Emery Ann, Zhou Shuntai, Pollom Elizabeth, Swanstrom Ronald
Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2017 Feb 28;91(6). doi: 10.1128/JVI.02515-16. Print 2017 Mar 15.
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 (/) is the most commonly used acceptor (about 50%) and A3 () 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 and 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 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. During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing 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.
全长人类免疫缺陷病毒1型（HIV-1）RNA可作为基因组或信使核糖核酸（mRNA），或者该RNA利用4个供体和10个受体进行剪接，从而在两个大小类别（1.8 kb和4 kb）中产生50多种生理相关转录本。我们开发了一种使用引物ID标签深度测序的检测方法来定量HIV-1剪接。使用实验室菌株NL4-3，我们发现A5（/）是最常用的受体（约50%），而A3（）是最少使用的（约3%）。当剪接到受体A1或A2后激活供体D2或D3时会产生两个小外显子，D2和D3的高频率使用显著减少了和转录本的数量。我们观察到剪接到受体A1和A2的温度敏感性存在明显模式。此外，A1近端保守结构的破坏导致所有利用A1的转录本减少了10倍。对一组B亚型传播/奠基者病毒的分析表明，剪接模式是保守的，但使用情况存在惊人的变异性。一个C亚型分离株情况类似，而一个猴免疫缺陷病毒（SIV）分离株则显示出显著差异。我们还观察到从一个转录本上的下游供体到另一个不同转录本上的上游受体的反式剪接，在1.8 kb RNA读数的0.3%中检测到这种情况。当内含子保留在4 kb大小类别中时，有几个剪接抑制实例。这些结果证明了该检测方法的实用性，并确定了HIV-1剪接调控的新实例。在HIV-1复制过程中，会产生50多种保守的剪接RNA变体。这里描述的剪接检测方法利用了深度测序技术的新进展，并结合引物ID标签的互补DNA（cDNA）引物，以在允许监测甚至低频剪接变体的深度上有效定量HIV-1剪接。我们已使用该检测方法来研究HIV-1剪接的几个特征，并确定这些剪接模式不同调控机制的新实例。这种剪接检测方法可用于详细探索HIV-1剪接是如何被调控的，并且以适度的通量，可用于筛选改变这些相对保守剪接模式的结构元件、小分子和宿主因子。
