Biology subjects

Danthi, P.

Publications and source records attributed to Danthi, P..

At least 19 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↗

bioRxiv2026

The reovirus σ3 protein induces proteasomal degradation of IKKβ to modulate the host response to infection

Reovirus strains differ in their capacity to modulate host antiviral signaling, but the viral determinants underlying these differences have remained unclear. In this study, we identify the outer capsid protein {sigma}3 as the viral determinant responsible for inducing the loss of the NF-{kappa}B kinase, IKK{beta}. We demonstrate that introduction of the {sigma}3 encoding gene segment of strain T3A in the background of strain T3DL recapitulates IKK loss observed during T3A infection. Our results indicate that de novo expression of T3A {sigma}3 during infection is necessary to mediate the loss of IKK. Further, the presence of T3A {sigma}3 alone is sufficient. We show that T3A {sigma}3 interacts with the IKK complex and targets IKK{beta} for degradation through a Cullin-RING ligase-proteasome dependent mechanism. Loss of IKK{beta} results in impaired NF-{kappa}B activation and reduced IFN-{beta} expression. Consequently, the T3DL/T3A S4 monoreassortant exhibits a replication advantage compared to T3DL. Collectively, these findings uncover a previously uncharacterized mechanism by which reovirus {sigma}3 suppresses innate antiviral signaling through targeted degradation of IKK{beta}, providing new insight into reovirus mediated immune modulation. IMPORTANCESuccessful viral infection and replication requires that the virus suppress host immune defenses, particularly innate immunity, which relies on signaling cascades to produce antiviral cytokines. Many viruses encode proteins to target key signaling molecules, such as those in the NF-{kappa}B pathway, but the exact mechanisms of evasion vary across viral families and even between closely related strains. In this study, we uncover a novel function of the reovirus outer capsid protein {sigma}3 to promote IKK{beta} degradation. Our findings reveal that {sigma}3 associates with a complex of proteins that includes IKK{beta}, a central kinase in the NF-{kappa}B pathway, to mediate its degradation via the proteasomal pathway. {sigma}3 mediated IKK{beta} loss decreases type I IFN expression during infection and enhances viral replication. Coupled with previous observations that {sigma}3 from other reovirus strains antagonizes NF-{kappa}B by altering its transcriptional activity, this work demonstrates that this conserved viral protein adopts strain-specific strategies to subvert host defenses.

microbiology↗

bioRxiv2025

Viral evolution of T1L mammalian orthoreovirus enhances breast cancer stem-like cell killing

BackgroundBreast cancer is the second leading cause of death in women in the US. Among the different subtypes of breast cancer, estrogen receptor positive (ER+) has a more favorable prognosis; however, patients can experience cancer recurrence 10 years or more after initial diagnosis. Breast cancer stem-like cells (BCSCs) are slowly proliferating cells that drive metastasis and resistance to therapies that target rapidly proliferating tumor cells. BCSC self-renewal and survival pathways make them difficult to eliminate; no selective therapeutics currently exist to target them. Our research aims to use mammalian orthoreovirus (MRV) as an oncolytic agent to eliminate BCSCs. MethodsWe evaluated the effects of three different MRV strains (T3D, T1L and R2) under both 2D and 3D culture conditions (which enrich for BCSCs) in parental and paclitaxel resistant (TaxR) MCF7 cells using cell viability assays, flow cytometry, western blotting, and tumorsphere assays. We identified differentially expressed genes in response to T1L by RNA sequencing. Novel MRV strains were developed by serial passaging of the parental T1L strain in TaxR BCSCs, leading to the isolation and sequencing of three distinct MRV clones. The novel MRV clones were assessed for their oncolytic effects using the aforementioned techniques alongside inhibitor-based studies targeting cell death pathways. ResultsNotably, at least two clones (T1L SP B and T1L SP C) demonstrated enhanced oncolytic potency compared to the parent T1L strain against MCF7 TaxR BCSCs, even though their infectivity and viral protein synthesis was reduced. Whole genome sequencing of the viral clones identified changes in viral protein sequences that correlate with enhanced potency against BCSCs. T1L, T1L SP B and T1L SP C induced apoptosis; however, T1L SP B may induce a more immunogenic form of cell death in the TaxR cells, as indicated by the increased release of extracellular ATP (eATP), a damage-associated molecular pattern (DAMP), which is associated with immunogenic cell death. ConclusionsIn summary, we developed novel oncolytic viruses promote oncolysis in therapy-resistant BCSCs. We found that T1L SP B induces apoptosis in a more immunogenic manner, highlighting the potential of these viruses to eliminate BCSCs and stimulate the immune system.

cancer biology↗

bioRxiv2025

Virion encapsidation and cell attachment function of the reovirus attachment protein is influenced by its structural flexibility

