FIG. 8.

3B precursor-linked RNA is produced by EG PV and converted to VPg-linked RNA prior to the hydantoin-sensitive step. Interrogation of protein linkage to WT and EG mutant RNA was analyzed by RNA immunoprecipitation and Northern blotting. (A) RNA isolated from replicon RNA-transfected cells. HeLa cells were transfected with WT, EG, or Y3F RNA and held at 37°C for 10 h. Total RNA was isolated from transfected HeLa cells and immunoprecipitated using antibodies against VPg, 3C, 3D, or HCV NS5A (as a negative control). The immunoprecipitated RNA was separated on a 0.6% agarose gel containing 0.8 M formaldehyde, transferred to a nylon membrane, and hybridized with a ³²P-labeled DNA probe. The hybridized DNA probe was visualized by phosphorimaging. Shown is a phosphorimage after a 1-day exposure. In vitro-transcribed RNA is shown as a reference. (B) RNA isolated at early times postinfection. HeLa cells were infected with WT PV (MOI, 10) and held at 37°C for 0, 4, or 5 h. As a positive control, EG PV (MOI, 10)-infected HeLa cells were incubated at 37°C for 10 h. Total RNA was isolated from infected HeLa cells and immunoprecipitated using antibodies against VPg, 3C, or HCV NS5A. The immunoprecipitated RNA was separated as described above. In vitro-transcribed RNA is shown as a reference. (C) RNA isolated from HeLa cells infected with WT PV or EG PV (MOI, 10) in the presence (+Hyd) or absence (−Hyd) of hydantoin at 37°C for 6 h (WT PV) or 8 h (EG PV). Total RNA was immunoprecipitated using antibodies against VPg and 3C. The immunoprecipitated RNA was separated as described above. WT or EG PV RNA used for immunoprecipitation is shown in lanes 1 to 4. (D) RNA isolated from purified virus particles. HeLa cells were infected with WT or EG PV and held at 37°C until CPE. Cells were harvested, and WT and EG PV was purified by precipitation using 8 to 10% PEG 8000 followed by centrifugation through a 30% sucrose cushion. Viral RNA was isolated from purified viruses and immunoprecipitated using antibodies against VPg, 3C, 3D, or HCV NS5A. The immunoprecipitated RNA was separated as described above. Total RNA purified from Y3F mutant RNA-transfected cells was used as a negative control.