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Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors
Journal of Virological Methods 73 (1998) 31 – 39
Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors William C. Manning *, John E. Murphy, Douglas J. Jolly, Steven J. Mento, Robert O. Ralston 1 Chiron Corporation, 4560 Horton Street, Emery6ille, CA 94608, USA Received 24 November 1997; received in revised form 4 February 1998; accepted 4 February 1998
Abstract A new method of producing vesicular stomatitis virus (VSV) G protein pseudotyped retroviral vectors is described. In this method, stocks of VSV-G pseudotyped vector were reproducibly obtained by infecting an en6 − , human, retroviral vector producer cell line with a recombinant murine cytomegalovirus (CMV) which expresses VSV-G protein. The recombinant murine CMV, RMCMVG, expressed VSV-G protein under transcriptional control of the human CMV immediate-early promoter. RMCMVG, like murine CMV, can infect human cells, but the infection is limited to the expression of the viral immediate-early genes; no productive replication of murine CMV occurs. Recombinant murine CMV vector infection of non-permissive cells may be useful in situations where high levels of gene expression are desired without concomitant viral vector replication. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant murine cytomegalovirus; Vesicular stomatitis virus G protein pseudotyped retroviral vectors
1. Introduction The most commonly used retroviral vectors for gene therapy are derived from murine leukemia * Corresponding author. Tel.: + 1 510 9234044; fax: +1 510 9232586; e-mail: william – [email protected] 1 Present address: GeneMedicine, The Woodlands, TX 77381.
virus (MLV). MLV vectors have been used to transduce a wide variety of cell types. Their ability to infect and transduce a cell, however, depends on the cell’s expression of the appropriate MLV receptor (Kavanaugh et al., 1994). The interaction of the viral envelope glycoprotein with its cellular receptor determines both the host range of the virus and its efficiency of infection. In a process referred to as pseudotyping, the host range of a
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00034-2
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retrovirus can be altered by replacing its envelope glycoprotein with an envelope glycoprotein from another virus. The vesicular stomatitis virus (VSV) G envelope protein has been used to pseudotype retroviral vectors (Rose and Gallione, 1981, Emi et al., 1991, Yee et al., 1994). VSV-G protein pseudotyped retroviral vectors have several properties which make them attractive vectors for gene therapy. In contrast to retroviral vectors bearing the MLV envelope which rely on the interaction of their envelope glycoprotein with a specific cellular receptor for infection, VSV-G pseudotyped vectors infect cells through a fusion event which is thought to be mediated by the interaction of VSV-G protein with a ubiquitous phospholipid (Florkiewicz and Rose, 1984, Mastromarino et al., 1987). This allows VSV-G pseudotyped vectors to infect cells from a wide range of species (Lin et al., 1994). Furthermore, VSV-G pseudotyped virions are less labile physically than MLV envelope-bearing virions and can be concentrated by centrifugation to high titer without loss of infectivity (Burns et al., 1993). Currently, VSV-G pseudotyped vectors are produced by transient transfection of an en6 − producer cell line with a plasmid expressing VSV-G protein. Since this system is based on transfection, it is not reproducible or suitable for large-scale manufacture. Attempts to generate producer lines for VSV-G pseudotyped vectors have been difficult due to the cytotoxicity of the G protein. Recently, an inducible system for the production of VSV-G pseudotyped retroviral vectors has been described (Yang et al., 1995). The suitability of this system for large-scale production of vector remains to be determined. The novel approach described here uses a viral vector to deliver the VSV-G protein to the retroviral vector producer cell line. The herpesvirus murine cytomegalovirus (MCMV) was chosen as the viral vector for several reasons. In cell culture, MCMV productive replication is limited to murine fibroblasts. MCMV, however, can infect many non-murine cells, including human cell lines, but viral transcription and gene expression are limited to the immediate-early class of genes and no productive viral replication occurs
(Walker and Hudson, 1987). Use of a recombinant MCMV which expresses VSV-G under transcriptional control of an immediate-early promoter takes advantage of the natural host specificity of the virus and permits VSV-G expression in human cell lines without concomitant MCMV replication. Previous studies have demonstrated that it is possible to construct recombinant MCMV which expresses foreign genes (Manning and Mocarski, 1988, Manning et al., 1992, Stoddart et al., 1994, Vieira et al., 1994). One of these recombinant viruses, RM427, expressed lacZ under transcriptional control of the human CMV (HCMV) immediate-early promoter. In RM427, lacZ was expressed as an immediate-early gene showing that the HCMV immediate-early promoter functioned with normal kinetics in the MCMV genome (Manning et al., 1992). A recombinant MCMV, RMCMVG, was constructed that expressed VSV-G protein under transcriptional control of the HCMV immediateearly promoter. RMCMVG infection of a human en6 − retrovirus producer cell line resulted in the production of VSV-G pseudotyped retroviral vectors.
2. Methods
2.1. Viruses and cell lines Wild-type MCMV (Smith strain K181+ ; Stoddart et al., 1994), RM427 (Manning et al., 1992) and RMCMVG were grown and titered on NIH3T3 cells. NIH3T3, HeLa, 293, and 293GP (Irwin et al., 1994), a 293 line containing MLV gag and pol but lacking an en6 gene, cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum.
