5066 Nucleic Acids Research, 2012, Vol. 40, No. 11 viral genome of betacoronavirus MHV. These include the polypyrimidine tract-binding protein (PTB) (27,28) with the leader sequence, hnRNP A1 (27,29,30) and hnRNP Q (31) with the 30-UTR. More recently, poly(A)-binding protein (PABP), hnRNP Q and glutamyl-prolyl-tRNA synthetase (EPRS) were found to play a role in corona- virus RNA synthesis through their interaction with the 30-UTR of alphacoronavirus TGEV (32). In addition, interaction of viral proteins with host proteins, such as the recently identiﬁed interaction between coronavirus nsp14 and DDX1 (33), may also play important enhance- ment functions in coronavirus replication and infection cycles. In this study, we describe the interaction of a cellular protein, MADP1 (zinc ﬁnger CCHC-type and RNA binding motif 1) with the 50-UTR of IBV and SARS- CoV, using yeast-based three hybrid screen (34) and RNA-binding assays. Subsequently, the RNA-binding domain of MADP1 and the RNA secondary structure re- sponsible for the interaction were mapped and deﬁned. Using indirect immunoﬂuorescence, we conﬁrmed that MADP1, despite being reported as a nuclear protein (35), was detected in the cytoplasm of virus-infected cells and partially co-localized with the RTCs. Upon silencing of MADP1 using siRNA, viral RNA synthesis on general has been affected, resulting in a lower replication efﬁ- ciency and infectivity. MATERIALS AND METHODS Over-expression of Flag-tagged proteins All wild-type and mutant MADP1 expressing constructs were based on the vector pXJ40Flag which contains both the CMV and T7 promoter and all expressed proteins were N-terminally tagged with the Flag epitope. For the over-expression of the wild-type and mutant MADP1 proteins, H1299 cells grown to 100% conﬂuency were infected with recombinant Vaccinia-T7 virus for 2 h (h), and the constructs were transfected into the infected cells using Effectene Transfection Reagent (Qiagen). Cells were lysed with lysis buffer [140 mM NaCl, 10 mM Tris (pH 8.0), 1% NP-40] 22 h post-transfection. Biotin-RNA pull-down assay Template DNA was ampliﬁed from plasmid DNA encoding the 50 end of IBV genome with various sets of primers targeting different regions of the 50-UTR (Tables 1 and 2), with the sense primers containing the T7 promoter sequence (36). Biotinylated RNAs were in vitro transcribed with T7 RNA polymerase (Roche Applied Science) in the presence of Biotin RNA Labeling Mix (Roche Applied Science) at 37 C for 2 h. Template DNAs were removed by digestion with RNase-free DNase I (Roche Applied Science) and the labeled RNAs puriﬁed with UltraPure Phenol:Chloroform:Isoamyl Alcohol (Invitrogen) then solubilized in nuclease-free water. Biotinylated RNA at 0.1 mM was incubated with cell lysates over-expressing EGFP, Flag-tagged MADP1 or its mutant proteins, respectively, in the presence of 10 mM dithiothreitol (DTT), 100 mg/ml yeast tRNA (Ambion) and 1 U/ml Protector RNase Inhibitor (Roche Applied Science) in a ﬁnal volume of 200 ml at room tem- perature for 30 min. The mixtures were incubated with 40 ml (50% slurry) of streptavidin agarose beads (Sigma Aldrich) at room temperature for 30 min. The beads were collected by centrifugation and washed three times with RNase P (RP) buffer (50 mM KCl, 1 mM MgCl2, 10 mM HEPES, pH 8.0), suspended in 25 ml of sodium dodecyl sulfate (SDS) sample buffer with 100 mM DTT. Bound proteins were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and detected with appropri- ate antibodies. Indirect immunoﬂuorescence microscopy and bromo-UTP labeling African green monkey kidney cells (Vero) grown to 50% conﬂuency in four-chamber glass slides were transfected to over-express Flag-tagged MADP1 or vector control using Effectene for 16 h. Transfected cells were infected with wild-type IBV or mock-infected with Vero cell lysate (Vero cells with serum-free medium subjected to three freeze–thaw cycles at minus 80 C and room temperature, respectively) for 1 h. Infection was allowed to progress for 2 h after virus removal and the cells were treated with actinomycin D at 15 mg/ml (Sigma Aldrich) for 4 h; 1 mM of BrUTP (Sigma Aldrich) was transfected into the cells with SuperFECT (Qiagen) for 3 h. Cells were ﬁxed at 10 h post-infection with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton-X 100 for 10 min. Treated cells were blocked in 10% goat serum, stained with primary antibodies mouse anti-BrdU and rabbit anti-Flag (Sigma Aldrich) and subsequently probed with AlexaFluor 488 anti-rabbit and 594 anti-mouse (Invitrogen) antibodies. Images were captured with Olympus Fluoview Upright Confocal microscope using a sequential laser scanning protocol. Viral growth assays H1299 cells grown to 30% conﬂuency were transfected with 100 nM of either siEGFP (50-GCAACGUGACCC UGAAGUUCdTdT-30) or siMadp1 (50-CAAUGACUU GUACCGGAUAdTdT-30) using DharmaFECT 2 siRNA Transfection Reagent (Dharmacon) for 72 h. Cells were infected with recombinant IBV-Luc at a multi- plicity of infectivity of 1 (MOI & 1) and incubated for 2 h at 37 C, 5% CO2. The virus-containing medium was replaced with fresh serum-free medium and the cells were either harvested immediately (0 h) or continued to be incubated at 37 C until speciﬁc time points post infection (4, 8, 12, 16, 20 or 24 h). Infected cells were subjected to lysis, either through three freeze–thaw cycles (at 80 C and room temperature, respectively) without removal of media, or using lysis buffer after removal of media. Fireﬂy luciferase activity which was used as an indication of viral activity for the recombinant virus was measured using Luciferase Assay System (Promega) according to manu- facturer’s instructions using the cell lysates. An end-point dilution assay, the 50% tissue culture infectious dose (TCID50), the amount of virus that will produce