5074 Nucleic Acids Research, 2012, Vol. 40, No. 11 Figure 7. Over-expression of siRNA-resistant MADP1 enhances IBV replication in stable MADP1-knockdown cells. (A) Northern blot analysis of H1299 cells stably transfected with either negative control shRNA (shNC) or short hairpin RNA to MADP1 (shMadp1). Total RNA was prepared from the two G418-selected stable clones and separated on 1% agarose gel and probed by a Dig-labeled speciﬁc MADP1probe. (B) Northern blot analysis of IBV RNAs in IBV-infected shNC and shMadp1 stable clones. Cells were infected with IBV-Luc at a multiplicity of 1, harvested at 0, 8, 10, 12, 14, 16 and 20 h post-infection, respectively. Total RNA was prepared, separated on 1% agarose gel and probed by a Dig-labeled speciﬁc probe. (C) Western blot analysis of IBV N protein expression in IBV-infected siMadp1 cells transfected with wild-type or mutant MADP1. shMadp1 stable cell clone was transfected with constructs expressing Flag-tagged wild-type MADP1 (FM) and its triple mutant [FM(YVF)], siRNA-resistant MADP1 (FMmut) and its triple mutant [FMmut(YVF)], negative controls EGFP (E), and empty vector control (F), respectively, using Lipofectamine 2000 (Invitrogen). Cells were then infected with IBV-Luc virus at a multiplicity of 1, and harvested at 19 h post-infection for western blot. Over-expressed Madp1 and IBV N were detected with speciﬁc antibodies, and actin was detected with a commercial antibody as a loading (Santa Cruz). (D) Fireﬂy luciferase activities of cell lysates from above were normalized against vector-transfected control (F, treated as 100%), and expressed as a percentage to that in the control cells. In addition to the differences in the secondary structures between the coronaviruses, there was a lack of sequence similarity as well (Figure 8B). DISCUSSION Previous studies on the involvement of host proteins in viral RNA synthesis have revealed a number of proteins which are able to interact with the UTRs of viral genomes (29,30,32,40–43). Some of these proteins may also interact with other viral proteins as well (30,40). Our attempts to identify host proteins involved in this early process of the coronavirus life cycle yielded MADP1. This protein was shown to be localized to the nucleoplasm but excluded from the nucleolus, but its role in RNA splicing remains to be determined (37). MADP1 contains two conserved RNA-binding domains, the RNA recognition motif (RRM) 1 and universal minicircle sequence binding protein (UMSBP) domains (a zinc ﬁnger CCHC-type) (35). The former was determined to be the domain respon- sible for the interaction between MADP1 and IBV 50-UTR. The MADP1 RRM 1 domain interacts with nucleic acid residues via aromatic and hydrophobic side chains at its active site, which in the case supplied by phenylalanine 55 and valine 53, respectively. Tyrosine 13 may have acted as an anchor for the phosphate backbone via electrostatic interactions. In this study, interaction between MADP1 and the SARS-CoV and IBV 50-UTR was initially identiﬁed by a yeast-based three hybrid screen and subsequently con- ﬁrmed using an in vitro RNA pull-down assay with IBV 50-UTR. A deeper look at the details of this interaction revealed that the RNA recognition motif, but not the zinc ﬁnger motif, of MADP1, is responsible for the interaction. This interaction is also shown to be speciﬁc and stem–loop I of IBV 50-UTR is essential for the interaction to occur. Although MADP1 was reported to be a nuclear protein (35), it could be detected in the cytoplasm of IBV-infected cells and partially overlaps with the de novo synthesized viral RNA, which marks the location of the RTCs in infected cells in the presence of actinomycin D. Silencing of MADP1 resulted in a marked reduction in syncytium formation upon IBV infection. A closer examination revealed that the synthesis of both genome- (gRNA) and