Biology subjects

Snyder, A. J.

Publications and source records attributed to Snyder, A. J..

9 recordsLinked to original sources

bioRxiv2026

Viral Capsid-Membrane Interactions Propel Non-Brownian Movements of Non-enveloped Reoviruses during Entry

Understanding how non-enveloped viruses breach host cell membranes is critical for developing strategies to block viral entry, a key step in infection. Despite extensive study, how viral capsids and host lipid membranes dynamically cooperate during membrane penetration remains poorly defined. Here, using reovirus as a model non-enveloped virus and planar model membranes, we identify previously unrecognized non-Brownian membrane motions of infectious subvirion particles (ISVPs) by single-virus tracking. We then integrate experiments with computational modeling to dissect the stepwise, processive capsid-membrane interactions encoded in these distinct dynamics. We show that ISVP motion transitions from an initial phase of directed translocation to progressively confined diffusion. This behavior reflects a multistep entry mechanism in which initial capsid-membrane contact triggers release of the membrane-active 1N peptide. As 1N accumulates within the bilayer, it generates membrane-associated viral retention sites that promote further virus adsorption and increasingly constrain particle mobility. By directly visualizing these motion signatures, we resolve transient and cooperative capsid-membrane interactions that are difficult to capture using conventional biochemical approaches. Together, these findings provide new insight into early membrane penetration events of non-enveloped viruses. SIGNIFICANCE STATEMENTNon-enveloped viruses must penetrate host cell membranes to initiate infection, yet the dynamic mechanisms by which viral capsids engage and remodel lipid membranes remain poorly understood. By directly visualizing the motion of individual reovirus particles on model membranes, we uncover previously unrecognized non-Brownian dynamics that encode stepwise capsid-membrane interactions during viral entry. Integrating single-virus tracking with computational modeling reveals how peptide-mediated membrane remodeling feeds back to regulate viral engagement. This work provides the first direct visualization of processive, stepwise capsid-membrane interactions during non-enveloped virus entry and establishes viral particle dynamics as a quantitative readout for dissecting membrane penetration mechanisms.

biophysics↗

bioRxiv2025

NKTR-255, a polymer-conjugated IL-15, synergizes with CAR-T cell therapy to activate endogenous anti-tumor immunity and improve tumor control

CAR-T cells have yet to show widespread efficacy in solid tumors due in part to their poor persistence and loss of function in the tumor microenvironment. Further, heterogenous expression of most CAR target antigens in solid tumors can lead to escape of antigen-null tumors that resist CAR-T killing. Strategies to cooperatively boost both CAR-T and endogenous anti-tumor immunity could curb tumor escape and may be critical for achieving durable efficacy in cancer patients. NKTR-255 is a polymer-conjugated IL-15 with extended half-life that can boost endogenous T and NK cells, as well as CD19 CAR-T activity in B cell malignancies. However, whether NKTR-255 is sufficient to overcome CAR-T dysfunction in the suppressive solid tumor microenvironment, and how NKTR-255 and CAR-Ts together re-shape endogenous anti-tumor immunity, is not known. Using an autochthonous mouse model of ROR1+ lung adenocarcinoma, we show that NKTR-255 significantly boosted accumulation, reduced exhaustion, and improved function of tumor-infiltrating CAR-T cells. Compared with NKTR-255 or CAR-T treatment alone, combination of NKTR-255 and CAR-T therapy synergistically increased tumor-infiltrating CD11b+ cytotoxic NK cells, activated dendritic cells, and endogenous tumor-specific T cells that preserved a PD-1+Tcf1+ stem-like phenotype. Consequently, NKTR-255 and CAR-T combination therapy induced complete elimination of ROR1+ tumor and significantly improved survival, with enhanced tumor control dependent on activity of both CAR-Ts and endogenous T cells. Altogether, our data suggest that combining NKTR-255 with CAR-T therapy is a promising strategy to enhance both CAR-T and endogenous anti-tumor immunity to promote coordinated control of aggressive tumors.

immunology↗

bioRxiv2025

MCM8/9 and FANCD2 interact within a shared pathway in response to replication stress caused by DNA crosslinks