The reovirus {sigma}1 attachment protein mediates virus interaction with host cell receptors that is critical for cell entry. Reovirus tropism is controlled by properties of {sigma}1. {sigma}1 is present as trimers that are held within turrets at the icosahedral vertices of reovirus virions. However, because {sigma}1 has not been visualized on reovirus virions in high resolution structures and because the fulllength structure of purified {sigma}1 protein has not been solved, it is not clear how {sigma}1 is presented on virions. What properties of {sigma}1 are essential for its incorporation on virions is also not known. In this study, we used ColabFold to model the structure of reovirus serotype 1 (T1) and serotype 3 (T3) {sigma}1 proteins. We find that these proteins fold into similar structures with regions of flexibility between the head and body domains of {sigma}1. We also predicted the structures of chimeric {sigma}1 proteins comprised of domain swaps between T1 and T3 {sigma}1 proteins. Our analyses indicate that chimeric proteins with mismatched body and head domain have increased flexibility in this region. Characterization of particles expressing such chimeric {sigma}1 proteins demonstrated that deviation from the flexibility of parental {sigma}1 leads to a reduction in {sigma}1 incorporation on to the virion. Further, we find that even when incorporation is not affected, virus attachment to host cell receptors is influenced by altered {sigma}1 flexibility. Finally, our work demonstrates that 1 protein impacts the encapsidation pattern and receptor engagement properties of {sigma}1 and that this effect is influenced by properties of the N-terminal portion of {sigma}1. ImportanceAttachment to host cell receptors is a critical step in initiation of virus infection. Some viruses attach to cellular receptors via dedicated viral proteins. Both the number of attachment factors present on the virus and whether they are present on the virus particle in the correct form can influence cell attachment. Here, using reovirus as a model, we use a protein structure prediction algorithm to model the as yet unknown structure of full-length reovirus attachment protein {sigma}1. We find predicted regions of flexibility in the protein and identify how this flexibility is regulated. We find that the flexibility of {sigma}1 independently regulates whether it is stably incorporated into particles and if can efficiently interact with host receptors.

microbiology↗

bioRxiv2025

WDR81 represses IKK mediated expression of pro-survival genes to regulate apoptosis

Apoptosis is a common host response to virus infection. The extent and timing of apoptosis following infection is controlled by the balance between the strength of signals that activate death signaling and those that promote the survival of cells. In many cell types, infection with Mammalian orthoreovirus (reovirus) results in induction of cell death by apoptosis late in infection. In this study, we uncovered that WD repeat-containing protein 81 (WDR81) is required for apoptosis induction after reovirus infection. The requirement for WDR81 for apoptosis induction is not unique to reovirus because cells lacking WDR81 are also resistant to apoptosis induced by other agonists that trigger the extrinsic apoptotic pathway. We find that in cells deficient in WDR81, expression of several pro-survival genes is upregulated. The expression of these genes is controlled by the inhibitor of kB kinase complex (IKK)-Nuclear Factor of kB (NF{kappa}B) signaling pathway. When IKK signaling is blocked in WDR81-deficient cells, pro-survival gene expression is restored to normal levels and the cells regain their susceptibility to reovirus-induced death receptor-triggered apoptosis. Our work uncovers a new function for WDR81 in controlling apoptosis. Additionally, it reveals a previously unknown link between endosomally localized protein WDR81 and IKK-NF{kappa}B signaling. Author SummaryVirus infection often results in death of the infected cells. Cell death prior to generation of virus progeny limits the spread of infection to neighboring cells and therefore can be beneficial to the host. However, cell death might also cause tissue destruction and could contribute to viral disease. It is therefore important to understand how cell death is controlled. Here, we uncover a cell death regulating role for WDR81 - a cellular protein that has not been previously implicated in affecting cell death. We find that when this protein is absent, cells express a much greater level of survival signals. These survival signals prevent efficient induction of cell death. By investigating how these survival signals are expressed, we reveal a new link between WDR81 and NF{kappa}B, a well-known cellular survival pathway.

microbiology↗

bioRxiv2024

Cholesterol-Dependent Membrane Deformation by Metastable Viral Capsids Facilitates Entry

Non-enveloped viruses employ unique entry mechanisms to breach and infect host cells. Understanding these mechanisms is crucial for developing antiviral strategies. Prevailing perspective suggests that non-enveloped viruses release membrane lytic peptides to breach host membranes. However, the precise involvement of the viral capsid in this entry remains elusive. Our study presents direct observations elucidating the dynamically distinctive steps through which metastable reovirus capsids disrupt host lipid membranes as they uncoat into partially hydrophobic intermediate particles. Using both live cells and model membrane systems, our key finding is that reovirus capsids actively deform and permeabilize lipid membranes in a cholesterol-dependent process. Unlike membrane lytic peptides, these metastable viral capsids induce more extensive membrane perturbations, including budding, bridging between adjacent membranes, and complete rupture. Notably, cholesterol enhances subviral particle adsorption, resulting in the formation of pores equivalent to the capsid size. This cholesterol dependence is attributed to the lipid condensing effect, particularly prominent at intermediate cholesterol level. Furthermore, our results reveal a positive correlation between membrane disruption extent and efficiency of viral variants in establishing infection. This study unveils the crucial role of capsid-lipid interaction in non-enveloped virus entry, providing new insights into how cholesterol homeostasis influences virus infection dynamics.

