Biology subjects

Pager, C. T.

Publications and source records attributed to Pager, C. T..

7 recordsLinked to original sources

bioRxiv2026

Inosine misincorporation into mRNA triggers the integrated stress response and activates an innate immune gene expression signature

The inosine triphosphate pyrophosphatase (ITPase) enzyme restricts levels of the non-canonical nucleotides (deoxy)inosine triphosphate (dITP/ITP) and prevents their aberrant misincorporation into nucleic acids. ITPase deficiency is associated with dilated cardiomyopathy and epileptic encephalopathy in humans and is usually fatal in infancy. It leads to pronounced inosine misincorporation into RNA but the cellular consequences of this misincorporation are not well understood and the pathogenic basis of ITPase deficiency remains unknown. Here we show that cellular transfection of mRNA with inosine misincorporation activates the integrated stress response (ISR) with an innate immune gene expression signature. This stress response triggers stress granule formation and is modulated by the double stranded RNA sensor protein Kinase R (PKR). Inosine nucleoside treatment of ITPase-deficient cells leads to endogenous inosine misincorporation into mRNA and activation of the ISR. Further, differentiation of human ITPase-deficient induced pluripotent stem cells into neurons results in a low-level stress response. Thus, our study establishes inosine misincorporation into mRNA as an unappreciated form of cellular stress. This is normally prevented by the ITPase enzyme, with implications for the pathogenesis of ITPase deficiency.

molecular biology↗

bioRxiv2025

N-acetyltransferase 10 promotes mRNA stability of immune response factors to modulate Zika virus infection

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that poses serious risks to human health. In previous work, we used RNA immunoprecipitation and mass spectrometry to show that more than 30 distinct RNA modifications, or chemical moieties, were present on the RNA genome of ZIKV. Among these, N4-acetylcytosine (ac4C) was one of the most abundant modifications. In this study, we investigated the role of N-acetyltransferase 10 (NAT10), the writer enzyme that acetylates cytidine, in ZIKV gene expression. Using NAT10 knockout cell lines, RNA interference (RNAi), and overexpression rescue strategies, we found that loss of NAT10 led to increased levels of ZIKV protein and RNA. However, the production of infectious virus particles was not significantly affected. Interestingly, in NAT10-deficient cells compared to wild-type (WT) cells, ZIKV protein and RNA were detectable earlier during infection, suggesting that the loss of NAT10 facilitated increased viral replication. Despite this increase, ZIKV RNA was more rapidly degraded, although the accumulation of small flaviviral RNAs was not significantly altered by the absence of NAT10. Further analysis of key components of the innate immune response revealed that, in the absence of NAT10 and during early infection, STAT1, IFIT1, and MX1 mRNA transcripts were rapidly degraded, leading to reduced expression of the respective innate immune proteins. Taken together, our findings demonstrate that NAT10, the ac4C writer enzyme, modulates the stability of specific innate immune mRNAs and thereby plays a regulatory role in ZIKV infection dynamics. IMPORTANCERNA modifications are chemical groups that are deposited post-transcriptionally on RNA. In recent years, RNA modifications in viral RNAs have been shown to have profound effects on viral gene expression and hence viral function. Indeed, numerous studies have investigated the role of N6-methyladenosine and 5-methylcytosine RNA modifications and the respective enzymes that deposit (writer), remove (eraser) and facilitate (reader) function on viral infection. In this study we show how N4-acetyltransferase 10 (NAT10), a writer enzyme that acetylates cytidine to form N4-acetylcytidine (ac4C), affects Zika virus gene expression. Specifically, we found that NAT10 regulates viral infection kinetics by affecting the stability of select mRNAs involved in the innate immune response pathway. Our findings highlight another mode by which the innate immune response is robustly regulated in response to ZIKV infection.

microbiology↗

bioRxiv2024

Zika virus infection in a cell culture model reflects the transcriptomic signatures in patients.

