Published online 22 February 2012 Nucleic Acids Research, 2012, Vol. 40, No. 11 5065–5077 doi:10.1093/nar/gks165 Binding of the 50-untranslated region of coronavirus RNA to zinc finger CCHC-type and RNA-binding motif 1 enhances viral replication and transcription Yong Wah Tan1,2, Wanjin Hong2 and Ding Xiang Liu1,* 1School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 and 2Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673 Received August 24, 2011; Revised and Accepted January 31, 2012 ABSTRACT Coronaviruses RNA synthesis occurs in the cyto- plasm and is regulated by host cell proteins. In a screen based on a yeast three-hybrid system using the 50-untranslated region (50-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 subse- quently confirmed in coronavirus infectious bronchitis virus (IBV). The specificity of the inter- action between MADP1 and the 50-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 50-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 immuno- fluorescence. 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 coron- aviral RNA synthesis. INTRODUCTION During the replication of mammalian viruses, it is inevit- able for host proteins to be involved in the viral life cycles. In fact, coronaviruses require host proteins to aid in the stages from virus entry to progeny release. Entry of the virus particle into a host cell requires the recognition of speciﬁc cell surface proteins, which act as receptors for the virus spike (S) protein (1–6). Upon entry into host cells, the ribonucleocapsid uncoats and releases the 50-capped viral genome, a single-stranded positive-sense RNA. The genomic RNA ranges from 27 to 32 kb in length, is the largest known of its kind and is structurally similar to host mRNA (7). The replicase gene, which spans the 50 two-thirds of the genome, is translated by host ribosomes into two large polyproteins, pp1a and pp1ab via a frame- shift event (8–10). The polyproteins are autoproteo- lytically processed into a maximum of 16 nonstructural proteins (8,11–16), which are assembled into replication– transcription complexes, including the main enzyme RNA-dependent RNA polymerase (nsp12) (17,18). This complex is required for generating new full-length virus RNA in replication as well as subgenome-length RNAs to be used for translation of virus structural and accessory proteins. In addition to their role in RNA synthesis, these nonstructural proteins may have multiple functions, such as the suppression of host mRNA translation as well as mRNA degradation by nsp1 of SARS coronavirus (SARS-CoV; 19–21), which may play a role in the sup- pression of immune response mounted by the host upon infection. The replication–transcription complex (RTC), which is located on membrane bound vesicles in the cytoplasm (22), is required for genome replication through continu- ous transcription and subgenomic RNA synthesis via dis- continuous transcription (18,23,24). Apart from the replicase gene products, a viral structural protein, the nucleocapsid (N), is also required for efﬁcient viral RNA synthesis (25,26). The resulting genome-size transcripts are destined to be packaged into progeny virions while the subgenomic, positive-sense transcripts are being translated into four structural proteins, spike (S), nucleocapsid (N), membrane (M) and envelope (E) proteins, as well as other accessory proteins. In virus RNA synthesis, the replicase complex is indis- pensable but not an exclusive participant. Several host proteins have been identiﬁed to be able to interact with regulatory signals within the untranslated regions in the *To whom correspondence should be addressed. Tel: +65 63162862; Email: dxliu@ntu.edu.sg The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.