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 ؋ 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.Retroviral vectors are widely used in studies of gene structure and function in cultured cells and in animal models. Retroviral vectors have also been used for clinical applications, including human somatic cell gene therapy. A number of retroviral vectors have been developed; most are based on avian and mammalian retroviruses. The majority of these vectors are replication-defective derivatives of the murine leukemia virus (MLV). In general, MLV vectors lack all genes for the viral structural proteins that are required for viral replication. The viral genes are usually expressed either by cotransfection or by a packaging cell line that supplies the viral proteins in trans. There are replication-competent MLV vectors; however, the insert size is limited (71, 72). Replication-competent vectors based on avian sarcoma/leukosis viruses (ASLV) can accept larger inserts. Naturally occurring ASLV can have several different envelopes (subgroups A to E). The various ASLV envelopes are distinguished based on host range; none of these envelopes allows the ASLV (or the vectors derived from them) to efficiently infect mammalian cells.We developed the replication-competent chimeric retroviral vector RCASBP-M2C(4070A) by replacing the subgroup A env gene of the ASLV-based retroviral vector RCASBP(A) with the env-coding sequence of an amphotropic MLV (6). The original amphotropic RCASBP replicated poorly; passage of the virus selected for a variant that has a single change, P242I, in gp70. The adapted vector, RCASBP-M2C(4070A), replicates efficiently in chicken embryo fibroblasts (CEF) or in DF-1 cells (25, 64) and can efficiently transfer genes into cultured mammalian cells; however, the virus is replication defective in mammalian cells. The RCASBP-M2C(4070A) vector has advantages compared with replication-defective MLVbased vectors. Since the RCASBP-M2C(4070A) vector is replication competent in avian cells, it s...

Retroviruses Human Immunodeficiency Virus and Murine Leukemia Virus Are Enriched in Phosphoinositides

“…For MLV release assays, 200 ng of plasmid MLV Env-GFP encoding full-length Friend 57 MLV genome with a green fluorescent protein (GFP) insertion into the envelope protein (28, 51) was cotransfected together with plasmids encoding 5-phosphatase IV or catalytically inactive 5-phosphatase ⌬1 or with a vector control as described above. At 48 h posttransfection, the released virus infectivity was measured by titering serial dilutions of the culture supernatants onto target cells (TZM-bl [12] and DFJ8 [2] cell lines for HIV and MLV, respectively). Luciferase activity was measured 36 to 48 h postinfection in TZM-bl cell lysates to determine HIV infectivity.…”

The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing

“…Alternatively, these vectors have been pantropized by vesicular stomatitis virus glycoprotein. The replication-competent ASLV vector with a splice acceptor (RCAS) was developed to facilitate the production of high-titer virus in avian cells (11) and efficient infection of mammalian cells when the avian envelope gene is replaced with murine envelope genes, amphotropic or ecotropic (1,2). A transgenic mouse expressing the chicken receptor for the ASLV-A subgroup was established as a platform for in vivo infection of mammalian hosts with ASLV (13), and a similar system with the ASLV-C subgroup receptor (8) is in preparation.…”

Efficient method to optimize antibodies using avian leukosis virus display and eukaryotic cells

“…Although RCAS vectors can then infect the mammalian cells expressing the appropriate receptor, and integrate into the cells genome delivering and expressing a transgene, there are multiple blocks to new virus formation. RCAS-based viruses have also been designed to infect mammalian cells efficiently using the amphotropic MLV envelope glycoprotein, RCASBP-M2C(797-8), and deliver and express transgenes, but again are blocked from the production of new viruses (28). In future experiments, the RCASBP-M2C(797-8) virus will be tested for delivering and expressing membranebound scFv libraries in CHO and HEK293 mammalian cell lines, often used for large-scale protein production by infection and then cell surface selection using FACS.…”