Figure 7.

Complex formation between L1 ORF1p and A3C requires bridging RNA molecules. (A) ORF1p coprecipitates with ectopically expressed A3A and A3C. Immunoblot analysis of cell lysates isolated from HeLa cells that were consecutively transfected with pDK101 and A3A-HA, A3C-HA or the empty expression vector pcDNA3.1/Zeo(+) (vector), respectively, and their derived immunoprecipitates. The presence of HA-tagged A3A and A3C or L1 ORF1p in cell lysates and immunoprecipitates (IP) was demonstrated using α-HA and α-ORF1p antibodies, respectively. β-actin expression served as loading control. (B) RNase A treatment of the cell lysate abolishes the interaction between A3C and L1 ORF1p. Immunoblot analysis of cell lysates isolated from HeLa cells that were co-transfected with pDK101 and A3C-HA or the empty expression vectors (pcDNA3.1 + pCEP4), respectively (upper panel). The cleared cell lysate was split in half and RNase A was added to one half to a final concentration of 50 µg/ml. After incubation of RNase-treated and untreated lysates with α-HA Affinity Matrix Beads, immunoprecipitates were submitted to immunoblot analyses (lower panel). (C) RNA-binding pocket mutation R122A abolishes the A3C/L1 ORF1p interaction. Immunoblot analysis of cell lysates isolated from HeLa cells that were co-transfected with pDK101 and expression plasmids encoding A3C-WT, R122A, N177A or C97S/C100S mutant proteins, respectively (upper panel, cell types), and their derived immunoprecipitates (lower panel, IP). In contrast to R122A, RNA-binding pocket mutation N177A does only moderately affect A3C/ORF1p complex formation. While mutating the A3C dimerization site (W74A) abolishes A3C/ORF1p interaction almost entirely, the CDA double-mutant C97S/C100S still exhibits significant interaction with L1 ORF1p. Lysates and immunoprecipitates obtained from W74A- and C97S/C100S-expressing HeLa cells were loaded on a different gel than the remaining lysates and immunoprecipitates.