Capsid-targeted viral inactivation can eliminate the production of infectious murine leukemia virus in vitro

Capsid-targeted viral inactivation (CTVI), a promising gene-based antiviral strategy against retroviruses, was designed to disrupt the retroviral life cycle by incorporating a degradative enzyme (e.g., nuclease) into viral pa rticles during assembly, thereby reducing or eliminating the production of infectious virus. The experimental system used to develop the CTVI strategy for retroviruses is designed to block the production of infectious Moloney murine leukemia virus (Mo-MLV). Two nucleases, Escherichia coil ribonuleas e HI and Staphylococcus nuclease, have been shown to be tolerated by the ce ll as Mo-MLV Gag-nuclease fusion polyproteins and still be active in the vi ral particles. The goal of this study was to determine what cellular and vi ral factors limit CTVI in cultured cells. The avian DF-1 cell line greatly expanded our ability to test the antiviral efficacy of CTVI in long-term as says and to determine the mechanism(s) of CTVI action. The CTVI antiviral e ffect is dependent on the level of Mo-MLV Gag-nuclease fusion polyprotein e xpressed. The Mo-MLV Gag-nuclease polyproteins produce a long-term prophyla ctic antiviral effect after a low- or high-dose Mo-MLV challenge. The Mo-ML V Gag-nuclease fusions have a significant therapeutic effect (similar to 10 00-fold) on the production of infectious Mo-MLV. The therapeutic CTVI effec t can be improved by a second delivery of the CTVI fusion gene. Both the pr ophylactic and the therapeutic CTVI antiviral approaches can virtually elim inate the production of infectious Mo-MLv in vitro and are only limited by the number of cells in the population that do not express adequate levels o f the CTVI fusion polyprotein. (C) 2000 Academic Press.