Defective RNA packaging is responsible for low transduction efficiency of CAEV-based vectors.

Replication defective retroviral vectors are regularly used for transfer and expression of exogenous genes into dividing cells and in animals. Since lentiviruses are able to infect terminally differentiated and non-dividing cells, their use to produce replication defective vectors may overcome this limitation. We developed two replication-defective lentiviral vectors based on the genome of Caprine Arthritis Encephalitis Virus (CAEV). The first vector (pBNL2) carries the neo and lacZ marker genes. Neo gene is expressed from a genomic RNA and lacZ gene from a subgenomic RNA. The second vector (pCSHL) carries a single fusion gene encoding both phleomycin resistance and beta-galactosidase activity. Replication-competent CAEV was used as helper virus to provide the viral proteins for transcomplementation of these vectors. Our data demonstrated that the genomes of both vectors were packaged into CAEV virions and transduced into goat synovial membrane cells following infection. However, the vector titers remained 3 to 4 logs lower than those of CAEV. Further analysis showed a lack of accumulation of unspliced pBNL2 RNA into the cytoplasm of producer cells resulting in the packaging of pBNL2 sub-genomic RNA only. In contrast, RNA produced from pCSHL vector was correctly transported to the cytoplasm and more efficiently packaged than the pBNL2 sub-genomic RNA as revealed by slot-blot and quantitative RT/PCR analyses. However this higher packaging efficiency of pCSHL genome did not result in a higher transduction efficiency of lacZ gene.

Immortalization of caprine fibroblasts permissive for replication of small ruminant lentiviruses.

OBJECTIVE: To establish immortalized caprine fibroblastic cell lines permissive for replication of small ruminant lentiviruses. ANIMALS: Carpal synovial membrane explants collected aseptically from a surgically delivered fetus of a lentivirus-seronegative goat. PROCEDURE: Immortalization of goat embryonic fibroblasts was performed by DNA transfection with plasmids coding for simian virus 40 large T antigen. The generated cell lines were phenotypically characterized. Cytogenetics, growth pattern, and sensitivity to viral infection were studied. RESULTS: 3 cell lines, designated TIGEF, mMTSV-54, and mMTSV-93, were generated. They had a more rapid doubling time than did fibroblasts from which they were derived, and retained morphologic and phenotypic fibroblastic characteristics. They were immortalized but not transformed because tumor formation was not promoted after their s.c. injection into athymic nude mice. The 3 cell lines were susceptible to caprine arthritis-encephalitis virus and visna-maedi virus infections, and supported a complete virus replication cycle. CONCLUSIONS: Cultured caprine fibroblastic cells were immortalized, using simian virus 40 large T antigen. The TIGEF, mMTSV-54, and mMTSV-93 immortalized cell lines were permissive to in vitro small ruminant lentivirus replication. CLINICAL RELEVANCE: Because lentivirus detection, as well as detailed studies of host-lentivirus interactions, are hampered by differences in viral susceptibility of each primary culture, permanent cell lines are essential tools for such analysis.