Characterization and detection of artificial replication-competent lentivirus of altered host range.
Replication-competent lentivirus (RCL) may be generated during the production phase or subsequently after introduction of a lentiviral vector into target cells, potentially by homologous or nonhomologous recombination. Because most gene transfer of HIV-based vectors involves the use of high-titer vesicular stomatitis virus (VSV) G-pseudotyped particles, one particular concern would be the generation of an RCL of altered host range, i.e., one that has incorporated the VSV G envelope in cis configuration. We report here on the artificial generation and properties of such a virus, including its detection after biological amplification. Viral spread, beginning with a very low inoculum, takes several weeks in culture and is characterized by "autoinfection," resulting in multiple proviral copies per cell, higher levels of viral gene expression, and eventual cell death. After this initial amplification step, the RCL is easily detectable by standard p24 assay or by "marker-rescue" assay. For the latter, a 293T-based cell line that has an integrated replication-defective provirus encoding alkaline phosphatase (AP) was used and mobilization of AP-containing virus was detected by transduction of naïve cells. Replication-defective virus was not amplified nor detected, demonstrating assay specificity. These results suggest that these artificial RCLs of broad host range have slightly different biological properties compared to wild-type HIV but still spread and are readily detectable.
Segall
Harry I
HI
Center for Cell and Gene Therapy, Department of Molecular Virology and Microbiology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
Yoo
Eunsun
E
Sutton
Richard E
RE
D000469:Alkaline Phosphatase / Q000378:metabolism; D002460:Cell Line; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005819:Genetic Markers; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008957:Models, Genetic; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013997:Time Factors; D014161:Transduction, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014779:Virus Replication
D005819:Genetic Markers; D000469:Alkaline Phosphatase