Clever J L, Eckstein D A, Parslow T G
Departments of Pathology and of Microbiology and Immunology, University of California, San Francisco, California 94143, USA.
J Virol. 1999 Jan;73(1):101-9. doi: 10.1128/JVI.73.1.101-109.1999.
The efficient packaging of genomic RNA into virions of human immunodeficiency virus type 1 (HIV-1) is directed by cis-acting encapsidation signals, which have been mapped to particular RNA stem-loop structures near the 5' end of the genome. Earlier studies have shown that three such stem-loops, located adjacent to the major 5' splice donor, are required for optimal packaging; more recent reports further suggest a requirement for the TAR and poly(A) hairpins of the 5' R region. In the present study, we have compared the phenotypes that result from mutating these latter elements in the HIV-1 provirus. Using a single-round infectivity assay, we find that mutations which disrupt base pairing in either the TAR or poly(A) stems cause profound defects in both packaging and viral replication. Decreased genomic packaging in a given mutant was always accompanied by increased packaging of spliced viral RNAs. Compensatory mutations that restored base pairing also restored encapsidation, indicating that the secondary structures of the TAR and poly(A) stems, rather than their primary sequences, are important for packaging activity. Despite having normal RNA contents, however, viruses with compensatory mutations at the base of the TAR stem were severely replication defective, owing to a defect in proviral DNA synthesis. Our findings thus confirm that the HIV-1 TAR stem-loop is required for at least three essential viral functions (transcriptional activation, RNA packaging, and reverse transcription) and reveal that its packaging and reverse transcription activities can be dissociated genetically by mutations at the base of the TAR stem.
人类免疫缺陷病毒1型（HIV-1）基因组RNA高效包装进病毒粒子是由顺式作用的包装信号指导的，这些信号已被定位到基因组5'端附近特定的RNA茎环结构。早期研究表明，位于主要5'剪接供体附近的三个这样的茎环是最佳包装所必需的；最近的报道进一步表明5'R区域的TAR和聚腺苷酸发夹也有需求。在本研究中，我们比较了HIV-1前病毒中这些后述元件发生突变所产生的表型。使用单轮感染性测定，我们发现破坏TAR或聚腺苷酸茎中碱基配对的突变在包装和病毒复制方面都导致严重缺陷。给定突变体中基因组包装的减少总是伴随着剪接病毒RNA包装的增加。恢复碱基配对的补偿性突变也恢复了包装，表明TAR和聚腺苷酸茎的二级结构而非其一级序列对包装活性很重要。然而，尽管RNA含量正常，但TAR茎基部有补偿性突变的病毒由于前病毒DNA合成缺陷而严重复制缺陷。我们的发现因此证实HIV-1 TAR茎环至少对三种基本病毒功能（转录激活、RNA包装和逆转录）是必需的，并揭示其包装和逆转录活性可通过TAR茎基部的突变在遗传上解离。
