, A., Mirro, J., Haynes, J. G., Ernst, S. M., and Nagashima, K. (1994). Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E–p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein. J. Virol. 68, 1773–1781. 16. Kiernan, R. E., and Freed, E. O. (1998). Cleavage of the murine leukemia virus transmembrane env protein by human immunodeficiency virus type 1 protease: Transdominant inhibition by matrix mutations. J. Virol. 72, 9621–9627. 17. Miller, A. D., Garcia, J. V., von Suhr, N., Lynch, C. M., Wilson, C., and Eidem, M. V. (1991). Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65, 2220–2224. 18. Ott, D., Friedrich, R., and Rein, A. (1990). Sequence analysis of amphotropic and 10A1 murine leukemia viruses: Close relationship to mink cell focus-inducing viruses. J. Virol. 64, 757–766. 19. Stitz, J., Steidl, S., Merget-Millitzer, H., Konig, R., Muller, P., Nocken, F., Engelstadter, M., Bobkova, M., Schmitt, I., Kurth, R., Buchholz, C. J., and Cichutek, K. (2000). MLV-derived retroviral vectors selective for CD4-expressing cells and resistant to neutralization by sera from HIV-infected patients. Virology 267, 229–236.