The Fanconi anemia (FA) protein FANCD2, and MCM8/9 heterohexameric helicase complex are critical for maintaining genomic integrity in response to replication stress. However, the nature of their relationship remains unclear. Here, we show that MCM8/9 physically interacts and functionally cooperates with FANCD2 during the repair of DNA interstrand crosslinks (ICLs). Using immunofluorescence and co-immunoprecipitation studies, we show that MCM8/9 interacts with FANCD2 through its core domain, independently of DNA. FANCD2 is essential for the recruitment of MCM9 to ICL damage induced nuclear foci and acts downstream the FANCD2I monoubiquitination. Although MCM8/9 foci formation requires its intact ATPase activity, BRCv motif and HROB, these are not required for FANCD2 binding, highlighting a distinction between physical interaction and functional activation. Interestingly, FANCD2 foci formation increase in MCM8 or MCM9 knockout cells, suggesting that MCM8/9 functions to mitigate replication associate stress. {gamma}H2AX DNA damage assays and cell survival assays show that combined loss of MCM9 and FANCD2 do not cause any additive DNA damage beyond individual knockouts, indicating an epistatic relationship and suggests they function in the same DNA repair pathway. Together, our findings identify MCM8/9 as a downstream effector of the FA pathway critical for resolving ICL induced DNA damage. HighlightsO_LIMCM8/9 interacts and colocalize with FANCD2 upon ICL DNA damage. C_LIO_LIFANCD2 is essential for recruitment of MCM9 to DNA damage site. C_LIO_LIMCM8/9 is epistatic to FANCD2 and within the same DNA damage response pathway. C_LI

cell biology↗

bioRxiv2025

Modulating AP-1 enables CAR-T cells to establish an intratumoral PD-1+Tcf1+ stem-like reservoir and overcomes resistance to PD-1 axis blockade

PD-1+Tcf1+ stem-like cells are critical mediators of endogenous T cell responses to PD-1/PD-L1 blockade and are maintained by MHC-dependent interactions with professional antigen-presenting cells (APCs). Unlike conventional T cells, CAR-T cells are activated by intact antigen expressed on tumors, not by peptide/MHC expressed on APCs, restricting their activation to the hostile tumor microenvironment (TME) that may impair preservation of this critical stem-like subset. Indeed, in an autochthonous model of ROR1+ lung cancer that we developed, CAR-T cells targeting the tumor-associated antigen ROR1 were uniformly Tcf1+ prior to infusion but rapidly downregulated Tcf1 in vivo and became terminally exhausted, similar to observations in patients, resulting in faster attrition and no enhancement in response to PD-L1 blockade. We hypothesized that overexpression of AP-1 family transcription factors, which can regulate T cell exhaustion, could enable CAR-Ts to maintain this critical PD-1+Tcf1+ subset within tumors independently of APCs and sensitize them to PD-1/PD-L1 blockade. Overexpression of the AP-1 TF c-Jun, but not BATF, improved preservation of PD-1+Tcf1+ CAR-T cells within tumors in a cell-intrinsic manner that correlated with increased persistence deeper within tumors. Notably, c-Jun overexpression alone was insufficient to prevent CAR-T exhaustion in the lung TME, in contrast to prior work in xenograft models, with progressive CAR-T dysfunction correlated with PD-1-dependent downregulation of c-Jun. However, c-Jun overexpression dramatically sensitized CAR-Ts to PD-L1 blockade, which restored c-Jun levels in CAR-Ts, drove log-fold expansion of CAR-Ts within tumors, and induced nearly complete eradication of ROR1+ tumor in highly aggressive models of lung cancer. Altogether, our data show that combination with PD-L1 blockade is necessary to unleash the full potential of c-Jun-overexpressing CAR-T cells in aggressive solid tumors like lung cancer and suggest that strategies to enhance formation of intratumoral PD-1+Tcf1+ reservoirs can overcome CAR-T resistance to PD-1 blockade.

immunology↗

bioRxiv2024

Production of OSU G5P Porcine Rotavirus Expressing a Fluorescent Reporter by Reverse Genetics

