Infection of Human Cells with Murine Amphotropic Replication-Competent Retroviruses

Abstract

Replication of the murine wild-type 4070A amphotropic retrovirus and a recombinant amphotropic replication-competent retrovirus arising from the PA12 packaging cell line varied considerably among the primate cell types tested. Medium from infected primate fibroblasts and endothelial cells contained the highest viral titers [10⁴–10⁵ focus-forming units (ffu)/ml], while most hematopoietic cell lines, such as K562 and MOLT4, were associated with viral titers in the range of 10³–10⁴ ffu/ml. Interestingly, HTLV-1–transformed T cell lines (TJF-2 and HM) and primary tumor infiltrating lymphocytes (TIL) had very low viral titer (0–10¹ ffu/ml). The low production of virus was not due to low infectivity and, in contrast to the virus, retroviral vectors were expressed without difficulty. Because screening for replication-competent retrovirus (RCR) is an important component of human retroviral-mediated gene therapy clinical protocols, a variety of assays were tested for their ability to detect RCR in virus-exposed cell lines. A biologic assay (3T3 amplification) and polymerase chain reaction (PCR) for the 4070A viral envelope are effective screening methods for RCR, even in cell lines associated with low virus production.

Overview summary

Utilization of retroviral vectors in clinical trials requires careful screening of vector supernate to prevent accidental exposure to replication-competent retrovirus (RCR). Cornetta et al. evaluate the wild-type 4070A amphotropic virus and a RCR arising from the PA12 packaging cell line; they note signiflcant differences in virus production among the various primate cells tested. Their findings have implications for testing retroviral vectors intended for clinical use.