Identification and Characterization of Severe Acute Respiratory Syndrome Coronavirus Subgenomic RNAs

first recognized in Guangdong Province, China, in November 2002, and its causative agent was identified as novel coronavirus (SARS-CoV). Coronaviruses are the largest RNA viruses, containing a single-stranded, plus-sense RNA ranging from 27 kb to 31.5 kb in size. The two large open reading frames (ORFs) (1a and 1b) at the 5 -end of the genome encode the viral replicase and are translated directly from the genomic RNA, while 1b is expressed by –1 ribosomal frameshifting. The 3 -one third of the genome comprises the


INTRODUCTION
first recognized in Guangdong Province, China, in November 2002, and its causative agent was identified as novel coronavirus (SARS-CoV). [1][2][3] Coronaviruses are the largest RNA viruses, containing a single-stranded, plus-sense RNA ranging from 27 kb to 31.5 kb in size. The two large open reading frames (ORFs) (1a and 1b) at the 5 -end of the genome encode the viral replicase and are translated directly from the genomic RNA, while 1b is expressed by -1 ribosomal frameshifting. 4 The 3 -one third of the genome comprises the unknown function. These proteins are translated through 6-9 nested and 3 -coterminal subgenomic RNAs (sgRNAs). approximately 29,700 nucleotides. [5][6][7] Fourteen ORFs have been identified, of which 12 are located in the 3 -proximal one-third of the genome. 4,5 The exact mechanisms of expression of the 3 -proximal ORFs are unknown but on the analogy with other coronaviruses, these ORFs are predicted to be expressed through a set of sgRNAs. 6 Identification of SARS-CoV sgRNAs in infected cells and characterization of molecular details of the leader-body fusion in the sgRNAs will help elucidate the regulatory mechanism of SARS-CoV transcription and replication. This knowledge will be useful in the development of antiviral therapeutic agents and vaccine for treatment and prevention of this newly emerged disease.

IDENTIFICATION OF NOVEL SUBGENOMIC RNAS
Northern blot, RT-PCR, and DNA sequencing revealed the existence of ten subgenomic RNAs including two novel subgenomic RNAs named 2-1 and 3-1. The leader-body fusion site (ACGAgC) of subgenomic RNA 2-1 has one nucleotide mismatch (lowercase) with SARS-CoV leader core sequence (CS-L ) ACGAAC and is located inside the S gene, 384 nucleotides downstream from the authentic CS (ACGAAC) for mRNA 2/S. The second novel subgenomic RNA (3-1) corresponded to the 3b ORF that was predicted to be expressed from mRNA 3. The leader-body fusion site (AaGAAC) for subgenomic mRNA 3-1 is 10 nucleotides upstream of AUG start codon of ORF 3b and has a mismatch (lowercase) with the SARS-CoV leader core sequence (ACGAAC).
To determine whether these new subgenomic RNAs are functional messages, the 5´-end (containing leader sequence plus downstream 150 nucleotides in case of sgRNA 2-1 and 400 nucleotides in case of sgRNA 3-1) were fused with green fluorescent protein (GFP) gene, and sgRNA codon usage was indirectly determined by the expression of the reporter gene. Strong fluorescence was observed in cells transfected with the construct in which the ORF2b was fused in-frame with GFP ( Figure 1A). Western blot with anti-GFP downstream AUG by leaky scanning was also detected in cells transfected with in-frame and out-frame construct ( Figure 1B, upper panel).
Cells transfected with the ORF3b in-frame construct displayed weak fluorescence while no fluorescence was observed in cells transfected with the ORF3b out-frame construct ( Figure 1A). Western blot analysis confirmed the existence of a 42, kDa band corresponding to 3b-GFP fusion protein ( Figure 1B, lower panel). These results indicated that sgRNA 2-1 and 3-1 could function in the environment of the cell.   A single band of fusion protein was detected by Western blot in cells transfected with constructs containing the 5 -end of sgRNA 2, 2-1, 3, 3-1, 4, 6 and 7, whereas, in cells weight band was also detected that may result from a downstream AUG codon ( Figure  2B).
We also detected some minor low molecular weight bands in some cases, possibly subgenomic RNA8-GFP fusion construct displayed fair amount of fluorescence but in showed that nine out of ten subgenomic RNAs could be functional messages in vivo; however, existence of proteins encoded by these subgenomic in SARS-CoV infected cells is yet to be determined. In summary, ten subgenomic RNAs including two novel subgenomic RNAs (2-1/3-1) were identified in SARS-CoV infected cells by Northern blot, RT-PCR, and DNA sequencing. Nine out of 10 subgenomic RNAs were potentially functional messages in SARS-CoV infected cells. The initiator AUG codon of subgenomic RNA 8 (ORF8b) was inactive in our experimental set-up.
transfected with mRNA 5 and 9, beside the expected fusion protein band, a low molecular resulting from downstream AUG codons by leaky scanning. Cells transfected with Western blot only a 27 kDa band of wild-type GFP was detected. Taken together-we ,