Viral transcriptional regulators extensively rewire host pathways through diverse mechanisms

Viral transcriptional regulators (vTRs) reprogram host gene regulatory networks to promote replication, persistence, and immune evasion. Despite the identification of hundreds of vTRs in human viruses, how they rewire host pathways remains unclear. Here, we systematically profiled 95 vTRs from diverse human viruses across multiple functional assays. vTRs perturb immune, cell proliferation/death, and signaling pathways through various mechanisms; some bind DNA directly, others cooperate or antagonize human transcription factors (hTFs), and some remodel chromatin. vTRs can act as activators or repressors and recruit similar but not identical repertoires of proteins as hTFs. These findings reveal vTRs as versatile transcriptional modulators that converge on conserved host “pressure points” while diversifying across pathways to promote viral replication and persistence. Notably, many vTR dysregulate genes within autoimmune, neurological, and cardiovascular risk loci, revealing mechanistic links to disease. Together, we provide a comprehensive resource for understanding and targeting viral control of human transcription. Overall design: To determine the transcriptomic effects of vTRs in human cells, we performed bulk RNA barcoding and sequencing (BRB-seq) in HEK293T cells expressing individual vTRs. Expression vTR clones were generated via Gateway LR cloning into the pEZY3 constitutive expression vector (Addgene plasmid #18672). Following transformation, plasmids were extracted, and insertion was confirmed via PCR and gel electrophoresis. The full list of vTRs tested by BRB-seq is provided in Data S2. For BRB-seq experiments, HEK293T cells were cultured in DMEM media supplemented with 10% FBS and seeded in two 96-well plates at a density of 18,000 cells/well. The following day, cells were transfected in quadruplicate using Lipofectamine 3000 (Thermo Fisher Scientific) with 80 ng of the pEZY3-vTR plasmids, as well as empty pEZY3 vector controls. After 24 hours, cells were either infected with 4.0 × 106 CEID50/mL Sendai virus (VR-907, ATCC), or left uninfected. RNAs were harvested 2 days post transfection, a time point that we showed has minimal interferon pathway activation due to transfection. Sequencing libraries were prepared using the MERCURIUS DRUG-seq kit (Alithea Genomics) according to manufacturer's instructions and as described previously.24 Briefly, HEK293T cells were permeabilized using a cell lysis buffer on ice 15 min with periodic agitation, followed by centrifugation to clear cellular debris. Individual RNA-containing lysates were then reverse-transcribed using barcoded oligo-dT primers such that the resulting cDNA from each individual sample was uniquely barcode-labeled. cDNA from all samples in a single plate were then pooled and purified using the column-based Zymo Clean & Concentration Kit according to manufacturer's instructions (Zymo, D4014). The pooled cDNA was then cleared of non-incorporated primers via exonuclease digestion, followed by second strand synthesis and quantification of ds-cDNA via Qubit HS-DNA kit (Thermo Fisher). Tagmentation was then performed on 20 ng cDNA, followed by library indexing and amplification using standard Illumina NGS adapter sequences. Indexed libraries were sequenced at a depth of 5 million raw reads per sample on an Illumina Novaseq platform. Scripts to process sequencing data are deposited in https://github.com/FuxmanBass-lab/vTRs.