Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA
Capsid coding sequence is required for efficient replication of human rhinovirus 14 RNA
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DOI：
10.1128/jvi.70.3.1941-1952.1996
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发表时间：
1996-03-01
影响因子：
5.4
通讯作者：
Lemon, SM
中科院分区：
文献类型：
--
作者：
McKnight, KL;Lemon, SM
小核糖核酸病毒的正链RNA基因组复制机制仍不明确，但现有模型并未表明编码衣壳蛋白的序列起作用。然而，候选RNA复制子（ΔP1β -半乳糖苷酶和ΔP1荧光素酶）代表了人类鼻病毒14型病毒（HRV - 14）的序列，其中报告蛋白序列（分别为β -半乳糖苷酶或荧光素酶）取代了大部分P1衣壳编码区，尽管多聚蛋白能有效进行初次切割，但在转染的H1 - HeLa细胞中无法复制。为了确定RNA复制可能需要哪些P1序列，构建了从基因组中按读码框去除P1区域片段的HRV - 14突变体。缺失涉及P1区域5'端1489个核苷酸的突变体能够有效复制，而缺失涉及3'端1079个核苷酸的突变体则不能。将3'端P1序列重新引入不能复制的ΔP1荧光素酶构建体中，产生了一个新的候选复制子ΔP1荧光素酶/VP3，它能很好地复制并高效表达荧光素酶。辅助病毒反式提供的衣壳蛋白无法拯救不能复制的ΔP1荧光素酶基因组，但能够包装比基因组长度更长的ΔP1荧光素酶/VP3复制子。因此，3'端的P1衣壳编码序列具有一种先前未被认识的与HRV - 14 RNA复制相关的顺式作用功能。
Mechanisms by which the plus-sense RNA genomes of picornaviruses are replicated remain poorly defined, but existing models do not suggest a role for sequences encoding the capsid proteins. However, candidate RNA replicons (Delta P1 beta gal and Delta P1Luc), representing the sequence of human rhinovirus 14 virus (HRV-14) with reporter protein sequences (P-galactosidase or luciferase, respectively) replacing most of the P1 capsid-coding region, failed to replicate in transfected H1-HeLa cells despite efficient primary cleavage of the polyprotein. To determine which P1 sequences might be required for RNA replication, HRV-14 mutants in which segments of the P1 region were removed in frame from the genome were constructed. Mutants with deletions involving the 5'-proximal 1,489 nucleotides of the P1 region replicated efficiently, while those with deletions involving the 3' 1,079 nucleotides did not. Reintroduction of the 3' P1 sequence into the nonreplicating Delta P1Luc construct resulted in a new candidate replicon, Delta P1Luc/VP3, which replicated well and expressed luciferase efficiently. Capsid proteins provided in trans by helper virus failed to rescue the nonreplicating Delta P1Luc genome but were able to package the larger-than-genome-length Delta P1Luc/VP3 replicon. Thus, a 3'-distal P1 capsid-coding sequence has a previously unrecognized cis-active function related to replication of HRV-14 RNA.