Nucleic Acids Research, 2012, Vol. 40, No. 11 5075 Figure 8. Comparison of the 50 UTR of IBV, SARS-CoV and HCoV-OC43. (A) Interaction of the 50 UTR from IBV, SARS-CoV and HCoV-OC43 with MADP1. Total cell lysates prepared from H1299 cells over-expressing Flag-tagged MADP1 were mixed with biotinylated RNA probes of full-length 50 UTRs of the three coronaviruses. The RNA–protein complexes were puriﬁed with streptavidin beads, resolved by SDS–PAGE and probed with antibody to Flag tag for the presence of Flag-tagged MADP1 protein. (B) The predicted secondary structures of stem–loop I from IBV, SARS-CoV and HCoV-OC43. subgenome-length RNAs (sgRNA) was compromised, resulting in a drastic reduction of viral structural protein expression and release of viral progeny (titers), hence the overall reduction of viral infectivity in the cells. Across different coronaviruses, the leader sequence situated in the extreme 50 end of the genome, is composed of stem–loops I and II. Mutations introduced into either stem–loop I or II resulted in non-viable viruses, impaired (sense and anti-sense) sgRNA synthesis, but not the full-length gRNA synthesis (38,44). It was, however, observed in this study that silencing of MADP1 did render an impact on gRNA synthesis, although to a lesser extent compared to sgRNA synthesis. This might have been due to a secondary effect of decreased sgRNA synthesis, as proteins encoded by sgRNAs may enhance viral RNA synthesis (45). The predicted structure of stem–loop II indicated a strong secondary interaction, which is highly conserved across different groups of coronaviruses. The predicted stem–loop I structure, on the other hand, appears to fold into a hairpin of low thermodynamic sta- bility, shows a wider sequence variation and is characterized by the presence of bulges, non-canonical base pairing as well as a prevalence of A-U base pairing (46). It has been shown in MHV that the structural liabil- ity of stem–loop I is a critical driving force in the 50- and 30-UTR interaction (44). Comparing the predicted stem– loop I structures of IBV to SARS-CoV and HCoV-OC43 (Figure 8B), it was noted that there exists a difference in the loop sequence. In addition, IBV stem–loop I has a shorter stem and the absence of bulges, although the structure may be as unstable thermodynamically as that of SARS-CoV and HCoV-OC43, due to the extremely high prevalence of weak base pairing between A and U as well as the presence of a non-canonical base pair at the base of the stem (46). Hence, sequence and structural dif- ferences may be one of the possible explanations for the observation of a weaker binding between MADP1 and SARS-CoV or HCoV-OC43 50-UTR than with IBV 50-UTR. In fact, the relatively weaker binding of MADP1 to the stem–loop I restoring mutant (50-UTR 2M2) demonstrated in this study supports that primary sequences in the 50-UTR may play a certain role in this interaction. Most studies on host involvement in coronaviral RNA synthesis were so far performed using MHV (29–31,42,43). Identiﬁcation of the interaction between MADP1 and 50-UTR as well as its functional involvement in corona- virus replication, in this study, therefore may represent the ﬁrst host protein identiﬁed to play a role in viral RNA synthesis by interacting with the 50-UTR of the viral RNA in a gammacoronavirus. The functional implication of the interaction between MADP1 and IBV 50-UTR may be extended to the rest of the members of the coronavirus family. In the case of hnRNP A1, it was initially reported to be functionally important for viral RNA syn- thesis for group II virus MHV (29,30). Subsequently, its involvement in viral RNA synthesis was also conﬁrmed in TGEV, a group I coronavirus (32). In this study, we have shown that betacoronaviruses HCoV-OC43, SARS-CoV and gammacoronavirus IBV can bind to MADP1, albeit