An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA
An intact TAR element and cytoplasmic localization are necessary for efficient packaging of human immunodeficiency virus type 1 genomic RNA
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DOI：
10.1128/jvi.73.5.4127-4135.1999
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发表时间：
1999-05-01
影响因子：
5.4
通讯作者：
Rekosh, D
中科院分区：
文献类型：
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作者：
Helga-Maria, C;Hammarskjöld, ML;Rekosh, D
虽然大多数定义人类免疫缺陷病毒1型（HIV-1）基因组RNA包装信号的报道都集中在主要5'剪接位点的下游区域，但其他人认为剪接位点的上游序列也可能起重要作用。在这项研究中，我们直接研究了HIV-1 TAR区域在RNA包装中所起的作用。对于这些实验，我们使用了前病毒表达系统，其在很大程度上不依赖于达特的转录激活。这使我们能够创建有效表达TAR中携带突变的RNA的构建体，并确定这些RNA被包装的能力。我们的结果表明，TAR中序列的缺失显著降低了病毒RNA被包装的能力。通过使用位于整个TAR结构中的一系列错义突变进一步检查RNA包装中对TAR序列的需求。先前显示影响达特反式激活的TAR突变，例如上环区中的G31 U或凸起中的UCU至AAG（核苷酸[nt] 22至24），对RNA包装没有任何影响。破坏TAR茎紧挨着凸起下面的部分的突变也几乎没有影响。相比之下，在TAR茎的下部含有突变的构建体中观察到对RNA包装的显著影响。改变nt 5至9、10至15、44至49或50至54的点突变都使RNA包装减少11至25倍。然而，恢复茎结构的补偿性双突变能够恢复包装。这些结果表明，RNA包装需要完整的下茎结构，而不是特定的序列。我们的研究结果还表明，RNA分子保留在细胞核内不能被包装，除非它们被运输到细胞质的Rev/Rev反应元件或梅森-辉瑞猴病毒组成型运输元件。
Although most reports defining the human immunodeficiency virus type 1 (HIV-1) genomic RNA packaging signal have focused on the region downstream of the major 5' splice site, others have suggested that sequences upstream of the splice site may also play an important role, In this study we have directly examined the role played by the HIV-1 TAR region in RNA packaging. For these experiments we used a proviral expression system that is largely independent of Tat for transcriptional activation. This allowed us to create constructs that efficiently expressed RNAs carrying mutations in TAR and to determine the ability of these RNAs to be packaged, Our results indicate that loss of sequences in TAR significantly reduce the ability of a viral RNA to be packaged. The requirement for TAR sequences in RNA packaging was further examined by using a series of missense mutations positioned throughout the entire TAR structure. TAR mutations previously shown to influence Tat transactivation, such as G31U in the upper loop region or UCU to AAG in the bulge (nucleotides [nt] 22 to 24), failed to have any effect on RNA packaging. Mutations which disrupted the portion of the TAR stem immediately below the bulge also had little effect. In contrast, dramatic effects on RNA packaging were observed with constructs containing mutations in the lower portion of the TAR stem, Point mutations which altered nt 5 to 9, 10 to 15, 44 to 49, or 50 to 54 all reduced RNA packaging 11- to 25-fold. However, compensatory double mutations which restored the stem structure were able to restore packaging. These results indicate that an intact lower stem structure, rather than a specific sequence, is required for RNA packaging. Our results also showed that RNA molecules retained within the nucleus cannot be packaged, unless they are transported to the cytoplasm by either Rev/Rev response element or the Mason-Pfizer monkey virus constitutive transport element.