FIG. 1.
Replication of SIN replicons in BHK-21 cells using helpers with different 5′ ends. (A) Experimental protocol proposed for tricomponent genome SIN propagation. Transfection of BHK-21 cells by replicon and two-helper RNAs results in the release of viral particles with three different genomes. These particles can be used for the next round of infection. (B) Schematic representation of replicon and helper genomes and helper 5′ UTRs (based on predicted secondary structure). DH-BB- and DH-BBdelSL2-based helpers had the 5′ tRNAAsp sequence derived from the naturally occurring SIN DI RNA (see reference 1 for details); BB- and BBdelSL2-based helpers had natural SIN 5′ UTRs. All of the helpers contained a deletion of nucleotides 426 to 7334 of the SIN genome, and delSL2 indicates an additional deletion of nucleotides 47 to 152 of the SIN genome. A total of 107 BHK-21 cells were cotransfected with SINrep/LacZ or SINrep/GFP replicons and indicated pairs of helper RNAs. Harvested virus particles were used for the next round of infection of 3 × 106 BHK-21 cells at the indicated MOIs (measured in infectious units [inf.u]/cell) in 100-mm-diameter dishes. Viruses were harvested at 40 h postinfection and were used further without concentrating. Titers refer to unconcentrated harvested virus-containing media. N.A. stands for not applicable; the passage could not be performed at an MOI of 10 or 1 infectious units/cell due to low titers of packaged replicons harvested after the previous passage. All of the experiments were performed multiple times and generated very reproducible titers. (C) Replication of replicon and helper RNAs in the cells after their cotransfection or infection (passage 2 [P2]) with tricomponent genome viruses. Autoradiographs of dried gels with RNAs metabolically labeled with [3H]uridine. RNAs were transfected using the conditions described in the text or were infected at an MOI of 3 or 30 infectious units/cell for the samples of replicons packaged with 5′ tRNA UTR- or SIN 5′ UTR-containing helpers, respectively. RNA labeling was performed between 4 and 8 h posttransfection or 12 to 16 h postinfection in the presence of ActD and [3H]uridine (20 mCi/ml). RNAs were isolated from the cells by using TRIzol reagent, denatured with glyoxal in dimethyl sulfoxide, and analyzed by agarose gel electrophoresis using previously described conditions (1). SINrep/LacZ and BB/C plus BB/Gl helpers, lanes 1 and 5; SINrep/LacZ and BBdelSL2/C plus BBdelSL2/Gl helpers, lanes 2 and 6; SINrep/GFP and BB/C plus BB/Gl helpers, lanes 3 and 7; SINrep/GFP and BBdelSL2/C plus BBdelSL2/Gl helpers, lanes 4 and 8; SINrep/LacZ and DH-BB/C plus DH-BB/Gl helpers, lanes 9 and 13; SINrep/LacZ and DH-BBdelSL2/C plus DH-BBdelSL2/Gl helpers, lanes 10 and 14; SINrep/GFP and DH-BB/C plus DH-BB/Gl helpers, lanes 11 and 15; SINrep/GFP and DH-BBdelSL2/C plus DH-BBdelSL2/Gl helpers, lanes 12 and 16. Co-transf., cotransfection.