Rotaviruses are a significant cause of severe, potentially life-threatening gastroenteritis in the young of many economically important animals. Although vaccines against porcine rotavirus exist, both live oral and inactivated, their effectiveness in preventing gastroenteritis is less than ideal. Thus, a need remains for the development of new generations of porcine rotavirus vaccines. The Ohio State University (OSU) rotavirus strain represents a Rotavirus A species with a G5P[7] genotype, the genotype most frequently associated with rotavirus disease in piglets. Using complete genome sequences that were determined by Nanopore sequencing, we developed a robust reverse genetics system enabling recovery of recombinant (r)OSU rotavirus. Although rOSU grew to high titers ([~]107 plaque-forming units/ml), its growth kinetics were modestly decreased in comparison to laboratory-adapted OSU virus. The reverse genetics system was used to generate rOSU rotavirus that served as an expression vector for foreign protein. Specifically, by engineering a fused NSP3-2A-UnaG open reading frame into the segment 7 RNA, we produced genetically stable rOSU that expressed the fluorescent UnaG protein as a functional separate product. Together, these findings raise the possibility of producing improved live oral porcine rotavirus vaccines through reverse genetics-based modification or combination porcine rotavirus vaccines that can express neutralizing antigens of other porcine enteric diseases.

microbiology↗

bioRxiv2021

A CRISPR-Cas9 screen reveals a role for WD repeat-containing protein 81 (WDR81) in the entry of late penetrating viruses

Successful initiation of infection by many different viruses requires their uptake into the endosomal compartment. While some viruses exit this compartment early, others must reach the degradative, acidic environment of the late endosome. Mammalian orthoreovirus (reovirus) is one such late penetrating virus. To identify host factors that are important for reovirus infection, we performed a CRISPR-Cas9 knockout (KO) screen that targets over 20,000 genes in fibroblasts derived from the embryos of C57/BL6 mice. We identified seven genes (WDR81, WDR91, RAB7, CCZ1, CTSL, GNPTAB, and SLC35A1) that were required for the induction of cell death by reovirus. Notably, CRISPR-mediated KO of WD repeat-containing protein 81 (WDR81) rendered cells resistant to reovirus infection. Susceptibility to reovirus infection was restored by complementing KO cells with human WDR81. Although the absence of WDR81 did not affect viral attachment efficiency or uptake into the endosomal compartments for initial disassembly, it delayed viral gene expression and diminished infectious virus production. Consistent with the role of WDR81 in impacting the maturation of endosomes, WDR81-deficiency led to the accumulation of reovirus particles in dead-end compartments. Though WDR81 was dispensable for infection by VSV (vesicular stomatitis virus), which exits the endosomal system at an early stage, it was required for VSV-EBO GP (VSV that expresses the Ebolavirus glycoprotein), which must reach the late endosome to initiate infection. These results reveal a broad, previously unappreciated role for WDR81 in promoting the replication of late penetrating viruses. AUTHOR SUMMARYViruses are obligate intracellular parasites that require the contributions of numerous host factors to complete the viral life cycle. Thus, the host-pathogen interaction can regulate cell death signaling and virus entry, replication, assembly, and egress. Functional genetic screens are useful tools to identify host factors that are important for establishing infection. Such information can also be used to understand cell biology. Notably, genome-scale CRISPR-Cas9 knockout screens are robust due to their specificity and the loss of host gene expression. Mammalian orthoreovirus (reovirus) is a tractable model system to investigate the pathogenesis of neurotropic and cardiotropic viruses. Using a CRISPR-Cas9 screen, we identified WD repeat-containing protein 81 (WDR81) as an essential host factor for reovirus infection of murine cells. Ablation of WDR81 blocked a late step in the viral entry pathway. Further, our work indicates that WDR81 is required for the entry of vesicular stomatitis virus that expresses the Ebolavirus glycoprotein.

