| SARS coronavirus (SARS-CoV) is a novel human coronavirus and is responsible for SARS outbreak in several regions of the world in 2003. The genome of SARS-CoV is about 30,000 nucleotides in length and sequence analysis revealed that nucleotides 1-72 contain a RNA leader sequence preceding an untranslated region (UTR). The nucleocapsid (N) protein of SARS-CoV is a major virion structural protein. For some coronavirus, such as mouse hepatitis virus (MHV), N protein associates with both viral genomic RNA and subgenomic mRNAs , and has structural and nonstructural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation. During these process, a high-affinity, specific interaction between the serine-arginine rich (SR-rich) region of the N protein and a conserved RNA sequence (UCUAA) present at the 5'-ends of MHV mRNA. We found that, for the SARS-CoV, there is not only a SR-rich region in the N protein but also a UCUAA consensus sequence in the leader sequence.In this study, we obtained the N gene by PCR from the cDNA of SARS-CoV and cloned the N and its fragments into expression vector pET30a. Then the proteins were expressed in E. coli and purified by affinity chromatography and gel filtration chromatography. The RNAleader sequence was synthesized by in vitro transcription and labeled by DIG The RNA-protein interaction was detected by Northwestern RNA blot assay. It showed that in vitro, SARS N protein could bind with leader sequence and a high-affinity, specific interaction took place between the SR-rich region of the N protein and the conserved RNA sequence (UCUAA). Our current results provided some, insight into the understanding of assembly of the SARS-CoV particles, which is an essential step for viral replication cycle. This work also provides the basis for future studies on the roles of N protein involved in genome replication, transcription > translation and virus encapsulation.
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