Development and characterization of an Fv-1-sensitive retrovirus-packaging system: single-hit titration kinetics...

We have constructed an RNA-packaging-deficient mutant of N-tropic murine leukemia virus WN1802N by removal of 330 nucleotides located between the upstream long terminal repeat and the start of the gag gene region. Transfection into mink CCL64 cells produced a cell line capable of packaging retrovirus vectors into ecotropic, Fv-1 N-tropic virions. Using retrovirus vectors that confer resistance to the antibiotic G418, we demonstrated that the magnitude of restriction in BALB/3T3 and SIM.R cells (both Fv-1b/b) and in RFM/3T3 cells (Fv-1nr/nr) is approximately 100-fold compared with that in AKR or NIH 3T3 cells (both Fv-1n/n). Furthermore, titration kinetics were single hit in restrictive cells. Colonies of antibiotic-resistant cells recovered after infection of genotypically restrictive cultures were phenotypically restrictive when reinfected, ruling out selection of stably nonrestrictive subpopulations. These results suggest that the ability to infect some fraction of cells in a genotypically restrictive culture does not require specific abrogation and that multihit kinetics may not be an essential feature of Fv-1 restriction.