Identification And Characterization Of Severe Acute Respiratory Syndrome Associated Coronavirus Subgenomic RNAs

The expression of genomic information of severe acute respiratory syndrome coronavirus (SARS-CoV) involves synthesis of a nested set of subgenomic RNAs (sgRNAs) by discontinuous transcription. In SARS-CoV-infected cells, 10 subgenomic RNAs were identified by Northern blot and RT-PCR, which were predicted to be functional in the expression of 12 open reading frames located in the 3'-one third of the genome. Two novel subgenomic RNAs (sgRNAs) were identified by RT-PCR and named 2-1 and 3-1, respectively. The leader-body fusion site (ACGAgC) of subgenomic RNA 2-1 has one nucleotide mismatch (lower case) with the core sequence (CS) ACGAAC in the leader TRS (TRS-L) of SARS-CoV, and is located inside the S gene, 384 nucleotides downstream to authentic CS (ACGAAC) for mRNA 2/S. Translation of subgenomic mRNA 2-1 could result in the synthesis of a truncated S protein (named S') missing the N-terminal 143 amino acids. The second novel subgenomic RNA (3-1) corresponded to the 3b ORF that was predicted to be expressed from mRNA 3. The existence of subgenomic RNA 3-1 may indicate that the ORF 3b could be expressed from a separate mRNA other than 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 one nucleotid mismatch (lower case) with the CS of SARS-CoV TRS-L. Both mRNA 2-1 and 3-1, used a variant of TRS that has one nucleotide mismatch with the core sequence (ACGAAC) in TRS-L, but are identical to the body TRS. Sequence analysis of the leader-body fusion sites of each subgenomic RNAs showed that the junction sequences and the corresponding transcription regulatory sequence (TRS) are unique for each species of subgenomic RNA and consistent after virus passages. Co-existence of both positive and negative-strands of SARS-CoV subgenomic RNAs and evidence forderivation of subgenomic mRNA core sequence from body core sequence favors the model of discontinuous transcription during negative-strand synthesis. In this study, a subpopulation of mRNA 3-1 was identified, which contains additional four nucleotides (UCC A) at the leader-body junction site. Although transcription of this subgenomic RNA could represent a rare event for SARS-CoV, it did render more evidence for the use of non-canonical transcriptional signals in synthesis of subgenomic RNAs. Three models were proposed to explain the synthesis of this rare RNA:1. The AAA is used as transcription regulating signal and the complementarity between CS-L and CS-B takes place in the AAA region during template switch step.2. The interaction and complementarity between CS-L and CS-B is the same as that of mRNA 3-1 but the RNA polymerase can slide-back 4 nucleotides on the leader template to start transcription from downstream sequence.3. The interaction and complementarity between CS-L and CS-B is the same as that of mRNA 3-1 but extended complementarity in upstream sequence results in the formation of a loop-like structure, which is removed, by some sort of proof-reading mechanism in normal 3-1 subgenomic RNA, However a subpopulation of subgenomic RNAs has escaped unedited.10 subgenomic RNAs (including two minor subgenomic RNAs) have been identified in coronavirus infected cells but only five (2a/S, 3a, 4/E, 5/M, 9/N) are known to be expressed. We fused the 5'-ends of the subgenomic RNAs with GFP, expression of subgenomic RNAs was indirectly measured by florescent microscopy and Western blot. Significant differences in level of expression of different subgenomic RNAs were observed. Relatively high level of reporter gene expression was observed from the 2-1, 3, 4/E, 5/M, 6 and 9/N mRNA-GFP fusion constructs, whereas low level of reporter gene expression was observed from subgenomic RNAs 2/S, 3-1, 7 and 8 fusion constructs. No significant relationship was found between Kozak context of AUG initiator codon, length, G+C contents, secondary structure of 5'-untranslated region and level of expression. The SARS-CoV may control relative abundance of structural and nonstructural proteins indirectly at the level of transcription or alternatively, relative abundance of proteins is controlled by cis or trans acting elements.Taken together, these results gain more insight into the molecular mechanisms of genome expression and subgenomic transcription of SARS-CoV.