Biology subjects

Beckham, J. D.

Publications and source records attributed to Beckham, J. D..

7 recordsLinked to original sources

bioRxiv2026

Structure informed xrRNA2 mutations in Zika virus provide a roadmap for vaccine development

Flaviviruses like Zika virus (ZIKV), contain RNA tertiary structures within the 3 untranslated region (UTR) that halt the 5-to-3 RNA exonuclease, Xrn1. Halting of Xrn1 at the two RNA structures, termed exonuclease-resistantRNA1 and 2 (xrRNA1 and xrRNA2), results in the formation of subgenomic flavivirus RNAs (sfRNA) that support viral pathogenesis. While the role of the flavivirus xrRNA1 in pathogenesis is well characterized, the role of the flavivirus xrRNA2 structure is not well studied. Using xrRNA crystal structure data, we inserted structure-informed mutations in ZIKV xrRNA2 to disrupt tertiary folding independent of significant sequence changes, evaluate sfRNA production, and define pathogenesis in a murine model of ZIKV infection. Compared to our prior work with ZIKV xrRNA1, we found that ZIKV xrRNA2 is under increased selection pressure to maintain sfRNA production resulting in multiple targeted mutations in xrRNA2 junctional region to induce a stable mutant. Using three targeted xrRNA junctional mutations termed ZIKV X2.L1, we found that the resulting ZIKV clone exhibits attenuated cell death in cultures and decreased viral growth in tissue cultures. In a murine model of ZIKV infection, mice inoculated with ZIKV X2.L1 exhibit significantly decreased symptomatic infection, improved survival, decreased end-organ infection in the brain, and continued robust neutralizing antibody responses to ZIKV. Despite attenuation, serum from ZIKV X2.L1-infected mice or mice vaccinated with ZIKV X2.L1, exhibited 100% protection from lethal ZIKV challenge. These studies show that RNA structure-informed mutations provide a robust model for flavivirus attenuation and vaccine design. Additional studies defining the mechanisms of robust neutralizing antibody responses and flavivirus-specific vaccine development are needed to continue the development of this novel vaccine platform approach for medically important flavivirus infections. Author summaryZika virus is a member of the Orthoflavivirus (referred to as flavivirus) genus that exhibit conserved RNA structures in the 3 untranslated region of the viral RNA genome. Two concerned RNA structures, termed exonuclease-resistant RNA 1 and 2, are important to support the ability of the virus to cause disease. While the first RNA structure is well studied, less is known about the role of exonuclease-resistant RNA 2 in the flavivirus infection. Using reverse genetics, we made mutations in the Zika virus exonuclease-resistant RNA 2 structure and studied how this mutant Zika virus was weakened or attenuated. We found that the mutant Zika virus clone exhibits reduced virus replication, reduced ability to kill cells, and decreased virulence in mouse models of Zika virus disease. Using this mutant virus as a potential vaccine candidate, we found that Zika virus with mutations in the exonuclease-resistant RNA 2 structure provide complete protection from lethal Zika virus challenge. These data suggest that targeting the second exonuclease resistant RNA structure in flaviviruses is a viable platform for the development of vaccine candidates for this important group of viruses.

microbiology↗

bioRxiv2025

Myeloid cell IL-15 production in the brain supports Bystander CD8+ T-Cell Neuropathic Immune Responses following Virus infection

The central nervous system (CNS) includes a uniquely regulated immune response that supports homeostasis, response to injury, and response to pathogens. Recent work has shown that virus-associated immune responses in the CNS may contribute to neuronal injury and long-term outcomes such as neurocognitive decline. However, the fundamental mechanisms that regulate acute infiltration of immune cells from vascular compartments into the CNS are not well defined. Using an attenuated Venezuelan equine encephalitis virus TC83 (referred to as TC83) to inoculate using olfactory and intracranial injections, we show that infection in the CNS and olfactory pathways results in rapid infiltration of both CD4+ and CD8+ T-cells as early as 3- and 5-days post-infection. CNS-infiltrating CD8+ T-cells exhibit a bystander, memory phenotype (CD49d+, Tbet+, NKG2D+, Eomes+), are cytotoxic, and are recruited independent of antigen specific responses. We show that infiltration of CD8+ bystander T-cells is supported by microglia and infiltrating macrophage expression of IL-15 and interferon expression in the CNS. These innate antiviral immune signals support activation of bystander CD8+ T-cells in the CNS that contribute to tissue injury independent of virus replication at early time points post-infection. These data support a mechanism by IL-15 stimulates bystander memory CD8+ T-cells to enter the CNS and contribute to injury independent of antigen-specific stimulation. ImportancePrior studies have shown that virus infections in the respiratory and olfactory nerve pathways can result in long term injury in the brain. However, the mechanisms that link virus infection in the olfactory neurons and brain injury are not understood. We show that virus infection of olfactory neurons results in immune stimulation in the brain of resident immune cells to release a cytokine called IL-15 and interferon. This results in infiltration and activation of non-specific T-cells that cause injury of neurons in the brain. This may be an important mechanism by which respiratory viruses and other viruses cause inflammation and injury in the brain.

immunology↗

bioRxiv2023

Zika virus dumbbell-1 structure is critical for sfRNA presence and cytopathic effect during infection

