Knock-down of vIRF-3 expression in JSC-1 cells by retroviral shRNA is associated with reduced proliferation. KSHV⁺ JSC-1 cells as well as KSHV⁻ BJAB and INA-6 cells were transduced with VSV-G pseudotyped retrovirus harvested from the supernatant of transfected GP2–293 cells and kept under puromycin selection for up to 7 days. Data from 3 independent experiments are shown. (A) Western blot performed using monoclonal antibody against vIRF-3 (3G7). JSC-1 cells were harvested 3 days after transduction with retroviral shRNA expression constructs or mock treatment. Equal amounts of total cell protein (5 × 10⁵ cells) were loaded per lane and separated on 8% discontinuous SDS-PAGE. Expression of sh976 targeted at vIRF-3 resulted in an about 80% reduction of vIRF-3 expression. Expression of actin, however, remained unchanged. (B) Expansion of vector-carrying cells as quantified via EGFP expression analysis by FACS; the percentage of EGFP-expressing cells was determined 24 hours and 4 days after retrovirus transduction. Cells were maintained in media containing puromycin. Cells transduced with retroviral vector and thus EGFP⁺ and puromycin resistant continued to proliferate in the case of sh-random (left). However, when vIRF-3 expression was knocked down by sh976, the percentage of EGFP⁺ transduced cells in the culture declined despite puromycin selection (right). (C) PEL cells (2 independent experiments represented by 2 pairs of dotted lines with ● and 2 solid lines with ●), BJAB cells (■), and cIRF-4⁺ INA-6 cells (*) were transduced with retroviral particles expressing sh976 targeted at vIRF-3 (solid lines) or nonsense shRNA (interrupted lines). Cells were kept under puromycin selection and the relative growth of EGFP-expressing cells was monitored for 7 days by FACS analysis. Whereas JSC-1 cells transduced with nonsense shRNA–expressing retrovirus continued to grow under puromycin selection (● connected by dotted lines), JSC-1 cells expressing the shRNA targeted at vIRF-3 failed to expand (solid lines with ●). The KSHV⁻ cell lines BJAB and INA-6 were used as a control (■ and *, respectively). BJAB and INA-6 cells continued to proliferate with both nonsense (■ or * and continuous lines) and shRNA (■ and * and dotted lines). p.i. indicates postinfection.