2.2. Plasmids and preparation of recombinant 6irus Plasmid cloning was by standard techniques (Maniatis et al., 1982). pON401 contains the HindIII L fragment of MCMV (Manning and Mocarski, 1988). pCMV-G contains the VSV-G
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Fig. 1. Structure of recombinant plasmids and viruses. A diagram of MCMV HindIII L is shown (Mercer et al., 1983). The HindIII and HpaI sites are indicated. The solid bar represents sequences contained in pON401. The immediate-early genes ie1 and ie2, their direction of transcription, and splicing patterns (Keil et al., 1987, Messerle et al., 1991) are indicated by arrows below the diagram. The hatched box indicates the position of the MCMV enhancer region. Below the map, the positions of the lacZ and VSV-G insertions in RM427 and RMCMVG are diagrammed. The shaded boxes represent the HCMV promoter-enhancers.
protein under transcriptional control of the HCMV ie1 promoter-enhancer (provided by Steve Chang, Chiron Corporation). To construct pON401-CMVG, the 2665-bp HincII fragment of pCMV-G containing VSV-G was cloned between the HpaI sites of pON401 (Fig. 1). pMLP-G contains the VSV-G protein under transcriptional control of the adenovirus major late promoter (provided by Steve Chang, Chiron Corporation). Protocols for the production and isolation of recombinant MCMV have been described (Manning and Mocarski, 1988). Briefly, NIH3T3 cells were co-transfected with 15 mg of linearized pON401-CMVG and RM427 viral DNA by cal-
cium phosphate precipitation. The amount of RM427 DNA used in the transfection was determined empirically for each viral DNA preparation. The co-transfections were harvested when cytopathic effect was complete, approximately 5 days after transfection. To isolate lacZ − recombinants, a limiting dilution assay was done. Briefly, 96-well tissue culture plates were seeded with NIH3T3 cells (1× 104 cells/well). Recombinant virus pools arising from the co-transfections were titered, and then used to infect 96-well plates at a dilution which would contain approximately 75 plaque-forming units (PFU) per 96-well plate. When cytopathic effect
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was evident (about 7 days after infection), 4methylumbelliferyl-b-D-galactoside (Sigma) was added to all wells at a final concentration of 150 mg/ml. At various times after infection (3 – 24 h) the plate was put on a UV light box and photographed. Wells containing parental lacZ + virus (RM427) would fluoresce. Uninfected wells, or wells infected by a lacZ − recombinant, would not fluoresce. Virus from non-fluorescing wells with cytopathic effect were expanded and analyzed by DNA blot analysis.
2.3. DNA analysis Viral DNA was prepared from purified virions as described (Vieira et al., 1994) and subjected to DNA blot analysis using standard techniques (Maniatis et al., 1982).
2.4. Isolation of infected cell proteins and immunoblotting Cells were infected with virus at a multiplicity of infection (MOI) of 10. To prepare total cell protein, cells were rinsed with phosphate buffered saline and then lysed in NP40 lysis buffer (100 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 0.5% NP40, 0.5% deoxycholate) supplemented with aprotinin (2 mg/ml), pepstatin (1 mg/ml), leupeptin (2 mg/ml), and Peflabloc SC (1 mg/ml). All protease inhibitors were purchased from Boehringer Mannheim. Plates were incubated on ice for 5 min. Cells were harvested by scraping, placed in 1.5 ml microcentrifuge tubes, and spun in a microfuge at high speed for 10 min at 4°C. Supernatants were collected and stored at − 80°C until analyzed. Infected cell proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels (Novex), transferred to nitrocellulose, and subjected to Western blot analysis. The primary antibody was a mouse monoclonal to VSV-G glycoprotein (Sigma ImmunoChemicals) used at a dilution of 1:100000. Peroxidase-conjugated goat anti-mouse (Boehringer Mannheim) at a dilution of 1:30000 was used as the secondary antibody. Blots were developed using ECL (Amersham) following manufacturer’s instructions.
2.5. Producer cells, retro6iral 6ector production, and titration To make the cell line for use in the production of pseudotyped vector, 293GP cells were infected with N2 LacZ, an MLV-based, VSV-G pseudotyped retroviral vector (Irwin et al., 1994). N2 LacZ contains the genes for b-galactosidase (lacZ), under transcriptional control of the MLV LTR (long terminal repeat unit), and neomycin phosphotransferase (neor), driven by the SV40 promoter. After infection, G418 resistant colonies were selected. Staining of the resulting neor cell lines with Bluogal (Gibco BRL) showed that all cells were blue. To produce VSV-G pseudotyped retrovirus, 10 cm plates of 293GP (N2 LacZ) cells were infected with RMCMVG at the desired MOI in a total volume of 2 ml. Two hours after infection, the cells were washed twice, and then incubated in 2 ml of medium until harvested; 24 or 48 h after infection, supernatants were filtered (0.45 mm pore) and stored at −80°C until titered. Supernatants were titered on HeLa cells. The day before infection 10 cm plates were seeded with 2× 106 cells. On day 1, plates were infected with 100 ml of supernatant in 2 ml of media containing Polybrene (Sigma; 8 mg/ml). After a 2 h incubation, the plates were washed, and incubated overnight with fresh media. On day 2, plates infected with supernantants from RMCMVG infected cells were split 1:10 and 1:100. Those from mock or wild-type MCMV infected supernatants were split 1:2. On day 3, G418 (Sigma; 1 mg/ml) was added to the media. Ten to 14 days later, colonies were stained and counted. Estimations of titer were also done by infecting 293 cells with supernatant and then staining for b-galactosidase activity 48 h after infection.
