Yang, K. J. | bioRxiv Science

bioRxiv2022

High-Throughput Discovery and Characterization of Viral Transcriptional Effectors in Human Cells

Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature and high sequence divergence, it is unclear which viral proteins can affect transcription and which specific sequences contribute to this function. Using a high-throughput assay, we measured the transcriptional regulatory potential of over 60,000 protein tiles across [~]1,500 proteins from 11 coronaviruses and all nine human herpesviruses. We discovered hundreds of new transcriptional effector domains, including a conserved repression domain in all coronavirus Spike homologs, dual activation-repression domains in VIRFs, and an activation domain in six herpesvirus homologs of the single-stranded DNA-binding protein that we show is important for viral replication and late gene expression in KSHV. For the effector domains we identified, we investigated their mechanisms via high-throughput sequence and chemical perturbations, pinpointing sequence motifs essential for function. This work massively expands viral protein annotations, serving as a springboard for studying their biological and health implications and providing new candidates for compact gene regulation tools.

systems biology↗

bioRxiv2021

A Two-tiered Functional Screen Identifies Herpesviral Transcriptional Modifiers and their Essential Domains

While traditional methods for studying large DNA viruses allow the creation of individual mutants, CRISPR/Cas9 can be used to rapidly create thousands of mutant dsDNA viruses in parallel. Here, we used this approach to study the human oncogenic Kaposis sarcoma-associated herpesvirus (KSHV). We designed a sgRNA library containing all possible ~22,000 guides targeting the genome of KSHV - one cut site approximately every 8 base pairs - enabling the pooled screening of the entire genome. We used this tool to phenotype all possible Cas9-targeted viruses for transcription of KSHV late genes, which is required to produce structural components of the viral capsid. By performing targeted deep sequencing of the viral genome to distinguish between knock-out and in-frame alleles created by Cas9, we discovered a novel hit, ORF46 - and more specifically its DNA binding domain - is required for viral DNA replication. Our pooled Cas9 tiling screen followed by targeted deep viral sequencing represents a two-tiered screening paradigm that may be widely applicable to dsDNA viruses.

genomics↗