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Viruses display large variability across all stages of their life cycle, including entry, gene expression, replication, assembly, and egress. We previously reported that the immediate early adenovirus (AdV) E1A transcripts accumulate in human lung epithelial A549 cancer cells with high variability, mostly independent of the number of incoming viral genomes, but somewhat correlated to the cell cycle state at the time of inoculation. Here, we leveraged the classical Luria-Delbrück fluctuation analysis to address whether infection variability primarily arises from the cell state or stochastic noise. The E1A expression was measured by the expression of green fluorescent protein (GFP) from the endogenous E1A promoter in AdV-C5_E1A-FS2A-GFP and found to be highly correlated with the viral plaque formation, indicating reliability of the reporter virus. As an ensemble, randomly picked clonal A549 cell isolates displayed significantly higher coefficients of variation in the E1A expression than technical noise, indicating a phenotypic variability larger than noise. The underlying cell state determining infection variability was maintained for at least 9 weeks of cell cultivation. Our results indicate that preexisting cell states tune adenovirus infection in favor of the cell or the virus. These findings have implications for antiviral strategies and gene therapy applications.IMPORTANCEViral infections are known for their variability. Underlying mechanisms are still incompletely understood but have been associated with particular cell states, for example, the eukaryotic cell division cycle in DNA virus infections. A cell state is the collective of biochemical, morphological, and contextual features owing to particular conditions or at random. It affects how intrinsic or extrinsic cues trigger a response, such as cell division or anti-viral state. Here, we provide evidence that cell states with a built-in memory confer high or low susceptibility of clonal human epithelial cells to adenovirus infection. Results are reminiscent of the Luria-Delbrück fluctuation test with bacteriophage infections back in 1943, which demonstrated that mutations, in the absence of selective pressure prior to infection, cause infection resistance rather than being a consequence of infection. Our findings of dynamic cell states conferring adenovirus infection susceptibility uncover new challenges for the prediction and treatment of viral infections., Competing Interests: The authors declare no conflict of interest.

Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Introduction: The occurrence and progression of lung adenocarcinoma (LUAD) impair T-cell immune responses, causing immune escape and subsequently affecting the efficacy of immunotherapy in patients. Aurora kinase A (AURKA) is upregulated in varying cancers, but its role in LUAD immune escape is elusive. This work attempted to explore molecular mechanisms of AURKA regulation in LUAD immune escape., Methods: Through bioinformatics analysis, AURKA level in LUAD was evaluated, and potential upstream transcription factors of AURKA were predicted using hTFtarget. ETS variant transcription factor 4 (ETV4) expression in LUAD was analyzed through The Cancer Genome Atlas. Pearson's correlation analysis was then utilized to test the correlation between AURKA and ETV4. Interaction and binding between AURKA and ETV4 were validated through dual-luciferase assay and chromatin immunoprecipitation. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) tested relative mRNA expression of AURKA and ETV4 in LUAD cells, cell counting kit-8 assayed cell viability, and Western blot analysis was conducted to determine the protein level of programmed death-ligand 1 (PD-L1). Coculture of LUAD cells with activated CD8+ T cells was carried out, and an LDH assay was used to assess the cytotoxicity of CD8+ T cells against LUAD cells. Interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) levels in the coculture system were assessed by enzyme-linked immunosorbent assay (ELISA). Western blot assessed protein levels of JAK2, p-JAK2, STAT3, and p-STAT3., Results: Compared to normal tissues, AURKA and ETV4 were upregulated in tumor tissues, and AURKA presented a negative association with CD8+ T-cell immune infiltration but a positive association with PD-L1. qRT-PCR unveiled significantly upregulated mRNA of AURKA and ETV4 in LUAD cells compared to normal lung epithelial cells. Knockdown of AURKA significantly decreased cell viability and PD-L1 protein level in LUAD cells, enhanced cytotoxicity of CD8+ T cells against LUAD cells and IFN-γ, IL-2, and TNF-α expression, while overexpression of AURKA yielded opposite results. Furthermore, the knockdown of ETV4 could reverse the oncogenic characteristics of cells caused by AURKA overexpression., Conclusion: Our study illustrated that ETV4/AURKA axis promoted PD-L1 expression, suppressed CD8+ T-cell activity, and mediated immune escape in LUAD by regulating the JAK2/STAT3 signaling pathway., (© 2024 S. Karger AG, Basel.)