Figure 1

Isolation and characterization of retrovirus-resistant clones. (A) Strategy used to isolate MLV-resistant clones. Transcriptional repression of cellular genes flanking the mutagenic vector was induced in ten independent pools of SILENCE cells by withdrawal of doxycycline for 72 h. The cells were then challenged with a VSV-G pseudotyped MLV vector, CMMP-CD4 [VSV-G], encoding human CD4, at a MOI of approximately 1.0 CD4-transducing units. Infected cells were depleted from the population by magnetic separation using an iron-conjugated CD4 antibody. Uninfected cells were allowed to recover in medium containing doxycycline for a minimum of 72 h, prior to a subsequent round of infection and selection. After five rounds of infection and selection, the 6B pool was considered to be relatively resistant to MLV infection, based on the resultant numbers of CD4-expressing cells. Individual cell clones were derived from this pool by limiting dilution and virus resistance was verified by challenging with a VSV-G pseudotyped MLV vector, VGIP3-Luc [VSV-G] encoding firefly luciferase. (B) Resistance of two mutant cell clones to infection by MLV and HIV-1 vectors. Wild type CHO-K1 and the 6/B-6 and 6/B-7 mutant CHO-K1 cell clones were challenged with VSV-G pseudotyped vectors encoding firefly luciferase: VGIP3-Luc or NL43 R + E- Luc. Cell viability was also monitored using the CTG assay. The ratios of firefly luciferase activity: cell viability were determined and compared to that of wild type CHO-K1 cells (defined as 100% infection). The data shown is the average mean of three independent experiments each performed with triplicate samples. Error bars indicate the standard deviation. The data was analyzed using an unpaired T-test, ***P value <0.0001.