Nucleic Acids Research, 2012, Vol. 40, No. 11 5067 Table 1. Nucleotide sequences of primers used to amplify DNA templates for in vitro transcription Primer name T7_i1-27 pT_i507-528R T7_i528-506 pT_i1-29 T7_i30-51 pT_i99-80 T7_i141-162 pT_i140-121 T7_i1-27_SL1dsmut T7_i1-26_SL1rsmut pT_EGFP_F T7_EGFP_510-528R T7_i27106-27125 LDX30 Sequence 50-TGTAATACGACTCACTATAGGACTTAAGATAGATATTAATATATATCT-30 50-TTTTTTTTTTTTTTTTTTTTTGTTGTCACTGTCTATTGTATGT-30 50-TGTAATACGACTCACTATAGGGTTGTCACTGTCTATTGTATGTC-30 50-TTTTTTTTTTTTTTTTTTTTTACTTAAGATAGATATTAATATATATGTAT-30 50-TGTAATACGACTCACTATAGGTACACTAGCCTTGCGCTAGATT-30 50-TTTTTTTTTTTTTTTTTTTTTCCTATGAGGACCAGCTGTAG-30 50-TGTAATACGACTCACTATAGGGCCACCTGTCAGGTTTTTGTTA-30 5’-TTTTTTTTTTTTTTTTTTTTTCAGGTGCCATCCAGGGCACT-30 50-TGTAATACGACTGAGTATAGGACTTAAGATACTTATTAATATATATGT-30 50-TGTAATACGACTCACTATAGGACTTAAGATACTTATTAATATATAAG-3’ 50-TTTTTTTTTTTTTTTTTTTTTATGGTGAGCAAGGGCGAGG-30 50-TGTAATACGACTCACTATAGGGCTGCCGTCCTCGATGTTG-30 50-TGTAATACGACTCACTATAGGGTAACATAATGGACCTGTTG-30 50-TGATGCCGGCCACGATGCGTC-30 Table 2. Primer pairs used in ampliﬁcation of DNA templates for in vitro transcription Fragment IBV 50-UTR (+) 50-UTR 1 50-UTR 2 50-UTR 3 50-UTR 4 50-UTR 2M1 50-UTR 2M2 IBV 30-UTR EGFP Sense primer T7_i1-27 T7_i1-27 T7_i1-27 T7_i30-51 T7_141-162 T7_i1-27_SL1dsmut T7_i1-26_SL1rsmut T7_i27106-27125 T7_EGFP_510-528R Anti-sense primer pT_i507-528R pT_i140-121 pT_i99-80 pT_140-121 pT_i507-528R pT_i99-80 pT_i99-80 LDX30 pT_EGFP_F pathological change in 50% of inoculated cell culture, of the infected cells was used as a measurement of virus titer. The TCID50 of the infected cells at each time point was determined by using the freeze–thawed infected cells. For each sample, a 10-fold serial dilution was performed and ﬁve wells of Vero cells on 96-well plates were infected with each dilution. The numbers of infected wells were collated and TCID50 of each sample was calculated using the Reed–Muench method (5). Reverse transcription-polymerase chain reaction determination of the replication and sub-genomic transcription efﬁciency of IBV Total RNAs were prepared from the infected cells at their speciﬁed time points using Trizol Reagent (Invitrogen) Reverse transcription (RT) was performed with Expand reverse transcriptase (Roche) according to the manufac- turer’s instructions using the sense primer IBV leader (50-26CTATTACACTAGCCTTGCGCT46-30) for the de- tection of negative-stranded subgenomic RNA (sgRNA) and the antisense primer IBV24803-R (50-24803CTCTGG ATCCAATAACCTAC24784-30) for the detection of positive-stranded sgRNA. Both primers were then used for PCR. If transcription of subgenomic mRNAs did occur, a 415-bp PCR product corresponding to the 50-terminal region of subgenomic mRNA 5 and a 648-bp fragment corresponding to the 50-terminal region of subgenomic mRNA 4 would be expected. Similarly, RT was carried out with the sense primer IBV14931-F (50-14931GCTTATCCACTAGTACATC14949-30) for the detection of negative-stranded genomic RNA. Sense primer IBV14931-F and the antisense primer IBV15600-R (50-15600CTTCTCGCACTTCTGCACTAGCA15578-30) were used for PCR. If replication of viral RNA occurred, a 670-bp PCR fragment would be expected. Construction and selection of stable knockdown cell lines Oligonucleotides were designed based on siMadp1 sequence and cloned into pSilencer 2.1 Neo (Ambion) ac- cording to manufacturer’s instructions. Negative control silencer construct was supplied with the cloning kit. Constructs pSilencer-NC (negative control) and pSilencer-Madp1 were transfected into H1299 cells with Effectene Transfection Reagent. Transfected cells were selected with 500 mg/ml G418 (Sigma) and the selected clones were subjected to screening for MADP1 knockdown efﬁciency. Selected H1299-shNC and H1299- shMadp1 stable cell lines were maintained in media con- taining 500 mg/ml of G418. RESULTS Yeast three-hybrid screening for human proteins that could interact with SARS-CoV UTRs In order to ﬁnd candidate host proteins that may be involved in the replication and transcription of corona- virus RNA, a yeast-based three-hybrid (34) screen against a human cDNA library using the 50-UTR of SARS-CoV RNA as bait was performed. Screens were also performed using the negative sense 50-UTR and 30-UTR as bait. Each screen yielded about six to eight colonies which were sequenced and non-sense sequences of the candidates were eliminated. In total, the screen identiﬁed three candidates, MADP1, HAX1 and riboso- mal protein L27a as binding partners to SARS-CoV positive sense 50-UTR, negative sense 50-UTR and negative sense 30-UTR, respectively. Although it was inter- esting to ﬁnd ribosomal protein L27a interacting with the