3. Results and discussion
3.1. Construction of RMCMVG RMCMVG was isolated following co-transfection of NIH3T3 cells with RM427 viral DNA and pON401-CMVG (Fig. 1). RM427, which is lacZ+, was chosen as the parental virus rather than
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Fig. 2. DNA blot analysis of recombinant viruses. DNA from parental RM427 and three isolates of RMCMVG was digested with HindIII, and separated on a 0.7% agarose gel. After transfer to nitrocellulose, the blot was hybridized with a 32P-labeled NcoI fragment from pCMV-G as indicated in the diagram. The 5.7 kbp and 4.3 kbp HindIII fragments expected for RMCMVG and the 3.9 kbp fragment expected for RM427 are indicated.
wild-type MCMV to allow the use of lacZ phenotype as a means of detecting recombinant virus. Recombination between pON401-CMVG and RM427 DNA would result in the replacement of VSV-G for lacZ, yielding lacZ − recombinants. After screening eight 96-well plates for lacZ − recombinants, six wells were identified that contained lacZ − virus. Three of these were grown up and plaque purified. These represented recombinants arising from two independent cotransfections. Viral DNA was prepared from the three isolates and subjected to DNA blot analysis. As shown in Fig. 2, all three recombinant viruses contained the 5.7 kbp and 4.3 kbp HindIII bands predicted for a VSV-G recombi-
nant. Isolate D3 was not yet plaque-purified and contained the 3.9 kbp RM427 HindIII fragment in addition to the two HindIII fragments predicted for RMCMVG. Isolate D9 was grown up and stock titers of approximately 1× 108 PFU/ ml were obtained. This was identical to titers obtained with stocks of wild-type MCMV, indicating that VSV-G protein expression does not interfere with MCMV replication in cell culture. In sharp contrast to wild-type MCMV, RMCMVG plaques demonstrated a marked syncytial phenotype (Fig. 3). The syncytial phenotype of RMCMVG was not surprising given that transfection of cells with plasmids expressing VSV-G leads to syncytium formation.
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Fig. 3. RMCMVG plaques exhibit a syncytium phenotype. NIH3T3 cells were infected with wild-type MCMV (A) or RMCMVG (B) at a dilution where individual plaques could be seen. The magnification was ×200.
3.2. RMCMVG expresses VSV-G protein in permissi6e and non-permissi6e cells To test the ability of RMCMVG to express VSV-G in murine cells, NIH3T3 cells were infected with the three isolates of RMCMVG and subjected to immunoblot analysis. As demonstrated in Fig. 4, NIH3T3 cells infected with each of the three isolates expressed abundant amounts of VSV-G protein by 24 h after infection. Cells infected with the parent virus, RM427, did not show any VSV-G expression. As a positive control, a lane containing VSV-G protein produced after transfection of 293 cells with pMLP-G was included on the gel. The two proteins of slightly different molecular weight that are recognized by the VSV-G monoclonal antibody may represent differently glycosylated forms of VSV-G. The same two bands are seen in the lane containing lysate from pMLP-G transfected cells indicating that the appearance of the two forms of VSV-G is not related to VSV-G expression in the MCMV genome. To determine whether RMCMVG also expressed VSV-G protein in non-permissive human cells, 293 cells were infected with RMCMVG and protein was harvested at 6, 24, and 48 h after infection. As shown in Fig. 5, VSV-G protein expression was detected as early as 6 h postinfection, but peaked at 24 h postinfection.
Fig. 4. RMCMVG expresses VSV-G protein in NIH3T3 cells. NIH3T3 cells were infected with RMCMVG isolates D3, D8, and D9 at an MOI of 10. At 24 h after infection, cell lysates were prepared and subjected to immunoblot analysis using a monoclonal antibody against VSV-G protein. Mock infected and RM427 infected cells are included as controls. The last lane contains VSV-G expressed in 293 cells after transfection with the expression vector pMLP-G. The position of VSV-G is indicated by arrows.
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Fig. 5. RMCMVG expresses VSV-G protein in human 293 cells. The 293 cells were mock infected or infected with RMCMVG at an MOI of 10. At 6, 24, and 48 h after infection, cells were lysed and analyzed by immunoblot analysis as described in Fig. 4. The position of VSV-G protein is indicated by arrows.
3.3. RMCMVG can be used to generate VSV-G pseudotyped retro6iral 6ectors Since it was clear that RMCMVG infection led to the expression of VSV-G in human cell lines, the use of RMCMVG infection to produce VSVG pseudotyped retroviral vectors was examined. 293GP (N2 LacZ) cells were infected with either Table 1 VSV-G pseudotyped vector production from 293GP (N2 LacZ) cells following infection with RMCMVG or wild-type MCMV Virus
RMCMVG
Wild-type MCMV
Mock a
MOIa
1 10 20 1 10 20
Titer (CFU/ml)b 24 h
48 h
9.0×103 2.3×104 4.2×104 NDc ND ND ND
2.0×102 4.0×103 1.2×104 ND ND ND ND
Multiplicity of infection used to infect the 293GP (N2 LacZ) cells. b Supernatants were harvested from 293GP (N2 LacZ) cells infected with either RMCMVG or wild-type MCMV at 24 h and 48 h postinfection. CFU titers were obtained as described in Section 2.c ND, no colonies detected.
wild-type MCMV or RMCMVG. Supernants were collected from cells at 24 h and 48 h after infection, and titered. As shown in Table 1, peak titers of approximately 1× 104 colony-forming units (CFU)/ml were found in the supernatants of cells 24 h after infection by RMCMVG at an MOI of 10 or 20. No CFU titers were found in the supernatants of mock infected cells or cells infected with wild-type MCMV. Since 293 cells are not permissive for MCMV replication, there should be no RMCMVG in the supernatants. When supernantants were titered for RMCMVG, titers of 200 PFU/ml were found. This small amount of MCMV is probably virus remaining from the initial infection. The small amount of residual RMCMVG in the VSV-G pseudotyped retroviral vector stocks is probably not important in most applications. It may be possible, however, to further reduce RMCMVG contamination by doing more stringent washes after RMCMVG infection, or by using a lower MOI. The titer of VSV-G pseudotyped vector obtained using this method was about 1–2 orders of magnitude lower than the titer obtained by transient transfection of a VSV-G expressing plasmid. To examine possible reasons for this difference in titer, the two methods were directly compared.