Zika virus (ZIKV), a re-emerging flavivirus, is associated with devasting developmental and neurological disease outcomes particularly in infants infected in utero. Towards understanding the molecular underpinnings of the unique ZIKV disease pathologies, numerous transcriptome-wide studies have been undertaken. Notably, these studies have overlooked the assimilation of RNA-seq analysis from ZIKV-infected patients with cell culture model systems. In this study we find that ZIKV-infection of human lung adenocarcinoma A549 cells, mirrored both the transcriptional and alternative splicing profiles from previously published RNA-seq data of peripheral blood mononuclear cells collected from pediatric patients during early acute, late acute, and convalescent phases of ZIKV infection. Our analyses show that ZIKV infection in cultured cells correlates with transcriptional changes in patients, while the overlap in alternative splicing profiles was not as extensive. Overall, our data indicate that cell culture model systems support dissection of select molecular changes detected in patients and establishes the groundwork for future studies elucidating the biological implications of alternative splicing during ZIKV infection.

microbiology↗

bioRxiv2023

Activation of ATF3 via the Integrated Stress Response Pathway Regulates Innate Immune and Autophagy Processes to Restrict Zika Virus

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus that can have devastating health consequences. The developmental and neurological effects from a ZIKV infection arise in part from the virus triggering cellular stress pathways and perturbing transcriptional programs. To date, the underlying mechanisms of transcriptional control directing viral restriction and virus-host interaction are understudied. Activating Transcription Factor 3 (ATF3) is a stress-induced transcriptional effector that modulates the expression of genes involved in a myriad of cellular processes, including inflammation and antiviral responses, to restore cellular homeostasis. While ATF3 is known to be upregulated during ZIKV infection, the mode by which ATF3 is activated and the specific role of ATF3 during ZIKV infection is unknown. In this study, we show via inhibitor and RNA interference approaches that ZIKV infection initiates the integrated stress response pathway to activate ATF4 which in turn induces ATF3 expression. Additionally, by using CRISPR-Cas9 system to delete ATF3, we found that ATF3 acts to limit ZIKV gene expression in A549 cells. We also determined that ATF3 enhances the expression of antiviral genes such as STAT1 and other components in the innate immunity pathway to induce an ATF3-dependent anti-ZIKV response. Our study reveals crosstalk between the integrated stress response and innate immune response pathways and highlights an important role for ATF3 in establishing an antiviral effect during ZIKV infection. ImportanceZIKV is a re-emerging mosquito-borne flavivirus that co-opts cellular mechanisms to support viral processes which can reprogram the host transcriptional profile. Such viral-directed transcriptional changes and the pro- or anti-viral outcomes remain understudied. We previously showed that ATF3, a stress-induced transcription factor, is significantly upregulated in ZIKV infected mammalian cells, along with other cellular and immune response genes. We now define the intracellular pathway responsible for ATF3 activation and elucidate the impact of ATF3 expression on ZIKV infection. We show that during ZIKV infection the integrated stress response pathway stimulates ATF3 which enhances the innate immune response to antagonize ZIKV infection. This study establishes a link between viral-induced stress response and transcriptional regulation of host defense pathways and thus expands our knowledge on virus-mediated transcriptional mechanisms and transcriptional control of interferon stimulated genes during ZIKV infection.

microbiology↗

bioRxiv2020

Programmable low-cost DNA-based platform for viral RNA detection

Viral detection is critical for controlling disease spread and progression. Recent emerging viral threats including Zika, Ebola, and the current COVID-19 outbreak highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches designed to mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show non-enzymatic detection of viral RNA to the attomole level, with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with a sample preparation step using either RNA extraction or isothermal pre-amplification. Our assay can be performed with minimal or no lab infrastructure, and is readily adaptable to detect other viruses. We demonstrate the adaptability of our method by quickly developing and testing DNA nanoswitches for detecting a fragment of SARS-CoV-2 RNA in human saliva. Given this versatility, we expect that further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.

bioengineering↗

bioRxiv2019

Asian Zika virus isolate significantly changes the transcriptional profile and alternative RNA splicing events in a neuroblastoma cell line