Zika virus (ZIKV) contains multiple conserved RNA structures in the viral 3 untranslated region (UTR), including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus genome replication and cytopathic effect (CPE). However, the role of the DB structure and the mechanism by which it contributes to viral pathogenesis is not known. Using recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones termed ZIKV-TL.PK, which disrupts DB-1 tertiary folding and ZIKV-p.2.5, which alters DB-1 secondary structure formation. In cell culture, we found that viral genome replication of both mutant clones is not significantly affected compared to ZIKV-WT, but viral CPE is considerably decreased. We investigated sub-genomic flaviviral RNA (sfRNA) formation by both DB-1 mutants following A549 infection and found both mutant clones have decreased levels of all sfRNA species compared to ZIKV-WT during infection. To investigate the mechanism of decreased CPE in our DB-1 mutant clones, we assayed ZIKV DB mutant-infected A549 cells for cell viability and caspase activation. We found that cell viability is significantly increased in DB-1 mutant-infected cells compared to ZIKV-WT due to reduced caspase 3 activation. We also show that replication of the ZIKV-P.2.5 mutant was significantly restricted by type I interferon treatment without altering interferon stimulated gene expression. Using a murine model of ZIKV infection, we show that both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality compared to ZIKV-WT virus due to tissue specific attenuation in ZIKV-DB viral replication in the brain tissue. Overall, our data show that the flavivirus DB-1 RNA structure is important for maintaining sfRNA levels during infection which supports caspase-3 dependent, viral cytopathic effect, type 1 interferon resistance, and viral pathogenesis in a mouse model.

microbiology↗

bioRxiv2020

Three-dimensional structure of a flavivirus dumbbell RNA reveals molecular details of an RNA regulator of replication

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5' and 3' untranslated regions (UTRs). The 3' UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs) that regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we used x-ray crystallography and structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

microbiology↗

bioRxiv2020

Disruption of Zika virus xrRNA1-dependent sfRNA1 production results in tissue-specific attenuated viral replication

Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths derived from the viral 3 untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of incomplete viral genome degradation of the 3UTR by host 5 to 3 exoribonuclease (Xrn1). The halting of Xrn1 in the 3UTR is due to two RNA pseudoknot structures in the 3UTR termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we rescued a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3 UTR structure. We found that virus which lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult IFNAR1-/- mice, we found that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to WT ZIKV infection. Despite thee attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.

microbiology↗

bioRxiv2020

Alpha-synuclein expression supports interferon stimulated gene expression in neurons

The protein alpha-synuclein (asyn) is predominantly expressed in neurons and is associated with neurodegenerative diseases like Parkinsons disease (PD); yet, a functional role for asyn in neurons is not clearly established. We have previously shown that asyn expression is up-regulated following viral infection in neurons and is critical for host immune responses to RNA virus infections. Here, we investigate the mechanisms underlying asyn-dependent immune responses to RNA virus infection in the brain. Using asyn knock-out (KO) mice and human neuronal models, we show that asyn is required for expression of the full repertoire of interferon-stimulated genes (ISGs) in neurons following acute RNA virus infection. Furthermore, treatment of asyn KO human neurons with poly I:C or type I interferon also fail to induce expression of the full complement of ISGs suggesting that asyn plays an important role in modulating neuronal innate immune responses. In brain tissue, asyn-dependent ISG expression is independent of microglia activation and supports activation of infiltrating lymphocytes following viral challenge. We also show that virus infections lead to accumulation of phosphorylated S129 asyn in human and non-human primate neuronal tissues. In a model of pS129 asyn pathology, we found that infection with West Nile virus increases microglia activation but does not significantly alter pS129 asyn pathology in the mouse model. Taken together, our results establish asyn as a novel, neuron-specific modulator of innate immunity by a mechanism that promotes interferon-stimulated gene expression and links responses to virus infection with formation of phosphorylated S129-asyn in neuronal tissue.

neuroscience↗

bioRxiv2019

Pregnancy alters innate immune responses to Zika virus infection in the genital tract

Recent outbreaks of Zika virus (ZIKV) have been associated with birth defects, including microcephaly and neurological impairment. However, the mechanisms which confer increased susceptibility to ZIKV during pregnancy remain unclear. We hypothesized that poor outcomes from ZIKV infection during pregnancy are due in part to pregnancy-induced alteration of innate immune cell frequencies and cytokine expression. To examine the impact of pregnancy on innate immune responses, we inoculated pregnant and non-pregnant female C57BL/6 mice with 5x105 FFU of ZIKV intravaginally. Innate immune cell frequencies and cytokine expression were measured by flow cytometry at day 3 post infection. Compared to non-pregnant mice, pregnant mice exhibited higher frequencies of uterine macrophages (CD68+) and tolerogenic dendritic cells (CD11c+ CD103+ and CD11c+ CD11b+). Additionally, ZIKV-infected pregnant mice had lower frequencies of CD45+ IL-12+ and CD11b+ IL-12+ cells in the uterus and spleen. These data show that pregnancy results in an altered innate immune response to ZIKV infection in the genital tract of mice and that pregnancy-associated immune modulation may play an important role in the severity of acute ZIKV infection.\n\nImportancePregnant females longer duration that viremia following infection with Zika virus but the mechanism of this is not established. Innate immune cellular responses are important for controlling virus infection and are important for development and maintenance of pregnancy. Thus, the acute immune response to Zika virus during pregnancy may be altered so that the pregnancy can be maintained. To examine this interaction, we utilized a mouse model of Zika virus infection during pregnancy using intravaginal inoculation. We found that following Zika virus infection, pregnant mice exhibited increased expression of tolerant or non-inflammatory dendritic cells. Additionally, we found that pregnant mice have significantly depressed ability to secrete the cytokine IL-12 from innate immune cells in the uterus and the spleen while maintaining MHCII expression. These findings show that pregnancy-induced changes in the innate immune cells are biased towards tolerance and can result in decreased antigen-dependent stimulation of immune responses.

immunology↗