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Fig. 6. Infection with RMCMVG leads to more expression of VSV-G than transient transfection of pCMV-G. The 293GP (N2 LacZ) cells were infected with RMCMVG (MOI 10) or transfected with pCMV-G (5 mg, calcium phosphate precipitation). At 24 h after infection/transfection, cells were lysed and subjected to immunoblot analysis as described in Fig. 4. Lysates were normalized for total protein concentration. The position of VSV-G protein is indicated by arrows.
Identical 60 mm plates of 293GP (N2 LacZ) cells were either transfected with pCMV-G (5 mg) or infected with RMCMVG (MOI of 10). At 24 h after transfection, or infection, the supernatants were titered, and the cells were lysed and assayed for VSV-G expression by immunoblot analysis. As shown in Fig. 6, there was more VSV-G in the cells infected by RMCMVG than in the cells transfected with pCMV-G. When the supernatants were titered, however, the transfected cells had a titer of about 106 transducing units/ml and the infected cells had a titer of about 104 transducing units/ml (data not shown). One possible explanation for the discrepancy between VSV-G protein expression and titer may be that the high level of VSV-G protein expressed by RMCMVG interferes with normal glycoprotein processing and transport to the cytoplasmic membrane. The cloning of VSV-G into the MCMV ie2 locus resulted in the juxtaposition of the MCMV and HCMV enhancers. This may be responsible for the very high level of VSV-G expression in RMCMVG. Moving VSV-G to another locus in the MCMV genome might result in less VSV-G protein expression, more efficient VSV-G maturation, and possibly increased pseudotyped vector titer. We are currently examining ways to increase the yield of VSV-G pseudotyped vector from our system.
The use of a recombinant MCMV vector in the production of VSV-G protein pseudotyped retroviral vectors highlights some of the features which make recombinant MCMV an attractive expression vector. One of these is the high level of gene expression achieved by using a CMV immediateearly promoter on a CMV genome in a non-permissive cell. In a non-permissive cell, this high level of gene expression is not associated with viral replication. A second benefit is delivery. Viral infection is more efficient than transfection in delivering a gene to a cell. MCMV vectors may be especially useful for the expression of proteins in cells which are not easily transfected by standard methods. The combination of high levels of expression and efficiency of delivery make recombinant MCMV an attractive expression vector.
Acknowledgements We thank Edward Mocarski for the generous gifts of MCMV (K181+ , RM427), and pON401. We also thank Steve Chang for providing the VSV-G expression vectors pCMV-G and pMLPG.
References Burns, J.C., Friedmann, T., Driever, W., Burrascano, M., Yee, J.K., 1993. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. U. S. A. 90, 8033 – 8037. Emi, N., Friedmann, T., Yee, J.K., 1991. Pseudotype formation of murine leukemia virus with the G protein of vesicular stomatitis virus. J. Virol. 65, 1202 – 1207. Florkiewicz, R.C., Rose, J.K., 1984. A cell line expressing vesicular stomatitis virus glycoprotein fuses at low pH. Science 225, 721 – 723. Irwin, M.J., Laube, L.S., Lee, V., Austin, M., Chada, S., Anderson, C.G., Townsend, K., Jolly, D.J., Warner, J.F., 1994. Direct injection of a recombinant retroviral vector induces human immunodeficiency virus-specific immune responses in mice and nonhuman primates. J. Virol. 68, 5036 – 5044. Kavanaugh, M.P., Miller, D.G., Zhang, W., Law, W., Kozak, S.L., Kabat, D., Miller, D.A., 1994. Cell-surface receptors for gibbon ape leukemia virus and amphotropic murine retrovirus are inducible sodium-dependent phosphate symporters. Proc. Natl. Acad. Sci. U. S. A. 91, 7071 – 7075.
W.C. Manning et al. / Journal of Virological Methods 73 (1998) 31–39 Keil, G.M., Ebeling, K.A., Koszinowski, U.H., 1987. Immediate early genes of murine cytomegalovirus: location, transcripts, and translation products. J. Virol. 61, 526–533. Lin, S., Gaiano, N., Culp, P., Burns, J.C., Friedmann, T., Yee, J.K., Hopkins, N., 1994. Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265, 666 – 668. Maniatis, T., Fritsch, E., Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Manning, W.C., Mocarski, E.S., 1988. Insertional mutagenesis of the murine cytomegalovirus genome: one prominent alpha gene (ie2 ) is dispensable for growth. Virology 167, 477 – 484. Manning, W.C., Stoddart, C.A., Lagenaur, L.A., Abenes, G.B., Mocarski, E.S., 1992. Cytomegalovirus determinant of replication in salivary glands. J. Virol. 66, 3794–3802. Mastromarino, P., Conti, C., Goldoni, P., Hauttecoeur, B., Orsi, N., 1987. Characterization of membrane components of the erythrocyte involved in vesicular stomatitis virus attachment and fusion at acidic pH. J. Gen. Virol. 68, 2359 – 2369. Mercer, J.A., Marks, J.R., Spector, D.H., 1983. Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith strain). Virology 129, 94 – 106. Messerle, M., Keil, G.M., Koszinowski, U.H., 1991. Structure and expression of the murine cytomegalovirus immediateearly gene 2. J. Virol. 65, 1638–1643.
