Adaptation of chimeric retroviruses in vitro and in vivo: isolation of avian retroviral vectors with extended host range

Abstract

We have designed and characterized two new replication-competent avian sarcoma/leukosis virus-based retroviral vectors with amphotropic and ecotropic host ranges. The amphotropic vector RCASBP-M2C(797-8), was obtained by passaging the chimeric retroviral vector RCASBP-M2C(4070A) (6) in chicken embryos. The ecotropic vector, RCASBP(Eco), was created by replacing the env-coding region in the retroviral vector RCASBP(A) with the env region from an ecotropic murine leukemia virus. It replicates efficiently in avian DFJ8 cells that express murine ecotropic receptor. For both vectors, permanent cell lines that produce viral stocks with titers of about 5 x 10(6) CFU/ml on mammalian cells can be easily established by passaging transfected avian cells. Some chimeric viruses, for example, RCASBP(Eco), replicate efficiently without modifications. For those chimeric viruses that do require modification, adaptation by passage in vitro or in vivo is a general strategy. This strategy has been used to prepare vectors with altered host range and could potentially be used to develop vectors that would be useful for targeted gene delivery.

FIG. 1

Schematic of structures of chimeric retroviral vector RCASBP(Eco) (A) and murine retroviral vector LRNL-J8 (B) LTR, long terminal repeat; Mo-MLV, Moloney MLV; SV40_E, simian virus 40 early promoter.

FIG. 2

Replication and adaptation of the ecotropic vector RCASBP(Eco1) in avian cells. DF-1 and DFJ8 cells were transfected with RCASBP(Eco1) plasmid DNA or infected with RCASBP(Eco1) virus and passaged every other day. At each passage of the transfected cells, samples of the culture medium were harvested and the level of virus production was quantified by p27 capture ELISA (see Materials and Methods). (A) Replication of unadapted virus. Cells were transfected with plasmid DNA. (B) Replication of the adapted virus. DF-1 and DFJ8 cells (two plates of each) were infected with cell culture medium harvested at passage 6 of the unadapted virus (A). (C) Replication of molecular clones of adapted RCASBP(Eco1). DFJ8 cells were transfected with plasmid DNAs of adapted RCASBP(Eco1) and, separately, with plasmid DNA of unadapted RCASBP(Eco1). Mock, uninfected cells; WT, wild-type (unadapted) vector. Values are averages of two independent determinations. OD₄₀₅, optical density at 405 nm.

FIG. 3

Changes in the amino acid sequence of the Env protein in molecular clones of adapted RCASBP(Eco1) viruses. Eco WT, wild-type ecotropic Env protein; Eco 23 to Eco 93, Env proteins in molecular clones of the adapted virus. RSV (Rous sarcoma virus), the envelope of the avian virus used to construct RCASBP(A), showing the sequence at the end of the transmembrane protein (TM). SP, signal peptide; SU, surface protein.

FIG. 4

Proteolytic processing of the pre-p15E in RCASBP(Eco73) virions. Virus particles produced by infected DFJ8 cells were recovered by ultracentrifugation. Viral proteins were fractionated on SDS–16% polyacrylamide gels and transferred to Immobilon P membranes (see Materials and Methods). Virion-associated transmembrane protein was detected with antibodies against p15E. MLV, wild-type amphotropic MLV (positive control); 4070A, RCASBP-M2C(4070A) (positive control); Eco 73, ecotropic vector RCASBP(Eco73).

FIG. 5

Cytotoxic effect of RCASBP-M2C(4070A) and chicken embryo-adapted viruses RCASBP-M2C(797) and RCASBP-M2C(808) in DF-1 cells. The cells were infected either with the cytotoxic amphotropic vector RCASBP-M2C (4070A) or with viruses recovered from two of the infected chicken embryos. The morphology of the infected cells at passage 4 is shown.

FIG. 6

Replication of chicken embryo-adapted amphotropic vectors in DF-1 cells. (A) p27 capture ELISA. The cells were infected with viruses obtained from infected chicken embryos or, separately, with the cytotoxic amphotropic virus RCASBP-M2C(4070A) and passaged every other day. At each passage, virus production was measured by quantitation of the p27 level in samples of the culture medium by ELISA. Values are averages of two independent determinations. (B) Virus was recovered from culture medium by ultracentrifugation, fractionated on SDS-gradient (5 to 20%) polyacrylamide gels, and detected by immunoblotting. Virion-associated Env and capsid proteins were analyzed by immunoblotting with antibodies against gp70 and p27. DF-1, supernatant from uninfected DF-1 cells; 4070A, supernatant from cells infected with RCASBP-M2C(4070A); 797 and 808, supernatant from cells infected with viruses recovered from chicken embryos. OD₄₀₅, optical density at 405 nm.

FIG. 7

Replication and cytotoxic effects of molecularly cloned chicken embryo-adapted amphotropic vectors in DF-1 cells. (A) p27 capture ELISA. DF-1 cells were transfected with plasmid DNA clones of chicken embryo-adapted vectors and passaged. Separately, the cells were transfected with cytotoxic amphotropic vector RCASBP-M2C(4070A). At passage 4, the amount of p27 capsid protein in the culture medium of the transfected cells was quantified by ELISA. Values are averages of two independent determinations. OD₄₀₅, optical density at 405 nm. (B) Cytotoxic effect in DF-1 cells at passage 4. 4070A, cells transfected with RCASBP-M2C (4070A); 797-1, 797-8, 808-1, 808-4, and 808-8, cells transfected with molecular clones of chicken embryo-adapted vectors.

FIG. 8

Structures of Env proteins encoded by wild-type amphotropic MLV (WT 4070A) and by molecular clones of the chicken embryo-adapted amphotropic vectors.

FIG. 9

Passage of cells infected with RCASBP-M2C(4070A)Puro and RCASBP-M2C(797-8)Puro. Infection was initiated by transfection. Cells were passaged, and the virus was titered at each passage (see Materials and Methods). The lower titer of the RCASBP-M2C(4070A)Puro virus was a result of death of the cells infected with this virus.