FIG. 3.
Expression of heterologous proteins by SIN replicons in the presence of replicating helper RNAs. (A) Schematic representation of SIN replicons without translational enhancer, SINrep/GFP and SINrep/LacZ; with a partial translational enhancer (SIN capsid-coding sequence with deleted amino acids 59 to 113), SINrep/Cdel/GFP, SINrep/Cdel/LacZ; and with complete capsid-coding sequence, SINrep/C/GFP and SINrep/C/LacZ. Both complete capsid and capsid with deletions were capable of cleaving themselves co- and posttranslationally. (B) BHK-21 cells were infected with GFP-expressing replicons packaged with 5′ tRNA UTR- or SIN 5′UTR-containing helpers at an MOI of 20 infectious units (inf.u)/cell. At 18 h postinfection, the expression of GFP was analyzed by flow cytometry on a Vantage fluorescence-activated cell sorter (Becton Dickinson). (C) A total of 5 × 105 BHK-21 cells were infected with GFP-expressing replicons packaged with DH-BB/C and DH-BB/Gl helpers (lanes 1, 2, and 3) or BB/C and BB/Gl helpers (lane 4) at an MOI of 20 infectious units/cell. Cells were harvested 30 h postinfection, and cell lysates equivalent to 105 cells were analyzed by gel electrophoresis. The gel was stained with Coomassie brilliant blue R-250. Lysate of cells infected with packaged SINrep/GFP replicon, lanes 1 and 4; SINrep/Cdel/GFP replicon, lane 2; SINrep/C/GFP replicon, lane 3. (D) BHK-21 cells were infected with β-galactosidase (β-gal.)-expressing replicons packaged with DH-BB/C and DH-BB/Gl helpers at an MOI of 20 infectious units/cell. Cells were harvested 30 h postinfection, and cell lysates were analyzed by gel electrophoresis. Accumulation of β-galactosidase was also tested using a Galacto-light Plus System (Applied Biosystems). The lysate of 5 × 104 cells was loaded on the gel. The gel was stained by Coomassie brilliant blue R-250. Lysate of uninfected cells, lane 1; cells infected with packaged SINrep/LacZ replicon, lane 2; SINrep/Cdel/LacZ replicon, lane 3; SINrep/C/LacZ replicon, lane 4.