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Rose, J.K., Gallione, C., 1981. Nucleotide sequences of the mRNA encoding the VSV G and M proteins as determined from cDNA clones containing the complete coding regions. J. Virol. 39, 519 – 528. Stoddart, C.A., Cardin, R.D., Boname, J.M., Manning, W.C., Abenes, G.B., Mocarski, E.S., 1994. Peripheral blood mononuclear phagocytes mediate dissemination of murine cytomegalovirus. J. Virol. 68, 6243 – 6253. Vieira, J., Farrell, H.E., Rawlinson, W.D., Mocarski, E.S., 1994. Genes in the HindIII J fragment of the murine cytomegalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette. J. Virol. 68, 4837 – 4846. Walker, D.G., Hudson, J.B., 1987. Analysis of immediateearly and early proteins of murine cytomegalovirus in permissive and nonpermissive cells. Arch. Virol. 92, 103 – 119. Yang, Y., Vanin, E.F., Whitt, M.A., Fornerod, M., Zwart, R., Schneiderman, R.D., Grosveld, G., Nienhuis, A.W., 1995. Inducible, high-level production of infectious murine leukemia retroviral vector particles pseudotyped with vesicular stomatitis virus G envelope protein. Hum. Gene Ther. 6, 1203 – 1213. Yee, J.K., Miyanohara, A., Laporte, P., Bouic, K., Burns, J.C., Friedmann, T., 1994. A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 91, 9564 – 9568.
Use of a recombinant murine cytomegalovirus expressing vesicular stomatitis virus G protein to pseudotype retroviral vectors William C. Manning *, John E. Murphy, Douglas J. Jolly, Steven J. Mento, Robert O. Ralston 1 Chiron Corporation, 4560 Horton Street, Emery6ille, CA 94608, USA Received 24 November 1997; received in revised form 4 February 1998; accepted 4 February 1998
Abstract A new method of producing vesicular stomatitis virus (VSV) G protein pseudotyped retroviral vectors is described. In this method, stocks of VSV-G pseudotyped vector were reproducibly obtained by infecting an en6 − , human, retroviral vector producer cell line with a recombinant murine cytomegalovirus (CMV) which expresses VSV-G protein. The recombinant murine CMV, RMCMVG, expressed VSV-G protein under transcriptional control of the human CMV immediate-early promoter. RMCMVG, like murine CMV, can infect human cells, but the infection is limited to the expression of the viral immediate-early genes; no productive replication of murine CMV occurs. Recombinant murine CMV vector infection of non-permissive cells may be useful in situations where high levels of gene expression are desired without concomitant viral vector replication. © 1998 Elsevier Science B.V. All rights reserved. Keywords: Recombinant murine cytomegalovirus; Vesicular stomatitis virus G protein pseudotyped retroviral vectors
1. Introduction The most commonly used retroviral vectors for gene therapy are derived from murine leukemia * Corresponding author. Tel.: + 1 510 9234044; fax: +1 510 9232586; e-mail: william – [email protected] 1 Present address: GeneMedicine, The Woodlands, TX 77381.
virus (MLV). MLV vectors have been used to transduce a wide variety of cell types. Their ability to infect and transduce a cell, however, depends on the cell’s expression of the appropriate MLV receptor (Kavanaugh et al., 1994). The interaction of the viral envelope glycoprotein with its cellular receptor determines both the host range of the virus and its efficiency of infection. In a process referred to as pseudotyping, the host range of a
0166-0934/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII S0166-0934(98)00034-2
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W.C. Manning et al. / Journal of Virological Methods 73 (1998) 31–39
retrovirus can be altered by replacing its envelope glycoprotein with an envelope glycoprotein from another virus. The vesicular stomatitis virus (VSV) G envelope protein has been used to pseudotype retroviral vectors (Rose and Gallione, 1981, Emi et al., 1991, Yee et al., 1994). VSV-G protein pseudotyped retroviral vectors have several properties which make them attractive vectors for gene therapy. In contrast to retroviral vectors bearing the MLV envelope which rely on the interaction of their envelope glycoprotein with a specific cellular receptor for infection, VSV-G pseudotyped vectors infect cells through a fusion event which is thought to be mediated by the interaction of VSV-G protein with a ubiquitous phospholipid (Florkiewicz and Rose, 1984, Mastromarino et al., 1987). This allows VSV-G pseudotyped vectors to infect cells from a wide range of species (Lin et al., 1994). Furthermore, VSV-G pseudotyped virions are less labile physically than MLV envelope-bearing virions and can be concentrated by centrifugation to high titer without loss of infectivity (Burns et al., 1993). Currently, VSV-G pseudotyped vectors are produced by transient transfection of an en6 − producer cell line with a plasmid expressing VSV-G protein. Since this system is based on transfection, it is not reproducible or suitable for large-scale manufacture. Attempts to generate producer lines for VSV-G pseudotyped vectors have been difficult due to the cytotoxicity of the G protein. Recently, an inducible system for the production of VSV-G pseudotyped retroviral vectors has been described (Yang et al., 1995). The suitability of this system for large-scale production of vector remains to be determined. The novel approach described here uses a viral vector to deliver the VSV-G protein to the retroviral vector producer cell line. The herpesvirus murine cytomegalovirus (MCMV) was chosen as the viral vector for several reasons. In cell culture, MCMV productive replication is limited to murine fibroblasts. MCMV, however, can infect many non-murine cells, including human cell lines, but viral transcription and gene expression are limited to the immediate-early class of genes and no productive viral replication occurs
(Walker and Hudson, 1987). Use of a recombinant MCMV which expresses VSV-G under transcriptional control of an immediate-early promoter takes advantage of the natural host specificity of the virus and permits VSV-G expression in human cell lines without concomitant MCMV replication. Previous studies have demonstrated that it is possible to construct recombinant MCMV which expresses foreign genes (Manning and Mocarski, 1988, Manning et al., 1992, Stoddart et al., 1994, Vieira et al., 1994). One of these recombinant viruses, RM427, expressed lacZ under transcriptional control of the human CMV (HCMV) immediate-early promoter. In RM427, lacZ was expressed as an immediate-early gene showing that the HCMV immediate-early promoter functioned with normal kinetics in the MCMV genome (Manning et al., 1992). A recombinant MCMV, RMCMVG, was constructed that expressed VSV-G protein under transcriptional control of the HCMV immediateearly promoter. RMCMVG infection of a human en6 − retrovirus producer cell line resulted in the production of VSV-G pseudotyped retroviral vectors.
