Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus
Functional analysis of the genomic and antigenomic promoters of human respiratory syncytial virus
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DOI：
10.1128/jvi.74.13.6006-6014.2000
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发表时间：
2000-07-01
影响因子：
5.4
通讯作者：
Peeples, ME
中科院分区：
文献类型：
--
作者：
Fearns, R;Collins, PL;Peeples, ME
利用基于质粒的微型复制子系统检测了呼吸道合胞病毒(RSV)转录和RNA复制过程中的启动子。基因组和反基因组的3‘端分别包含44个核苷酸(NT)前导区(Le)和155个核苷酸尾部补体(TRC)区，每个末端都应该包含一个RNA复制启动子。基因组3‘端也应该有转录启动子。用不同长度的TRC替换Le，表明TRC的3‘端36个核苷酸足以进行广泛的(但不是最大的)复制，当与转录基因开始(GS)信号并列时，该序列也能够指导转录。研究还表明，第一个基因的GS信号之前的Le区域可以被删除，但不会对转录起始产生影响或略有减少。因此，TRC有能力直接转录，即使它在性质上不这样做，它与基因组3‘端的部分序列同源性可能代表了在复制和转录中活跃的保守启动子的重要元件。将引入的TRC序列的长度递增到147个核苷酸，导致复制增加四倍，并几乎完全抑制转录。这两个效应是不相关的，这意味着转录和复制不是由共同的聚合酶介导的相互转化的过程，而是独立的过程。复制的增加是特定于TRC序列的，这意味着存在一个非必要的、促进复制的顺式作用元件。相反，对转录的抑制作用仅仅是由于基因组3‘端与GS信号之间的间距改变，这意味着转录酶将第一个GS信号识别为启动子元件。复制的增强或转录的抑制都不是由于3‘端和5’端之间碱基配对电位的增加，比较了Le和TRC启动子指导RNA合成的相对强度，发现它们非常相似。因此，这些发现强调了RSV抗原基因组启动子和基因组启动子之间高度的功能相似性，但进一步区分了启动子对转录和复制的要求。
The promoters involved in transcription and RNA replication by respiratory syncytial virus (RSV) were examined by using a plasmid-based minireplicon system. The 3' ends of the genome and antigenome, which, respectively, contain the 44-nucleotide (nt) leader (Le) and 155-nt trailer-complement (TrC) regions, should each contain a promoter for RNA replication. The 3' genome end also should have the promoter for transcription. Substitution for the Le with various lengths of TrC demonstrated that the 3'-terminal 36 nt of TrC are sufficient for extensive (but not maximal) replication and that when juxtaposed with a transcription gene-start (GS) signal, this sequence was also able to direct transcription. It was also shown that the region of Le immediately preceding the GS signal of the first gene could be deleted with either no effect or with a slight decrease in transcription initiation. Thus, the TrC is competent to direct transcription even though it does not do so in nature, and the partial sequence identity it shares with the 3' end of the genome likely represents the important elements of a conserved promoter active in both replication and transcription. Increasing the length of the introduced TrC sequence incrementally to 147 nt resulted in a fourfold increase in replication and a nearly complete inhibition of transcription. These two effects were unrelated, implying that transcription and replication are not interconvertible processes mediated by a common polymerase, but rather are independent processes. The increase in replication was specific to the TrC sequence, implying the presence of a nonessential, replication-enhancing cis-acting element. In contrast, the inhibitory effect on transcription was due solely to the altered spacing between the 3' end of the genome and GS signal, which implies that the transcriptase recognizes the first GS signal as a promoter element. Neither the enhancement of replication nor the inhibition of transcription was due to increased base-pairing potential between the 3' and 5' ends, The relative strengths of the Le and TrC promoters for directing RNA synthesis were compared and found to be very similar. Thus, these findings highlighted a high degree of functional similarity between the RSV antigenomic and genomic promoters, but provided a further distinction between promoter requirements for transcription and replication.