A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication
A conserved hairpin motif in the R-U5 region of the human immunodeficiency virus type 1 RNA genome is essential for replication
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DOI：
10.1128/jvi.71.3.2346-2356.1997
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发表时间：
1997-03-01
影响因子：
5.4
通讯作者：
Berkhout, B
中科院分区：
文献类型：
--
作者：
Das, AT;Klaver, B;Berkhout, B
人类免疫缺陷病毒（HIV） RNA基因组的未翻译先导区包含多个发夹基序。在RNA分子的3'端重复的前导的重复区域编码了众所周知的TAR发夹和单链环中具有聚腺苷化信号AAUAAA的第二个发夹结构[聚(a)发夹]。这种多(A)茎环结构及其热力学稳定性在HIV和猿猴免疫缺陷病毒分离株中得到很好的保守，尽管序列存在相当大的差异，这表明该RNA基序在病毒复制中具有生物学功能。与这一观点一致，我们证明了改变茎区稳定性或删除发夹上部的突变确实严重抑制了1型HIV的复制。而在碱基对茎的左侧或右侧的不稳定突变干扰病毒复制，双突变，允许形成新的碱基对，复制比两个单独的病毒突变更快。在长时间培养具有改变的发夹稳定性的病毒后，获得了具有额外突变的逆转病毒，这些突变恢复了poly(A)发夹的热力学稳定性。瞬时转染实验表明，5' poly(A)发夹突变不影响原病毒基因组的转录、病毒mrna的翻译和基因组rna的逆转录。我们发现病毒粒子的基因组RNA含量会因为poly(A)发夹的不稳定而减少，而不会因为poly(A)发夹结构的稳定或截断而减少。这些结果表明，基因组RNA分子5'端poly(A)发夹结构的形成对于将病毒基因组包装成病毒粒子和/或病毒粒子RNA的稳定性是必要的。
The untranslated leader region of the human immunodeficiency virus (HIV) RNA genome contains multiple hairpin motifs. The repeat region of the leader, which is reiterated at the 3' end of the RNA molecule, encodes the well-known TAR hairpin and a second hairpin structure with the polyadenylation signal AAUAAA in the single-stranded loop [the poly(A) hairpin]. The fact that this poly(A) stem-loop structure and its thermodynamic stability are well conserved among HIV and simian immunodeficiency virus isolates, despite considerable divergence in sequence, suggests a biological function for this RNA motif in viral replication. Consistent with this idea, we demonstrate that mutations that alter the stability of the stem region or delete the upper part of the hairpin do severely inhibit replication of HIV type 1. Whereas destabilizing mutations in either the left- or right-hand side of the base-paired stem interfere with virus replication, the double mutant, which allows the formation of new base pairs, replicates more rapidly than the two individual virus mutants. Upon prolonged culturing of viruses with an altered hairpin stability, revertant viruses were obtained with additional mutations that restore the thermodynamic stability of the poly(A) hairpin. Transient transfection experiments demonstrated that transcription of the proviral genomes, translation of the viral mRNAs, and reverse transcription of the genomic RNAs are not affected by mutation of the 5' poly(A) hairpin. We show that the genomic RNA content of the virions is reduced by destabilization of this poly(A) hairpin but not by stabilization or truncation of this structure, These results suggest that the formation of the poly(A) hairpin structure at the 5' end of the genomic RNA molecule is necessary for packaging of viral genomes into virions and/or stability of the virion RNA.