Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting
Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting
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DOI：
10.1128/jvi.74.6.2760-2769.2000
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发表时间：
2000-03-01
影响因子：
5.4
通讯作者：
Brown, PO
中科院分区：
文献类型：
--
作者：
Laurent, LC;Olsen, MN;Brown, PO
我们对人类免疫缺陷病毒1型（HIV-1）RNA基因组5'端1,000个核苷酸片段的功能特性进行了详细的定量分析。病毒基因组的这一区段含有几个重要的顺式作用序列，包括TAR、聚腺苷酸化、病毒att位点、负链引物结合位点和5'剪接供体序列，以及基质蛋白和衣壳蛋白N-末端一半的编码序列。使用遗传足迹技术定量测定134个独立插入突变（i）产生稳定病毒RNA，（ii）组装和释放含病毒RNA的病毒颗粒，以及（iii）进入宿主细胞、完成逆转录、进入宿主细胞核并通过整合在宿主基因组中产生前病毒的能力。所有的突变体都被构建和分析，大大减少了诱变研究中通常涉及的劳动。结果证实了在HIV基因组的该区域中存在几个先前已知的功能特征，并为几个新特征提供了证据，包括新近鉴定的顺式作用序列，这些顺式作用序列似乎有助于（i）稳定的病毒转录物的形成，（ii）病毒RNA包装，和（iii）病毒复制的早期步骤。结果还指出基质的N-末端部分在稳定的病毒RNA转录物的形成中的未预料到的反式作用。最后，与以前的报道相反，本研究的结果表明，基质和衣壳蛋白中的有害突变主要干扰病毒组装。
We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5' end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5' splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies, The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including nea ly identified cis-acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication, The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.