Abstract
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., long terminal repeat [LTR],
W, and Rev response element [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, W, and
RRE for long-term passage. Strikingly, RanDeL-seq identified a
previously unreported similar to 300-bp 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
untranscribed (UTR) termini, encompassing a large fraction of the
nonstructural genes. Collectively, RanDeL-seq provides a versatile
framework for generating viral deletion mutants, enabling discovery of
replication mechanisms and development of novel antiviral therapeutics,
particularly for emerging viral infections.
IMPORTANCE Recent studies have renewed interest in developing novel
antiviral therapeutics and vaccines based on defective interfering
particles (DIPs)-a subset of viral deletion mutants that conditionally
replicate. Identifying and engineering DIPs require that viral cis- and
trans-acting elements be accurately mapped. Here, we introduce a
high-throughput method (random deletion library sequencing
[RanDeL-seq]) to comprehensively map cis- and trans-acting elements
within a viral genome. RanDeL-seq identified essential cis elements in
HIV, including the obligate nature of the once-controversial viral
central polypurine tract (cPPT), and identified a new cis region
proximal to the Rev responsive element (RRE). RanDeL-seq also identified
regions of Zika virus required for replication and packaging. RanDeL-seq
is a versatile and comprehensive technique to rapidly map cis and trans
regions of a genome.
