Verification of Cct1 and Cct2 roles in modulating FHV replication in infected S2 cells. (A) RT-PCR validation of Cct1 or Cct2 RNAi-mediated knockdown. Drosophila S2 cells were treated with dsRNAs specific for LacZ (lane 1), Cct1 (lane 2), Cct2 (lane 3), or both Cct1 and Cct2 (lane 4) for 72 h, and gene-specific mRNA expression was examined by semi-quantitative RT-PCR as described in Fig. 2, except that only results from cDNA dilutions that produced submaximal signals are shown. (B) Total PC content in cells treated with the dsRNAs described above. PC levels were determined as in Fig. 3. (C) Viability of cells treated with dsRNAs described above determined by MTT assay. (D) FHV RNA accumulation in infected S2 cells after RNAi-mediated knockdown of Cct1, Cct2, or both. Mock-infected cells (lane 1), cells treated with the indicated dsRNA as described above or FHV RNA1 as a positive control (lanes 2-6), or treated with 50 μM miltefosine (lane 7), were infected with FHV and viral-specific RNAs or protein were analyzed by northern blotting or immunoblotting 12 h after infection, respectively. For viral RNA analysis, blots for positive-sense (+) and negative-sense (-) genomic RNA1 and subgenomic (+)RNA3 are shown. The decrease in (+)RNA1 accumulation in Cct1 knockdown cells (lane 4) was not consistently seen in all experiments. Ribosomal RNA (rRNA) bands detected by ethidium bromide staining are shown as loading controls. For protein analysis, blots for FHV protein A and the cellular loading control tubulin are shown. (E) Quantitative data for genomic (+)RNA1 and (-)RNA1, subgenomic (+)RNA3, and protein A accumulation in S2 cells treated with the indicated dsRNA or miltefosine after infection with FHV. Results are expressed as percentage of accumulation relative to LacZ dsRNA-treated control. P-value < 0.05* or 0.005**.