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Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites
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RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements.

Timothy Notton1,2, Joshua J Glazier1, Victoria R Saykally1

  • 1Gladstone Center for Cell Circuitry, Gladstone Institutes, San Francisco, California, USA.

Mbio
|January 20, 2021
PubMed
Summary

A new method, random deletion library sequencing (RanDeL-seq), comprehensively maps viral cis and trans elements. This technique identified critical, previously unknown elements in HIV and Zika virus, aiding antiviral development.

Keywords:
biotechnologyhuman immunodeficiency virussynthetic biology

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Area of Science:

  • Virology
  • Genomics
  • Molecular Biology

Background:

  • Noncoding genomic regions function as cis elements, crucial for viral processes like gene expression and encapsidation.
  • Mapping these viral cis elements traditionally relies on laborious deletion or mutant screening methods.
  • Accurate mapping of cis and trans elements is vital for understanding viral replication and developing antiviral therapies, including defective interfering particles (DIPs).

Purpose of the Study:

  • To introduce a high-throughput method, random deletion library sequencing (RanDeL-seq), for comprehensive, single-nucleotide resolution mapping of viral cis and trans elements.
  • To apply RanDeL-seq to identify essential cis-acting elements in HIV-1 and Zika virus (ZIKV).
  • To demonstrate the utility of RanDeL-seq in discovering novel viral regulatory regions and facilitating antiviral therapeutic development.

Main Methods:

  • Generation of variable-size random deletions in viral genomes via transposon integration, excision, and exonuclease chewback.
  • Barcoding of deletion mutants for high-throughput tracking using sequencing, termed RanDeL-seq.
  • Application of RanDeL-seq to screen large libraries of HIV-1 (>23,000 variants) and ZIKV (>90,000 variants).

Main Results:

  • RanDeL-seq successfully mapped HIV-1 cis and trans elements at single-nucleotide resolution, confirming known elements like the LTR, Ψ, and RRE.
  • The study revealed the central DNA flap (cPPT-CTS) in HIV-1 is as critical as LTR, Ψ, and RRE for long-term passage.
  • A novel, essential cis-acting region of approximately 300 bp downstream of the RRE in HIV-1 was identified, along with extensive cis-acting regions in ZIKV nonstructural genes.

Conclusions:

  • RanDeL-seq is a versatile and comprehensive technique for rapidly generating viral deletion libraries and mapping cis and trans elements.
  • The method identified critical cis elements in HIV-1, including the obligate nature of cPPT and a new region near the RRE.
  • RanDeL-seq revealed extensive cis-acting regions in ZIKV, essential for replication and packaging, highlighting its potential for discovering viral mechanisms and developing antivirals.