2. Methods
2.1. Viruses and cell lines Wild-type MCMV (Smith strain K181+ ; Stoddart et al., 1994), RM427 (Manning et al., 1992) and RMCMVG were grown and titered on NIH3T3 cells. NIH3T3, HeLa, 293, and 293GP (Irwin et al., 1994), a 293 line containing MLV gag and pol but lacking an en6 gene, cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum.
2.2. Plasmids and preparation of recombinant 6irus Plasmid cloning was by standard techniques (Maniatis et al., 1982). pON401 contains the HindIII L fragment of MCMV (Manning and Mocarski, 1988). pCMV-G contains the VSV-G
W.C. Manning et al. / Journal of Virological Methods 73 (1998) 31–39
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Fig. 1. Structure of recombinant plasmids and viruses. A diagram of MCMV HindIII L is shown (Mercer et al., 1983). The HindIII and HpaI sites are indicated. The solid bar represents sequences contained in pON401. The immediate-early genes ie1 and ie2, their direction of transcription, and splicing patterns (Keil et al., 1987, Messerle et al., 1991) are indicated by arrows below the diagram. The hatched box indicates the position of the MCMV enhancer region. Below the map, the positions of the lacZ and VSV-G insertions in RM427 and RMCMVG are diagrammed. The shaded boxes represent the HCMV promoter-enhancers.
protein under transcriptional control of the HCMV ie1 promoter-enhancer (provided by Steve Chang, Chiron Corporation). To construct pON401-CMVG, the 2665-bp HincII fragment of pCMV-G containing VSV-G was cloned between the HpaI sites of pON401 (Fig. 1). pMLP-G contains the VSV-G protein under transcriptional control of the adenovirus major late promoter (provided by Steve Chang, Chiron Corporation). Protocols for the production and isolation of recombinant MCMV have been described (Manning and Mocarski, 1988). Briefly, NIH3T3 cells were co-transfected with 15 mg of linearized pON401-CMVG and RM427 viral DNA by cal-
cium phosphate precipitation. The amount of RM427 DNA used in the transfection was determined empirically for each viral DNA preparation. The co-transfections were harvested when cytopathic effect was complete, approximately 5 days after transfection. To isolate lacZ − recombinants, a limiting dilution assay was done. Briefly, 96-well tissue culture plates were seeded with NIH3T3 cells (1× 104 cells/well). Recombinant virus pools arising from the co-transfections were titered, and then used to infect 96-well plates at a dilution which would contain approximately 75 plaque-forming units (PFU) per 96-well plate. When cytopathic effect
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was evident (about 7 days after infection), 4methylumbelliferyl-b-D-galactoside (Sigma) was added to all wells at a final concentration of 150 mg/ml. At various times after infection (3 – 24 h) the plate was put on a UV light box and photographed. Wells containing parental lacZ + virus (RM427) would fluoresce. Uninfected wells, or wells infected by a lacZ − recombinant, would not fluoresce. Virus from non-fluorescing wells with cytopathic effect were expanded and analyzed by DNA blot analysis.
2.3. DNA analysis Viral DNA was prepared from purified virions as described (Vieira et al., 1994) and subjected to DNA blot analysis using standard techniques (Maniatis et al., 1982).
2.4. Isolation of infected cell proteins and immunoblotting Cells were infected with virus at a multiplicity of infection (MOI) of 10. To prepare total cell protein, cells were rinsed with phosphate buffered saline and then lysed in NP40 lysis buffer (100 mM NaCl, 20 mM Tris pH 7.5, 1 mM EDTA, 0.5% NP40, 0.5% deoxycholate) supplemented with aprotinin (2 mg/ml), pepstatin (1 mg/ml), leupeptin (2 mg/ml), and Peflabloc SC (1 mg/ml). All protease inhibitors were purchased from Boehringer Mannheim. Plates were incubated on ice for 5 min. Cells were harvested by scraping, placed in 1.5 ml microcentrifuge tubes, and spun in a microfuge at high speed for 10 min at 4°C. Supernatants were collected and stored at − 80°C until analyzed. Infected cell proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels (Novex), transferred to nitrocellulose, and subjected to Western blot analysis. The primary antibody was a mouse monoclonal to VSV-G glycoprotein (Sigma ImmunoChemicals) used at a dilution of 1:100000. Peroxidase-conjugated goat anti-mouse (Boehringer Mannheim) at a dilution of 1:30000 was used as the secondary antibody. Blots were developed using ECL (Amersham) following manufacturer’s instructions.
