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

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], Ψ, 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, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼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

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

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], Ψ, 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, Ψ, and RRE for long-term passage. Strikingly, RanDeL-seq identified a previously unreported ∼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

EvryFlare. III. Temperature Evolution and Habitability Impacts of Dozens of Superflares Observed Simultaneously by Evryscope and TESS

Superflares may provide the dominant source of biologically relevant UV radiation to rocky habitable-zone M-dwarf planets (M-Earths), altering planetary atmospheres and conditions for surface life. The combined line and continuum flare emission has usually been approximated by a 9000 K blackbody. If superflares are hotter, then the UV emission may be 10 times higher than predicted from the optical. However, it is unknown for how long M-dwarf superflares reach temperatures above 9000 K. Only a handful of M-dwarf superflares have been recorded with multiwavelength high-cadence observations. We double the total number of events in the literature using simultaneous Evryscope and Transiting Exoplanet Survey Satellite observations to provide the first systematic exploration of the temperature evolution of M-dwarf superflares. We also increase the number of superflaring M dwarfs with published time-resolved blackbody evolution by ∼10×. We measure temperatures at 2 minutes cadence for 42 superflares from 27 K5-M5 dwarfs. We find superflare peak temperatures (defined as the mean of temperatures corresponding to flare FWHM) increase with flare energy and impulse. We find the amount of time flares emit at temperatures above 14,000 K depends on energy. We discover that 43% of the flares emit above 14,000 K, 23% emit above 20,000 K and 5% emit above 30,000 K. The largest and hottest flare briefly reached 42,000 K. Some do not reach 14,000 K. During superflares, we estimate M-Earths orbiting <200 Myr stars typically receive a top-of-atmosphere UV-C flux of ∼120 W m-2 and up to 103 W m-2, 100-1000 times the time-averaged X-ray and UV flux from Proxima Cen.WH acknowledges partial funding support of the Proxima Cen data and analysis through the Cycle 26 HST proposal GO 15651. WH, HC, NL, JR, and AG acknowledge funding support by the National Science Foundation CAREER grant 1555175, and the Research Corporation Scialog grants 23782 and 23822. HC is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-1144081. OF and DdS acknowledge support by the Spanish Ministerio de Econom´ıa y Competitividad (MINECO/FEDER, UE) under grants AYA2013-47447- C3-1-P, AYA2016-76012-C3-1-P, MDM-2014-0369 of ICCUB (Unidad de Excelencia ‘Mar´ıa de Maeztu’). The Evryscope was constructed under National Science Foundation/ATI grant AST-1407589. This paper includes data collected by the TESS mission. Funding for the TESS mission is provided by the NASA Explorer Program. This work has made use of data from the European Space Agency (ESA) mission Gaia (https://www.cosmos.esa.int/gaia), processed by the Gaia Data Processing and Analysis Consortium (DPAC, https://www.cosmos.esa.int/web/gaia/ dpac/consortium). Funding for the DPAC has been provided by national institutions, in particular the institutions participating in the Gaia Multilateral Agreement. This research made use of Astropy,5 a communitydeveloped core Python package for Astronomy (Astropy Collaboration et al. 2013; Price-Whelan et al. 2018), and the NumPy, SciPy, and Matplotlib Python modules (van der Walt et al. 2011; Virtanen et al. 2020; Hunter 2007). Facilities: CTIO:Evryscope, TES

Disrupting autorepression circuitry generates "open-loop lethality" to yield escape-resistant antiviral agents.

Across biological scales, gene-regulatory networks employ autorepression (negative feedback) to maintain homeostasis and minimize failure from aberrant expression. Here, we present a proof of concept that disrupting transcriptional negative feedback dysregulates viral gene expression to therapeutically inhibit replication and confers a high evolutionary barrier to resistance. We find that nucleic-acid decoys mimicking cis-regulatory sites act as "feedback disruptors," break homeostasis, and increase viral transcription factors to cytotoxic levels (termed "open-loop lethality"). Feedback disruptors against herpesviruses reduced viral replication &gt;2-logs without activating innate immunity, showed sub-nM IC50, synergized with standard-of-care antivirals, and inhibited virus replication in mice. In contrast to approved antivirals where resistance rapidly emerged, no feedback-disruptor escape mutants evolved in long-term cultures. For SARS-CoV-2, disruption of a putative feedback circuit also generated open-loop lethality, reducing viral titers by &gt;1-log. These results demonstrate that generating open-loop lethality, via negative-feedback disruption, may yield a class of antimicrobials with a high genetic barrier to resistance