Yong Wah Tan1,2, Wanjin Hong2 and Ding Xiang Liu1
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551
Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673
Published in Nucleic Acids Research, 22 February 2012 (Epub ahead of print)
Coronaviruses RNA synthesis occurs in the cytoplasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 5′-untranslated region (5′-UTR) of SARS coronavirus (SARS-CoV) RNA as bait against a human cDNA library derived from HeLa cells, we found a positive candidate cellular protein, zinc finger CCHC-type and RNA-binding motif 1 (MADP1), to be able to interact with this region of the SARS-CoV genome. This interaction was subsequently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the interaction between MADP1 and the 5′-UTR of IBV was investigated and confirmed by using an RNA pull-down assay. The RNA-binding domain was mapped to the N-terminal region of MADP1 and the protein binding sequence to stem–loop I of IBV 5′-UTR. MADP1 was found to be translocated to the cytoplasm and partially co-localized with the viral replicase/transcriptase complexes (RTCs) in IBV-infected cells, deviating from its usual nuclear localization in a normal cell using indirect immunofluorescence. Using small interfering RNA (siRNA) against MADP1, defective viral RNA synthesis was observed in the knockdown cells, therefore indicating the importance of the protein in coronaviral RNA synthesis.
Figure Legend: Knockdown of MADP1 by siRNA suppresses IBV infection.
(A) RT-PCR analysis of the effect of MADP1 knockdown on IBV RNA replication. H1299 cells were transfected twice with siRNA targeting either Madp1 (+siMadp1) or EGFP (-siMadp1) and infected with IBV-Luc recombinant virus three days after the first transfection. Samples were harvested at 4 h-intervals, and mock-infected cells were used as negative control. RT-PCR analyses of the mRNA levels of MADP1, the negative strand IBV RNA (gRNA(-)), (+) and (-) mRNAs 3 and 4 (sgRNA) and control GAPDH were carried out.
(B) Western blot analyses of the protein levels of Madp-1, viral proteins spike (S), nucleocapsid (N) and cellular protein actin for loading control.
(C) TCID50 of total virus produced by the virus infected cells showed that the silencing of Madp1 reduced virus titres by several folds.
(D) Luciferase gene activity measured for the cell lysate indicated a dramatic drop in viral activity in the Madp1 silenced H1299 cells
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