5072 Nucleic Acids Research, 2012, Vol. 40, No. 11 All mutants resulted in a reduction in RNA-binding afﬁnity for the biotinylated RNA molecule and the reduc- tion was most dramatic for triple residue mutant YVF (Figure 4C), implying cooperative binding demonstrated by the three residues. This ﬁnding conﬁrms that the MADP1 RRM is involved in the interaction with IBV 50-UTR. MADP1 is required for efﬁcient virus transcription To demonstrate the signiﬁcance of the interaction between MADP1 and IBV 50-UTR, an siRNA duplex designed to silence MADP1 expression (siMadp1) and a negative control siRNA targeting EGFP protein (siEGFP) were used in time course experiments. H1299 cells were trans- fected twice with the siRNA duplexes in 24-h interval and infected with IBV-Luc recombinant virus (39) 3 days after the ﬁrst transfection. IBV-infected samples were harvested at 0, 4, 8, 12, 16, 20 and 24 h post-infection, respectively, and the mock-infected cells were harvested at 24 h post-infection as a negative control. The levels of negative stranded genomic RNA and both the positive and negative stranded subgenomic RNA were assessed by RT-PCR (Figure 5A). Densitometric analyses identiﬁed a reduction between 40% and 80% of MADP1 mRNA was achieved by this siRNA which resulted in a reduction between 70% and 90% of negative stranded genomic viral RNA, 40–80% of negative stranded subgenomic viral RNA and 50–90% of positive stranded subge- nomic viral RNA. Western blot analysis also noted a reduction in the expression of viral structural genes, between 50% and 90% reduction for S and N proteins, with a reduction between 40% and 80% of MADP1 protein (Figure 5B). Virus titers as represented by the tissue culture infectious dose (Log10 TCID50) at each infection time point was reduced by a minimum of 3-fold and up to 10-fold compared to siEGFP-transfected cells beyond 4 h of infection (Figure 5C). Fireﬂy luciferase activity of cell lysates harvested at different time points showed a minimum of 50% reduction upon the silencing of MADP1, which supports further the observation that the total viral protein production was much reduced (Figure 5D). To eliminate the possibility that the phenotype observed in MADP1-silenced cells during IBV infection was due to an off-target effect of the siRNA duplex used, four add- itional siRNA duplexes targeting different regions of MADP1 were used in various combinations with siMadp1 (Figure 6) to check their effect on IBV infection, as illustrated by the expression of the luciferase gene (Figure 6B). All six combinations of ﬁve different siRNA duplexes resulted in a reduction in the luciferase activity of the infected cells by either 70% (siCombi 3 and 4), without siMadp1 or more than 90% (siCombi 1, 2, 5 and 6) with siMadp1, compared to negative control, siEGFP-transfected cells (Figure 6B). This implies that, in general, knocking down MADP1 with any siRNA results in a reduction of virus infection. Expression of a silencing-resistant mutant MADP1 in a stable MADP1-knockdown cell clone enhances IBV replication A stable cell clone expressing short hairpin RNA to MADP1 (shMadp1) was selected from H1299 cells and the madp1 mRNA level was conﬁrmed using northern blot (Figure 7A). The expression of MADP1 and the effect of MADP1-knockdown on IBV infection were tested by comparing with a G418-selected cell line without expression of shMadp1 (non-targeting control, shNC). The results showed that, in general, silencing of MADP1 with shRNA reduced the amount of viral mRNA production before 16 h post-infection (Figure 7B). The amount of virus mRNA is higher in shMadp1 cells compared to shNC cells beyond 16 h of infection as infec- tion in shNC cells progressed much faster and most cells died and detached (Figure 7B). The shMadp1 cell line was then transfected with con- structs expressing Flag-tagged wild type MADP1 (FM), triple residue mutant (FM(YVF)), two mRNA mutants resistant to silencing by siMadp1 based on wild-type MADP1 (FMmut) and the triple residue mutant (FMmut(YVF)), negative vector control (F) and EGFP (E), respectively. The two siRNA-resistant mutants were constructed by mutating the siRNA-targeting sequence with degenerate codons, so that the protein sequence of MADP1 was maintained. These transfected cells were sub- sequently infected with IBV-Luc and harvested at 19 h post-infection. Western blotting results showed an obvious increase in the amount of IBV N expression in cells over-expressing silencing-resistant wild-type MADP1 (FMmut) as well as a slight increase in cells over-expressing both normal triple residue mutant (FM(YVF)) and silencing-resistant triple residue mutant (FMmut(YVF)) (Figure 7C). An assessment of the luciferase activity of total cell lysates showed that over-expression of triple residue mutants FM(YVF) and FMmut(YVF) resulted in a slight increase of the luciferase activity in shMadp1 cells, whereas over-expression of silencing-resistant wild-type MADP1 (FMmut) resulted in a more drastic increase of the luciferase activity in shMadp1 cells (Figure 7D). MADP1 interacts weakly with human coronavirus OC43 (HCoV-OC43) 50-UTR It was noted that although MADP1 interacted with both SARS-CoV and IBV 50-UTR, the interaction was rather weak for the former. A comparison of the predicted stem– loop I structures from both coronaviruses indicated a marked difference in their primary sequence as well as the secondary structures. Hence, a third coronavirus, HCoV-OC43, whose stem–loop I which deviated further from IBV than SARS-CoV, was assessed for its binding to MADP1 (Figure 8A). It was found that the binding of MADP1 to the 50 UTR of HCoV-OC43 was as weak, if not weaker than SARS-CoV. It was also noted that the predicted stem–loop I structure of HCoV-OC43 contained a bulge which encompassed a larger area of the stem compared to SARS-CoV (Figure 8B). Bulges were con- spicuously absent from the IBV stem–loop I (Figure 8B).