5076 Nucleic Acids Research, 2012, Vol. 40, No. 11 with different afﬁnities. Due to the lack of a high contain- ment facility, the functional implication of the relatively weaker interaction between SARS-CoV 50-UTR and MADP1 was not further studied. It is, therefore, yet to be demonstrated if this weaker binding dictates less dependency on MADP1 in SARS-CoV RNA replication and infectivity. Current evidence indicates that MADP1 is compartmentalized in the nuclei of cultured cells (35), markedly differing from the cytoplasmic, perinuclear localization of the coronavirus RTCs (47–49). As there was no report on the possibility of MADP1 shuttling between the nucleus and cytoplasm, our observation using indirect immunoﬂuorescence that over-expressed MADP1 upon IBV infection became partially localized in the cytoplasm may represent a ﬁrst report that MADP1 could be localized outside the nucleus. This could have been achieved with either an existing shuttling mechanism used by a nuclear protein or the assistance of viral factors. For example, IBV N protein is known to enter the nucleus while maintaining a predominantly cyto- plasmic localization (50,51). Alternatively, binding of viral RNA may partially retain the newly synthesized MADP1 in the cytoplasm, as observed in this study. It was observed that over-expression of Flag-tagged MADP1 was unable to fully restore IBV infection in MADP1-knockdown cells, even though the expression level of the introduced MADP1 construct far surpassed the endogenous level, as observed by western blot analysis. Considering the fact that only 30% of cells were transfected and over-expressed MADP1 protein despite the presence of a higher level of the protein in the transfected cells, it is understandable that the ex- pression of viral proteins could not be restored after combining both transfected and untransfected cells. Interestingly, over-expression of silencing-sensitive MADP1 was unable to cause an increase in virus infec- tion, comparing to that was observed for silencing- resistant MADP1 (FMmut) in shMadp1 cells, even though their expression levels were comparable. This lends further support to the conclusion that MADP1 is actively involved in the replication and infectivity of IBV. Although the functional studies involving IBV, a chicken coronavirus, and a human protein, MADP1, were conducted using human and African green monkey cells, which were non-native. It is noteworthy that MADP1 (HomoloGene 12095) is conserved in humans (Homo sapiens), chimpanzees (Pan troglodytes), wolves (Canis lupus), cattle (Bos Taurus), mice (Mus musculus), rats (Rattus norvegicus) and chickens (Gallus gallus). The African green monkey genome is not available at NCBI, but an alignment search using basic local alignment search tool (BLAST) of the MADP1 amino acid sequence against the rhesus macaque (Macaca mulatta) RefSeq Protein library yields a 99% sequence similarity between the two species. The chicken homolog, on the other hand, bears 85% amino acid sequence similarity, but with an almost identical match in the N-terminal 120 amino acids, to the human MADP1 protein. As the predicted interaction domain lies in the N-terminus, it is highly likely that the homologs from other species could replace human MADP1 in the interaction studies. In conclusion, the involvement of MADP1 in corona- virus RNA synthesis and its signiﬁcance are demonstrated in this study in the tissue culture system. Further studies with an MADP1 knock-out animal system, which is cur- rently not available, would be required to conﬁrm further the involvement of MADP1 in coronavirus RNA synthesis. FUNDING A Biomedical Research Council grant (BMRC 08/1/22/ 19.589); and a grant from Nanyang Technological University (SUG M58080000), Singapore. Funding for open access charge: Agency for Science, Technology and Research (A*STAR), Singapore. Conﬂict of interest statement. None declared. REFERENCES 1. Hofmann,H., Pyrc,K., van der Hoek,L., Geier,M., Berkhout,B. and Pohlmann,S. (2005) Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc. Natl Acad. Sci. USA, 102, 7988–7993. 2. Li,W., Moore,M.J., Vasilieva,N., Sui,J., Wong,S.K., Berne,M.A., Somasundaran,M., Sullivan,J.L., Luzuriaga,K., Greenough,T.C. et al. (2003) Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426, 450–454. 3. Shulla,A., Heald-Sargent,T., Subramanya,G., Zhao,J., Perlman,S. and Gallagher,T. (2011) A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. J. Virol., 85, 873–882. 4. Wu,K., Li,W., Peng,G. and Li,F. (2009) Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor. Proc. Natl Acad. Sci. USA, 106, 19970–19974. 5. Yamada,Y. and Liu,D.X. (2009) Proteolytic activation of the spike protein at a novel RRRR/S motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells. J. Virol., 83, 8744–8758. 6. Yamada,Y., Liu,X.B., Fang,S.G., Tay,F.P. and Liu,D.X. (2009) Acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells. PLoS One, 4, e6130. 7. Brian,D.A. and Baric,R.S. (2005) Coronavirus genome structure and replication. Curr. Top. Microbiol. Immunol., 287, 1–30. 8. Plant,E.P. and Dinman,J.D. (2008) The role of programmed-1 ribosomal frameshifting in coronavirus propagation. Front. Biosci., 13, 4873–4881. 9. Wang,X., Wong,S.M. and Liu,D.X. (2006) Identiﬁcation of hepta- and octo-uridine stretches as sole signals for programmed +1 and -1 ribosomal frameshifting during translation of SARS-CoV ORF 3a variants. Nucleic Acids Res., 34, 1250–1260. 10. Namy,O., Moran,S.J., Stuart,D.I., Gilbert,R.J. and Brierley,I. (2006) A mechanical explanation of RNA pseudoknot function in programmed ribosomal frameshifting. Nature, 441, 244–247. 11. Ziebuhr,J., Snijder,E.J. and Gorbalenya,A.E. (2000) Virus-encoded proteinases and proteolytic processing in the Nidovirales. J. Gen. Virol., 81, 853–879. 12. Fang,S., Chen,B., Tay,F.P., Ng,B.S. and Liu,D.X. (2007) An arginine-to-proline mutation in a domain with undeﬁned functions within the helicase protein (Nsp13) is lethal to the coronavirus infectious bronchitis virus in cultured cells. Virology, 358, 136–147. 13. Fang,S.G., Shen,H., Wang,J., Tay,F.P. and Liu,D.X. (2008) Proteolytic processing of polyproteins 1a and 1ab between