Characterizing HIV-1 Splicing by Using Next- Generation Sequencing
Characterizing HIV-1 Splicing by Using Next- Generation Sequencing
复制标题
DOI：
10.1128/jvi.02515-16
复制
发表时间：
2017-03-01
影响因子：
5.4
通讯作者：
Swanstrom, Ronald
中科院分区：
文献类型：
--
作者：
Emery, Ann;Zhou, Shuntai;Swanstrom, Ronald
全长人类免疫缺陷病毒1型（HIV-1）RNA作为基因组或作为mRNA，或者该RNA使用4个供体和10个受体进行剪接，以产生两种大小类别（1.8 kb和4 kb）的50多种生理相关转录物。我们开发了一种使用引物ID标记的深度测序来定量HIV-1剪接的测定法。使用实验室菌株NL 4 -3，我们发现A5（env/ nef）是最常用的受体（约50%），A3（达特）使用最少（约3%）。当剪接到受体A1或A2之后激活供体D2或D3时，产生两个小外显子，并且D2和D3的高水平使用显著降低了vif和vpr转录物的量。我们观察到不同的模式的剪接受体A1和A2的温度敏感性。此外，破坏A1附近的保守结构导致所有使用A1的转录物减少10倍。对一组B亚型传播/创始病毒的分析表明，剪接模式是保守的，但使用的变异性令人惊讶。亚型C分离株是相似的，而猴免疫缺陷病毒（SIV）分离株显示出显着差异。我们还观察到从一个转录本上的下游供体到另一个转录本上的上游受体的反式剪接，我们在0.3%的1.8 kb RNA读段中检测到。当env内含子保留在4-kb大小的类别中时，有几个剪接抑制的例子。这些结果证明了该检测方法的实用性，并确定了HIV-1剪接调节的新例子。重要信息在HIV-1复制过程中，产生了50多种保守的剪接RNA变体。本文所述的剪接测定使用深度测序技术的新发展，结合Primer ID标记的cDNA引物，以有效地定量HIV-1剪接，其深度甚至允许监测低频剪接变体。我们已经用这种方法来检查HIV-1剪接的几个特点，并确定这些剪接模式的不同调节机制的新例子。这种剪接测定可用于详细探索HIV-1剪接是如何调节的，并且具有中等通量，可用于筛选改变这些相对保守的剪接模式的结构元件、小分子和宿主因子。
Full-length human immunodeficiency virus type 1 ( HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes ( 1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 ( env/ nef) is the most commonly used acceptor (about 50%) and A3 (tat) the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of vif and vpr transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/ founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the env intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation.IMPORTANCE During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deepsequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.