RanDeL-seq: A high-throughput method to map viral cis- and trans-acting elements

Abstract

<jats:title>Abstract</jats:title><jats:p>It has long been known that noncoding genomic regions can be obligate <jats:italic>cis</jats:italic> elements acted upon <jats:italic>in trans</jats:italic> by gene products. In viruses, <jats:italic>cis</jats:italic> 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 <jats:italic>cis</jats:italic> and <jats:italic>trans</jats:italic> 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., <jats:underline><jats:bold>Ran</jats:bold></jats:underline>dom-<jats:underline><jats:bold>De</jats:bold></jats:underline>letion <jats:underline><jats:bold>L</jats:bold></jats:underline>ibrary <jats:underline><jats:bold>seq</jats:bold></jats:underline>uencing, RanDeL-seq). Using RanDeL-seq, we generated and screened &gt;23,000 HIV-1 variants to generate a single-base resolution map of HIV-1’s <jats:italic>cis</jats:italic> and <jats:italic>trans</jats:italic> elements. The resulting landscape recapitulated HIV-1’s known <jats:italic>cis</jats:italic>-acting elements (i.e., LTR, Ψ, 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, Ψ, 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 &gt;90,000 variants of Zika virus (ZIKV). Unexpectedly, RanDeL-seq indicated that ZIKV’s <jats:italic>cis</jats:italic>-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, particularly for emerging viral infections.</jats:p><jats:sec><jats:title>Importance</jats:title><jats:p>Recent studies have renewed interest in developing novel antiviral therapeutics and vaccines based on defective interfering particles (DIPs)—a subset of viral deletion mutant that conditionally replicate. Identifying and engineering DIPs requires that viral <jats:italic>cis</jats:italic>- and <jats:italic>trans</jats:italic>-acting elements be accurately mapped. Here we introduce a high-throughput method (Random Deletion Library sequencing, RanDeL-seq) to comprehensively map <jats:italic>cis-</jats:italic> and <jats:italic>trans</jats:italic>-acting elements within a viral genome. RanDeL-seq identified essential cis elements in HIV, including the obligate nature of the once-controversial viral central poly-purine tract (cPPT) and identified a new <jats:italic>cis</jats:italic> 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.</jats:p></jats:sec>

Identification of a therapeutic interfering particle—A single-dose SARS-CoV-2 antiviral intervention with a high barrier to resistance

“…An engineered TIP RNA conditionally propagates and inhibits SARS-CoV-2 in cell culture Given the challenges of engineering TIPs for other viruses (Notton et al, 2021), we set out to construct a TIP with appropriate parameters. As a starting point, we used previous high-throughput analyses of the cis genetic elements required for efficient propagation of sub-genomic transcripts in other RNA viruses (Notton et al, 2021) and historical data from murine hepatitis virus (Baric et al, 1988;Makino et al, 1990), both of which argued that, at a minimum, the viral 5 0 and 3 0 UTRs were essential for conditional replication and construction of a TIP. Consequently, we designed two minimal sub-genomic synthetic constructs, encoding different lengths of the 5 0 and 3 0 regions of the viral genome and tested whether they met the threshold values to act as TIPs.…”

“…From an evolutionary perspective, it is also not immediately obvious why endogenous TIPs have not naturally evolved to limit virus infections and the considerations are complex, particularly in a virus's natural hosts (Daugherty and Malik, 2012). For lentiviruses such as HIV, the barrier appears more straightforward, as R 0 >1 variants appear to require recombination ''acrobatics'' since a cis element necessary for efficient transmission is within the TIP deletion and must be recovered and repositioned outside the deleted region (Notton et al, 2021). However, for flaviviruses there is evidence that natural DIPs arise and transmit through host populations (Aaskov et al, 2006), and historical hypotheses have asserted that such DIPs have biological fitness roles aiding the parent wild-type virus.…”