microbiology↗

bioRxiv2021

The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The M2 gene encodes outer capsid protein 1, which mediates host membrane penetration during reovirus entry. We infected newborn C57BL/6 mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L genetic background (T1L/T3DM2). T1L was non-lethal in wild-type mice, whereas greater than 90% of mice succumbed to T1L/T3DM2 infection. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions indicative of substantial inflammatory infiltrate. Lesions in T1L/T3DM2-infected mice contained necrotic cardiomyocytes with pyknotic debris, and extensive lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of small purulent lesions in a small subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 compared to T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a non-lethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis. IMPORTANCEReovirus is a non-enveloped virus with a segmented double-stranded RNA genome that serves as a model for studying viral myocarditis. The mechanisms by which reovirus drives myocarditis development are not fully elucidated. We found that substituting the M2 gene from strain type 3 Dearing (T3D) into an otherwise type 1 Lang (T1L) genetic background (T1L/T3DM2) was sufficient to convert the non-lethal T1L strain into a lethal infection in neonatal C57BL/6 mice. T1L/T3DM2 disseminated more efficiently and reached higher maximum titers than T1L in all organs tested, including the heart. T1L is mildly myocarditic and induced small areas of cardiac inflammation in a subset of mice. In contrast, hearts from mice infected with T1L/T3DM2 contained extensive cardiac inflammatory infiltration and more activated caspase-3-positive cells, which is indicative of apoptosis. Together, our findings identify the reovirus M2 gene as a new determinant of reovirus-induced myocarditis.

microbiology↗

bioRxiv2019

Selection and characterization of a reovirus mutant with improved thermostability

The environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol and sodium dodecyl sulfate) can render virions noninfectious. As such, discrete proteins are necessary to stabilize the dual layered structure of mammalian orthoreovirus (reovirus). The outer capsid participates in cell entry: (i) {sigma}3 is degraded to generate the infectious subviral particle and (ii) 1 facilitates membrane penetration and subsequent core delivery. 1-{sigma}3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (1 M258I and {sigma}3 S344P) within heat resistant strains. {sigma}3 S344P was sufficient to enhance capsid integrity and to reduce protease sensitivity. Moreover, these changes impaired replicative fitness in a reassortant background. This work reveals new details regarding the determinants of reovirus stability.\n\nSIGNIFICANCENonenveloped viruses rely on protein-protein interactions to shield their genomes from the environment. The capsid, or protective shell, must also disassemble during cell entry. In this work, we identified a determinant within mammalian orthoreovirus that regulates heat resistance, disassembly kinetics, and replicative fitness. Together, capsid function is balanced for optimal replication and for spread to a new host.

microbiology↗

bioRxiv2018

The reovirus μ1 protein contributes to the environmental stability of virions

The mammalian orthoreovirus (reovirus) outer capsid is composed of 200 1-{sigma}3 heterohexamers and a maximum of 12 {sigma}1 trimers. During cell entry, {sigma}3 is degraded by luminal or intracellular proteases to generate a metastable intermediate, called infectious subviral particle (ISVP). Prior to disassembly, {sigma}3 stabilizes the virion by capping 1. Reovirus fails to establish a productive infection when {sigma}3 degradation is prevented, suggesting proteolytic priming is required for entry. Once uncovered, ISVPs are converted to ISVP*s, which is accompanied by a 1 rearrangement. Nonetheless, whether {sigma}3 degradation can be bypassed for virions to adopt an altered conformation is undetermined. In this report, we utilized the T1L/T3D M2 reassortant, which encodes a mismatched outer capsid, to further investigate the determinants of reovirus stability. When 1-{sigma}3 were derived from different strains, virions resembled wild type in structure and protease sensitivity. Using heat as a surrogate for environmental assault, T1L/T3D M2 ISVPs were more susceptible to inactivation than wild type ISVPs. In contrast, virions of each strain were equally stable. Surprisingly, virion associated 1 rearranged into an ISVP*-like conformation concurrent with loss of infectivity. Despite the presence {sigma}3, a hyperstable variant of 1 also contributed to heat resistance. The dual layered architecture of reovirus allowed for differential sensitivity to inactivating agents; the inner capsid (core) displayed exceptional resistance to heating. Together, these findings reveal a previously undefined contribution from 1 in maintaining virion stability.

microbiology↗