FIG. 4.

BLM is not responsible for defective viral replication or concatemer formation. (A) Viral replication was assessed in HeLa cells and HeLa cells stably expressing shRNA against BLM (HeLa-shBLM cells). The shRNA sequence 5′-TGCCAATGACCAGGCGATC was inserted into the pSUPER.retro.puro vector (Orbigen), and Phoenix retroviral packaging cells were used to produce retrovirus. HeLa cells were transduced with retrovirus, and the HeLa-shBLM cells were selected and maintained in puromycin. Cells were infected with Ad5 (MOI of 10) or dl1004 (MOI of 3) in triplicate, and DNA was extracted for quantitative PCR at 4 and 30 hpi as previously described (35). Accumulation of viral DNA is represented as fold increase over input viral DNA, as determined at the 4-h time point, and error bars represent standard errors of the means (SEM) from the triplicate samples. (B) Immunoblotting of lysates from infected cells at the 4-h time point confirmed knockdown of BLM levels in HeLa-shBLM cells. GAPDH served as a loading control. (C) Concatemer formation was examined by PFGE analysis of DNA at 48 hpi with Ad5 (MOI of 25) and dl1004 (MOI of 50) as previously described (57). Viral DNA was visualized by staining the gel in ethidium bromide, and the position of the linear viral genome is indicated by an arrowhead. Formation of concatemers by dl1004 was not dependent on BLM.