Mutagenic analysis of the 3 ' cis-acting elements of the rubella virus genome

Thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) struct ures; SL1 and SL2 are both located in the E1 coding region, while SL3 and S L4 are within the 59-nt 3' untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis . RNase mapping indicated that SL2 and SL3 are in equilibrium between two c onformations, in the second of which the previously proposed CAL binding si te in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3' UTR with a RUB infectious clone, Robo302, revealed that most of the 3' UTR is required for viral viability except for the 3'-terminal 5 nt and the pol y(A) tract, although poly(A) was rapidly regenerated during subsequent repl ication, Maintenance of the overall SL3 structure, the 11-nt single-strande d sequence between SL3 and SSL4, and the sequences forming SL4 were all imp ortant for viral viability. Studies on the interaction between host factors and the 3' UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced crass-linking detected six host protei n species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, intera cting with the 3' UTR Site-directed mutagenesis of SL2 by nucleotide substi tutions showed that maintenance of SL2 stem rather than the U-U bulge was c ritical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants h aving the SL2 stem destabilized had much lower binding activity. However, a ll of these mutations gave rise to viable viruses when introduced into Robo 302, indicating that binding of CAL to SL2 is independent of viral viabilit y.