RanDeL-Seq: a High-Throughput Method to Map Viral cis- and trans-Acting Elements

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.