2.5. Producer cells, retro6iral 6ector production, and titration To make the cell line for use in the production of pseudotyped vector, 293GP cells were infected with N2 LacZ, an MLV-based, VSV-G pseudotyped retroviral vector (Irwin et al., 1994). N2 LacZ contains the genes for b-galactosidase (lacZ), under transcriptional control of the MLV LTR (long terminal repeat unit), and neomycin phosphotransferase (neor), driven by the SV40 promoter. After infection, G418 resistant colonies were selected. Staining of the resulting neor cell lines with Bluogal (Gibco BRL) showed that all cells were blue. To produce VSV-G pseudotyped retrovirus, 10 cm plates of 293GP (N2 LacZ) cells were infected with RMCMVG at the desired MOI in a total volume of 2 ml. Two hours after infection, the cells were washed twice, and then incubated in 2 ml of medium until harvested; 24 or 48 h after infection, supernatants were filtered (0.45 mm pore) and stored at −80°C until titered. Supernatants were titered on HeLa cells. The day before infection 10 cm plates were seeded with 2× 106 cells. On day 1, plates were infected with 100 ml of supernatant in 2 ml of media containing Polybrene (Sigma; 8 mg/ml). After a 2 h incubation, the plates were washed, and incubated overnight with fresh media. On day 2, plates infected with supernantants from RMCMVG infected cells were split 1:10 and 1:100. Those from mock or wild-type MCMV infected supernatants were split 1:2. On day 3, G418 (Sigma; 1 mg/ml) was added to the media. Ten to 14 days later, colonies were stained and counted. Estimations of titer were also done by infecting 293 cells with supernatant and then staining for b-galactosidase activity 48 h after infection.
3. Results and discussion
3.1. Construction of RMCMVG RMCMVG was isolated following co-transfection of NIH3T3 cells with RM427 viral DNA and pON401-CMVG (Fig. 1). RM427, which is lacZ+, was chosen as the parental virus rather than
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Fig. 2. DNA blot analysis of recombinant viruses. DNA from parental RM427 and three isolates of RMCMVG was digested with HindIII, and separated on a 0.7% agarose gel. After transfer to nitrocellulose, the blot was hybridized with a 32P-labeled NcoI fragment from pCMV-G as indicated in the diagram. The 5.7 kbp and 4.3 kbp HindIII fragments expected for RMCMVG and the 3.9 kbp fragment expected for RM427 are indicated.
wild-type MCMV to allow the use of lacZ phenotype as a means of detecting recombinant virus. Recombination between pON401-CMVG and RM427 DNA would result in the replacement of VSV-G for lacZ, yielding lacZ − recombinants. After screening eight 96-well plates for lacZ − recombinants, six wells were identified that contained lacZ − virus. Three of these were grown up and plaque purified. These represented recombinants arising from two independent cotransfections. Viral DNA was prepared from the three isolates and subjected to DNA blot analysis. As shown in Fig. 2, all three recombinant viruses contained the 5.7 kbp and 4.3 kbp HindIII bands predicted for a VSV-G recombi-
nant. Isolate D3 was not yet plaque-purified and contained the 3.9 kbp RM427 HindIII fragment in addition to the two HindIII fragments predicted for RMCMVG. Isolate D9 was grown up and stock titers of approximately 1× 108 PFU/ ml were obtained. This was identical to titers obtained with stocks of wild-type MCMV, indicating that VSV-G protein expression does not interfere with MCMV replication in cell culture. In sharp contrast to wild-type MCMV, RMCMVG plaques demonstrated a marked syncytial phenotype (Fig. 3). The syncytial phenotype of RMCMVG was not surprising given that transfection of cells with plasmids expressing VSV-G leads to syncytium formation.
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Fig. 3. RMCMVG plaques exhibit a syncytium phenotype. NIH3T3 cells were infected with wild-type MCMV (A) or RMCMVG (B) at a dilution where individual plaques could be seen. The magnification was ×200.
3.2. RMCMVG expresses VSV-G protein in permissi6e and non-permissi6e cells To test the ability of RMCMVG to express VSV-G in murine cells, NIH3T3 cells were infected with the three isolates of RMCMVG and subjected to immunoblot analysis. As demonstrated in Fig. 4, NIH3T3 cells infected with each of the three isolates expressed abundant amounts of VSV-G protein by 24 h after infection. Cells infected with the parent virus, RM427, did not show any VSV-G expression. As a positive control, a lane containing VSV-G protein produced after transfection of 293 cells with pMLP-G was included on the gel. The two proteins of slightly different molecular weight that are recognized by the VSV-G monoclonal antibody may represent differently glycosylated forms of VSV-G. The same two bands are seen in the lane containing lysate from pMLP-G transfected cells indicating that the appearance of the two forms of VSV-G is not related to VSV-G expression in the MCMV genome. To determine whether RMCMVG also expressed VSV-G protein in non-permissive human cells, 293 cells were infected with RMCMVG and protein was harvested at 6, 24, and 48 h after infection. As shown in Fig. 5, VSV-G protein expression was detected as early as 6 h postinfection, but peaked at 24 h postinfection.
