It has long been known that noncoding genomic regions can be obligate cis elements acted upon in trans by gene products. In viruses, cis elements regulate gene expression, encapsidation, and other maturation processes but mapping these elements relies on targeted iterative deletion or laborious prospecting for rare, spontaneously occurring mutants. Here, we introduce a method to comprehensively map viral cis and trans elements at single-nucleotide resolution by high-throughput random deletion. Variable-size deletions are randomly generated by transposon integration, excision, and exonuclease chewback, and then barcoded for tracking via sequencing (i.e., Random-Deletion Library sequencing, RanDeL-seq). Using RanDeL-seq, we generated and screened >23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s cis and trans elements. The resulting landscape recapitulated HIV-1’s known cis-acting elements (i.e., LTR, Y, and RRE) and surprisingly indicated that HIV-1’s central DNA flap (i.e., central polypurine tract, cPPT to central termination sequence, CTS) is as critical as the LTR, Y, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ~300bp region downstream of RRE extending to splice acceptor 7 that is equally critical for sustained viral passage. RanDeL-seq was also used to construct and screen a library of >90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s cis-acting regions are larger than the UTR termini, encompassing a large fraction of the non-structural genes. Collectively, RanDeL-seq provides a versatile framework for generating viral deletion mutants enabling discovery of replication mechanisms and development of novel antiviral therapeutics, including for emerging viral infections.

中文翻译：

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RanDeL-seq：高通量方法，可绘制病毒顺式和反式作用元件

早就知道，非编码基因组区域可以是基因产物反式作用的顺式顺式元件。在病毒中，顺式元件可调控基因表达，衣壳化和其他成熟过程，但对这些元件进行绘图则依赖于靶向的重复缺失或难得的，自发的突变体。在这里，我们介绍一种通过高通量随机缺失以单核苷酸分辨率全面映射病毒顺式和反式元件的方法。通过转座子整合，切除和核酸外切酶的回切随机产生可变大小的缺失，然后将其条形码化以通过测序进行跟踪（即，随机缺失文库测序，RanDeL-seq）。使用RanDeL-seq，我们生成并筛选了超过23,000个HIV-1变体，以生成HIV-1的顺式和反式元件的单碱基分辨率图。由此产生的景观概括了HIV-1的已知顺式作用元件（即LTR，Y和RRE），并出人意料地表明，HIV-1的中央DNA瓣（即中央多嘌呤束，中央终止序列的CPPT，CTS）同样重要作为长期通过的LTR，Y和RRE。引人注目的是，RanDeL-seq在RRE下游发现了一个以前未报道的〜300bp区域，该区域延伸至剪接受体7，这对于持续的病毒传代同样至关重要。RanDeL-seq也用于构建和筛选Zika病毒（ZIKV）> 90,000变体的库。出乎意料的是，RanDeL-seq表明ZIKV的顺式作用区比UTR末端大，涵盖了很大一部分非结构基因。总的来说，