【作者】 Snawar Hussain；

【导师】 郭德银；

【作者基本信息】 武汉大学 ， 生物化学， 2005， 博士

【摘要】 SARS冠状病毒亚基因组RNA的分析和鉴定 冠状病毒的一个重要特点是其独特的转录策略(strategy)——即通过不连续的转录合成一组嵌套(nested)的亚基因组RNA(sgRNA)使基因组3’端基因得到表达。转录过程中,每个亚基因组的合成都涉及到一个不连续的步骤,即一段由基因组5’端编码的前导RNA(leader RNA)与主体序列(body sequence)在不同的转录调节序列(transcription regulatory sequence TRS)相连。前导序列在转录时可以自由交换,因此前导序列和mRNA序列可以来源于两个不同的RNA分子,这一过程发生于亚基因组RNA负链RNA模板合成时。TRS在不连续转录时主体序列与前导序列的融合及不连续转录过程中起关键作用,含有一段高度保守的同一序列(consensus sequence CS)。在严重呼吸综合症(SARS)病毒(Sars-Cov)的感染细胞中,我们使用RT-PCR或Northern blot的方法检测到有12种亚基因组RNA,其可能参与表达3’端12个ORF,其中通过进一步的RT-PCR分析鉴定出2个未报道的亚基因组RNA,命名为2-1,3-1,这些亚基因组RNA集中在Sars-Cov基因组后1/3部分。亚基因组RNA存在于S基因内部,在S基因本身CS(ACGAAC)下游384个碱基处,它的前导序列与主体序列结合位点ACGAGC存在着与CS(ACGAAC)一个位点的错配,。 翻译亚基因组RNA 2-1可以得到一个截短的S蛋白(s’),其N端缺少了143个氨基酸。亚基因组RNA 3—1在3b区,预计可能从mRNA3开始表达,它的存在可能预示着ORF3b是从另外一个mRNA而不是从mRNA3开始表达的。亚基因组RNA 3—1的前导序列与主体序列的结合区AaGAAC在ORF3b起始序列AUG上游10个碱基处,与Sars-Cov冠状病毒CS序列同样存在一个碱基的错配。亚基因组RNA 2-1和3-1都与前导序列TRS有一个碱基的错配,但是与主体序列的TRS都是一致的。对不同亚基因组RNA主体序列和前导序列的结合区测序可以看到连接序列和相关的TRS序列都各有不同,而且在病毒的传代中保持相对的稳定性。亚基因组RNA正链和负链模板的共存和亚基因组RNA保守序列与主体序列保守区一致都可以支持不连续转录发生在从全长基因组RNA模板合成负链RNA时的假说模型。根据这一模型,RNA多聚酶在TRS暂停,然后跳跃更多还原

【Abstract】 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.更多还原