Biology subjects

Baker, S. F.

Publications and source records attributed to Baker, S. F..

7 recordsLinked to original sources

bioRxiv2026

Mapping global transcript isoform landscapes of influenza A virus infection and interferon stimulation reveals RNA-binding protein networks associated with immune remodeling

Infected cells rapidly adapt their transcriptional profile to upregulate genes that limit pathogen replication and promote inflammation. Viruses also induce widespread changes in host gene expression, and both host and virus further remodel the transcriptome through targeted mRNA degradation, modulation of alternative splicing, and alterations at transcript 5 and 3 prime ends. The complexity of the response to infection makes it difficult to understand which transcriptional changes are driven by the virus and which reflect the host antiviral program. Short-read RNA sequencing has been the primary tool for studying these changes, but its inability to resolve full-length transcript isoforms limits isoform switching-based analyses, narrowing our understanding of how transcript usage shapes immunity. Here we profiled the host transcriptome in response to interferon-{beta} treatment or influenza A virus infection using both short- and long-read RNA sequencing analyzed with the SQANTI3 pipeline. Because isoform switching and transcript abundance change are distinct regulatory events, we focused our analysis on differential transcript usage to uncover a layer of transcriptome remodeling that standard expression analysis cannot detect. Pathway analysis revealed distinct regulons for genes undergoing transcriptional upregulation compared with those undergoing isoform switching. These data provide a rich resource for querying the isoform changes underlying shared and distinct transcriptional programs triggered by viral infection or innate immune stimulation. To identify putative mechanisms coordinating these stimulation-dependent isoform changes, we integrated 232 eCLIP datasets and found a small set of RNA-binding proteins localized at these isoform switches, including DDX3X. Comparing the responses to interferon and to virus highlighted RNA-binding proteins associated with infection-dependent immune remodeling. Altogether, these results demonstrate the value of hybrid sequencing for deciphering the post-transcriptional regulatory code active during the antiviral response.

immunology↗

bioRxiv2026

A novel ISG15 transcript restricts influenza A virus infection

The interferon response orchestrates antiviral defense through the induction of interferon-stimulated genes (ISGs). Although nearly all human genes undergo alternative splicing, the identity and function of many ISG transcript isoforms remains poorly understood. Using long-read RNA sequencing of human cells that were undergoing an interferon response, we identified a previously unrecognized interferon-inducible ISG15 transcript. This non-canonical ISG15 (ncISG15) transcript contains a unique 5' untranslated region and encodes an N-terminally truncated protein in frame with ISG15. During interferon stimulation, ncISG15 produces an ISGylation pattern distinct from canonical ISG15 and potently inhibits influenza A virus polymerase activity. The influenza A viral interferon antagonist protein NS1 counteracts ncISG15-mediated restriction; however, endogenous ncISG15 executes modest antiviral activity against NS1-encoding viruses. Antiviral activity of ncISG15 is largely independent of its function as a cytokine, through ISG augmentation, or by ISGylation. This novel transcript adds a new layer of regulatory complexity to the ISG15-driven antiviral network.

microbiology↗

bioRxiv2025

Tandem split-GFP influenza A viruses for sensitive and accurate replication analyses

Reporter influenza A viruses (IAVs) are valuable tools for studying virus fitness, screening antivirals, and assessing host-virus interactions. However, the compact and segmented nature of the IAV genome presents major challenges for engineering genetically stable reporter viruses with minimal fitness defects. To address this, we developed a replication-competent IAV incorporating a tandem split-GFP reporter strategy. Specifically, we appended seven tandem repeats of the GFP11 peptide (GFP11x7) to the C-terminus of the PB2 or PA polymerase subunit. Viruses contained or lacked a self-cleaving 2A peptide between the viral and reporter genes to create polymerase fusion proteins or released GFP, respectively. These viruses are complemented in trans by cells expressing GFP1-10, allowing bright fluorescence with minimal disruption to viral function. The tandem GFP11 reporter viruses exhibit delayed replication kinetics, but are genetically stable over serial passages with a strong concordance between GFP signal and viral gene expression. We demonstrate their utility in high-throughput applications including fluorescence-based quantification of infection, neutralizing antibody titration, antiviral drug screening, and host factor identification, with results matching traditional assays but on an accelerated timeline. Furthermore, we engineered a battery of GFP1-10-expressing cell lines from multiple vertebrate species, illustrating the broad compatibility of this platform for comparative host studies. This system enables sensitive, scalable, and quantitative evaluation of influenza virus replication across diverse experimental contexts. The GFP11x7 reporter platform offers a versatile and robust tool for virology research and therapeutic screening, with potential for rapid adaptation to emerging IAV strains.