Alternative splicing of pre-mRNAs expands a single genetic blueprint to encode multiple functionally diverse protein isoforms. Viruses have previously been shown to interact with, depend on, and alter host splicing machinery. The consequences however incited by viral infection on the global alternative slicing (AS) landscape are under appreciated. Here we investigated the transcriptional and alternative splicing profile of neuronal cells infected with a contemporary Puerto Rican Zika virus (ZIKVPR) isolate, the prototypical Ugandan ZIKV (ZIKVMR) isolate and dengue virus 2 (DENV2). Our analyses revealed that ZIKVPR induced significantly more differential changes in expressed genes compared to ZIKVMR or DENV2, despite all three viruses showing equivalent infectivity and viral RNA levels. Consistent with the transcriptional profile, ZIKVPR induced a higher number of alternative splicing events compared to ZIKVMR or DENV2, and gene ontology analyses highlighted alternative splicing changes in genes associated with mRNA splicing. All three viruses modulated alternative splicing with ZIKVPR having the largest impact on splicing. ZIKV alteration of the transcriptomic landscape during infection caused changes in cellular RNA homeostasis, which might dysregulate neurodevelopment and function leading to neuropathologies such as microcephaly and Guillain-Barre syndrome associated with the ZIKV infection.

microbiology↗

bioRxiv2018

Zika Virus Subverts Stress Granules to Promote and Restrict Viral Gene Expression

Flaviviruses limit the cell stress response by preventing the formation of stress granules and utilize different proteins involved in the stress granule pathway to modulate viral gene expression. In this study, we investigated the formation of stress granules during Zika virus (ZIKV) infection and the role stress granule proteins play during the viral life cycle. Using immunofluorescence and confocal microscopy, we determined that ZIKV disrupted the formation of arsenite-induced stress granules and changed the subcellular distribution, but not the abundance or integrity of stress granule proteins. To investigate the role of different stress granule proteins in ZIKV infection we used target-specific siRNAs to deplete six proteins, namely Ataxin2, G3BP1, HuR, TIA-1, TIAR and YB-1. Depletion of TIA-1 and TIAR affected ZIKV protein and RNA levels, but viral titers did not change. Conversely, depletion of Ataxin2 and YB-1 decreased virion production despite having only a small effect on ZIKV protein and expression. Notably, however, depletion of G3BP1 and HuR decreased and increased ZIKV gene expression and virion production, respectively. Using an MR766 Gaussia luciferase reporter genome together with knockdown and overexpression assays, G3BP1 and HuR were found to modulate ZIKV replication. These data indicate that ZIKV disrupts the formation of stress granules by sequestering stress granule proteins required for replication, which is where G3BP1 functions to promote ZIKV infection, while HuR exhibits an antiviral effect. The consequence of ZIKV re-localizing and subverting select stress granule proteins might have broader consequences on cellular RNA homeostasis contributing to cellular gene dysregulation and ZIKV pathogenesis.\n\nImportanceIn response to viral infection, cellular translation is stalled, and translation initiation complexes, cellular mRNAs, and RNA binding proteins aggregate in stress granules. Because the assembly of stress granules antagonize translation of viral proteins, which is a critical step for single-stranded positive-sense RNA viruses to replicate the viral genome, many viruses have developed strategies to inhibit stress granule formation. In this study, we observed that Zika virus restricts the formation of stress granules likely by re-localizing specific stress granule proteins during infection. We also determined that specific stress granule proteins function to facilitate and limit Zika virus replication. This interaction of Zika virus with stress granule proteins is interesting, as many stress granule proteins are also known to function in neuronal granules, which are critical in neural development and function. Moreover, dysregulation of different stress granule proteins in neurons has been shown to play a role in the progression of neurodegenerative diseases. The likely consequences of Zika virus modulating stress granule assembly and subverting specific stress granule proteins are alterations to cellular mRNA transcription, splicing, RNA stability, and translation. Such changes in cellular ribostasis could have profound effects on neural development and contribute to the devastating developmental and neurological anomalies observed following intrauterine Zika virus infection. Our study provides new insights into virus-host interactions and the identification of the stress granule proteins that may contribute to the unusual pathogenesis associated with this reemerging arbovirus.

microbiology↗