Fig. 4. RMCMVG expresses VSV-G protein in NIH3T3 cells. NIH3T3 cells were infected with RMCMVG isolates D3, D8, and D9 at an MOI of 10. At 24 h after infection, cell lysates were prepared and subjected to immunoblot analysis using a monoclonal antibody against VSV-G protein. Mock infected and RM427 infected cells are included as controls. The last lane contains VSV-G expressed in 293 cells after transfection with the expression vector pMLP-G. The position of VSV-G is indicated by arrows.
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Fig. 5. RMCMVG expresses VSV-G protein in human 293 cells. The 293 cells were mock infected or infected with RMCMVG at an MOI of 10. At 6, 24, and 48 h after infection, cells were lysed and analyzed by immunoblot analysis as described in Fig. 4. The position of VSV-G protein is indicated by arrows.
3.3. RMCMVG can be used to generate VSV-G pseudotyped retro6iral 6ectors Since it was clear that RMCMVG infection led to the expression of VSV-G in human cell lines, the use of RMCMVG infection to produce VSVG pseudotyped retroviral vectors was examined. 293GP (N2 LacZ) cells were infected with either Table 1 VSV-G pseudotyped vector production from 293GP (N2 LacZ) cells following infection with RMCMVG or wild-type MCMV Virus
RMCMVG
Wild-type MCMV
Mock a
MOIa
1 10 20 1 10 20
Titer (CFU/ml)b 24 h
48 h
9.0×103 2.3×104 4.2×104 NDc ND ND ND
2.0×102 4.0×103 1.2×104 ND ND ND ND
Multiplicity of infection used to infect the 293GP (N2 LacZ) cells. b Supernatants were harvested from 293GP (N2 LacZ) cells infected with either RMCMVG or wild-type MCMV at 24 h and 48 h postinfection. CFU titers were obtained as described in Section 2.c ND, no colonies detected.
wild-type MCMV or RMCMVG. Supernants were collected from cells at 24 h and 48 h after infection, and titered. As shown in Table 1, peak titers of approximately 1× 104 colony-forming units (CFU)/ml were found in the supernatants of cells 24 h after infection by RMCMVG at an MOI of 10 or 20. No CFU titers were found in the supernatants of mock infected cells or cells infected with wild-type MCMV. Since 293 cells are not permissive for MCMV replication, there should be no RMCMVG in the supernatants. When supernantants were titered for RMCMVG, titers of 200 PFU/ml were found. This small amount of MCMV is probably virus remaining from the initial infection. The small amount of residual RMCMVG in the VSV-G pseudotyped retroviral vector stocks is probably not important in most applications. It may be possible, however, to further reduce RMCMVG contamination by doing more stringent washes after RMCMVG infection, or by using a lower MOI. The titer of VSV-G pseudotyped vector obtained using this method was about 1–2 orders of magnitude lower than the titer obtained by transient transfection of a VSV-G expressing plasmid. To examine possible reasons for this difference in titer, the two methods were directly compared.
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Fig. 6. Infection with RMCMVG leads to more expression of VSV-G than transient transfection of pCMV-G. The 293GP (N2 LacZ) cells were infected with RMCMVG (MOI 10) or transfected with pCMV-G (5 mg, calcium phosphate precipitation). At 24 h after infection/transfection, cells were lysed and subjected to immunoblot analysis as described in Fig. 4. Lysates were normalized for total protein concentration. The position of VSV-G protein is indicated by arrows.
Identical 60 mm plates of 293GP (N2 LacZ) cells were either transfected with pCMV-G (5 mg) or infected with RMCMVG (MOI of 10). At 24 h after transfection, or infection, the supernatants were titered, and the cells were lysed and assayed for VSV-G expression by immunoblot analysis. As shown in Fig. 6, there was more VSV-G in the cells infected by RMCMVG than in the cells transfected with pCMV-G. When the supernatants were titered, however, the transfected cells had a titer of about 106 transducing units/ml and the infected cells had a titer of about 104 transducing units/ml (data not shown). One possible explanation for the discrepancy between VSV-G protein expression and titer may be that the high level of VSV-G protein expressed by RMCMVG interferes with normal glycoprotein processing and transport to the cytoplasmic membrane. The cloning of VSV-G into the MCMV ie2 locus resulted in the juxtaposition of the MCMV and HCMV enhancers. This may be responsible for the very high level of VSV-G expression in RMCMVG. Moving VSV-G to another locus in the MCMV genome might result in less VSV-G protein expression, more efficient VSV-G maturation, and possibly increased pseudotyped vector titer. We are currently examining ways to increase the yield of VSV-G pseudotyped vector from our system.
The use of a recombinant MCMV vector in the production of VSV-G protein pseudotyped retroviral vectors highlights some of the features which make recombinant MCMV an attractive expression vector. One of these is the high level of gene expression achieved by using a CMV immediateearly promoter on a CMV genome in a non-permissive cell. In a non-permissive cell, this high level of gene expression is not associated with viral replication. A second benefit is delivery. Viral infection is more efficient than transfection in delivering a gene to a cell. MCMV vectors may be especially useful for the expression of proteins in cells which are not easily transfected by standard methods. The combination of high levels of expression and efficiency of delivery make recombinant MCMV an attractive expression vector.
Acknowledgements We thank Edward Mocarski for the generous gifts of MCMV (K181+ , RM427), and pON401. We also thank Steve Chang for providing the VSV-G expression vectors pCMV-G and pMLPG.
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