microbiology↗

bioRxiv2022

CXCR4 controls movement and degranulation of CD8+ T cells in the influenza-infected lung via differential effects on interaction and tissue scanning

Effector CD8+ T cell interactions are critical in controlling viral infection by directly killing infected cells but overabundant or sustained activation also exacerbates tissue damage. Chemokines promote the trafficking of effector CD8+ T cells into infected tissues, but we know little about how chemokines regulate the function of CD8+ T cells within tissues. Using a murine model of influenza A virus infection, we found that expression of the chemokine receptor CXCR4 by lung-infiltrating cytotoxic T cells correlated with the expression of the degranulation marker CD107a. Inhibition of CXCR4 reduced activation, adhesion, and degranulation of cytotoxic T cells in vitro and in vivo. Moreover, in live influenza-infected lung tissue, T cells stopped moving in lung regions with high levels of influenza antigen, and CXCR4 was essential for CD8+ T cells to execute this arrest signal fully. In contrast, CXCR4 increased the motility of CD8+ T cells in low-influenza areas of the lung. We also found that CXCR4 stimulated the effector function of lung-infiltrating cytotoxic T cells even after clearance of influenza virus, and inhibition of CXCR4 expedited the recovery of influenza-infected mice, despite delayed clearance of the replication-competent virus. Our results suggest that CXCR4 promotes the interaction strength of cytotoxic T cells in lung tissue through combined effects on T cell movement and interaction with virally infected target cells in influenza infected-lungs.

immunology↗

bioRxiv2020

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immuno-competitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the pro-viral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the anti-viral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR) that interacts with PB2. cMECR binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and lies solely within cMECR. Thus, alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme.

microbiology↗

bioRxiv2019

EPS8 facilitates uncoating of influenza A virus

All viruses balance interactions between cellular machinery co-opted to support replication and host factors deployed to halt the infection. We used gene correlation analysis to perform an unbiased screen for host factors involved in influenza A virus (FLUAV) infection. Our screen identified the cellular factor epidermal growth factor receptor pathway substrate 8 (EPS8) as the highest confidence pro-viral candidate. Knockout and overexpression of EPS8 confirmed its importance in enhancing FLUAV infection and titers. Loss of EPS8 did not affect virion attachment, uptake, or fusion. Rather, our data show that EPS8 specifically functions during virion uncoating. EPS8 physically associated with incoming virion components, and subsequent nuclear import of released ribonucleoprotein complexes was significantly delayed in the absence of EPS8. Our study identified EPS8 as a host factor important for uncoating, a crucial step of FLUAV infection during which the interface between the virus and host is still being discovered.

microbiology↗

bioRxiv2018

Differential splicing of ANP32A in birds alters its ability to stimulate RNA synthesis by restricted influenza polymerase

Adaptation of viruses to their host can result in specialization and a restricted host range. Species-specific polymorphisms in the influenza virus polymerase restrict its host range during transmission from birds to mammals. ANP32A was recently been identified as a cellular co-factor impacting polymerase adaption and activity. Avian influenza polymerases require ANP32A containing an insertion resulting from an exon duplication uniquely encoded in birds. Here we find that natural splice variants surrounding this exon create avian ANP32A proteins with distinct effects on polymerase activity. We demonstrate species-independent direct interactions between all ANP32A variants and the PB2 polymerase subunit. This interaction is enhanced in the presence of viral genomic RNA. In contrast, only avian ANP32A restored ribonucleoprotein complex assembly for a restricted polymerase by enhancing RNA synthesis. Our data suggest that ANP32A splicing variation amongst birds differentially impacts viral replication, polymerase adaption, and the potential of avian hosts to be reservoirs.

microbiology↗