biophysics↗

bioRxiv2023

Proteasome activity is required for reovirus entry into cells

Since viruses have limited coding capacity in their genomes, they use host cell machinery to complete virtually every stage of their replication cycle. Mammalian orthoreovirus (reovirus) is comprised of two concentric protein shells, the inner core and the outer capsid. Following attachment to its receptor, reovirus enters the cell by receptor-mediated endocytosis. Within endosomes reovirus utilizes host acid-dependent proteases to process the viral outer capsid. Specifically, the outer capsid protein {sigma}3 is degraded and 1 is cleaved to form the disassembly intermediate infectious subvirion particles (ISVPs). ISVPs undergo additional conformational changes into ISVP*s that release small peptides which mediate the penetration of endosomal membranes. Membrane penetration allows for delivery of the remaining viral core into the cytoplasm for subsequent gene expression. Here, we describe that the ubiquitin proteasome system controls an entry step of reovirus particles. We show that chemically inhibiting the proteasome blocks infection at a stage following ISVP formation but prior to transcriptional activation of cores. Specifically, inhibition of the proteasome prevents conformational changes in 1 characteristic of ISVP-to-ISVP* conversion. In the absence of these conformational changes, cores are unable to be delivered and become transcriptionally active, thereby blocking viral replication. This work highlights a previously unknown way in which reovirus relies on host factors for successful replication. IMPORTANCEDue to their limited genetic capacity, viruses are reliant on multiple host systems to replicate successfully. Mammalian orthoreovirus (reovirus) is commonly used as a model system for understanding host-virus interactions. In this study, we identify the host ubiquitin proteasome system as a regulator of reovirus entry. Inhibition of the proteasome using a chemical inhibitor blocks reovirus uncoating. Blocking these events reduces subsequent replication of the virus. This work identifies that additional host factors controls reovirus entry.

microbiology↗

bioRxiv2022

The reovirus σ1 attachment protein influences the stability of its entry intermediate

Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a {sigma}1 monoreassortant virus, we recently showed that {sigma}1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by {sigma}1 and whether the lower encapsidation level of {sigma}1 is connected to this property is unknown. To define a correlation between encapsidation of {sigma}1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in {sigma}1 or those that contain chimeric {sigma}1 molecules composed of {sigma}1 portions from Type 1 and Type 3 reovirus strains. We found that under most conditions where {sigma}1 encapsidation on the particle was lower, ISVPs displayed lower stability. By separating wild-type particles based on the level of encapsidated {sigma}1, we also found that lower {sigma}1 encapsidation leads to lower ISVP stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, {sigma}1 properties influence stability without influencing {sigma}1 encapsidation. These data indicate that {sigma}1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the {sigma}1 attachment protein on the properties of the reovirus capsid.

microbiology↗

bioRxiv2022

Early events in reovirus infection influence induction of innate immune response

Mammalian orthoreovirus (reovirus) is a double-stranded (ds)RNA virus which encapsidates its 10 genome segments within a double-layered viral particle. Reovirus infection triggers an antiviral response in host cells which serves to limit viral replication. This antiviral response is initiated by recognition of the incoming viral genome by host sensors present in the cytoplasm. However, how host sensors gain access to the reovirus genome is unclear as this dsRNA is protected by the viral particle proteins throughout infection. To initiate infection, reovirus particles are endocytosed and the outer viral particle layer is disassembled through the action of host proteases. This disassembly event is required for viral escape into the cytoplasm to begin replication. We show that even after disassembly is complete, endosomal proteases are required to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate at least some viral dsRNA genome is exposed and detectable during entry. We hypothesize that some proportion of reovirus particles remain trapped within endosomes, allowing for the breakdown of these particles and release of their genome. We show that rapidly uncoating mutants escape the endosome more rapidly and induce a diminished immune response. Further, we show that particles entering through dynamin-independent pathways evade detection by host sensors. Overall, our data provide new insight into how genomes from entering reovirus particles are detected by host cells. IMPORTANCEViruses must infect host cells to replicate, often killing the host cell in the process. However, hosts can activate defenses to limit viral replication and protect the organism. To trigger these host defenses to viral infections, host cells must first recognize they are infected. Mammalian orthoreovirus (reovirus) is a model system used to study host-virus interactions. This study identifies aspects of host and virus biology which determine the capacity of host cells to detect infection. Notably, entry of reovirus into host cells plays a critical role in determining the magnitude of immune response triggered during infection. Mutants of reovirus which can enter cells more rapidly are better at avoiding detection by the host. Additionally, reovirus can enter cells through multiple routes. Entry through some of these routes also helps reovirus evade detection.

microbiology↗

bioRxiv2021

The reovirus σ3 protein inhibits NF-κB-dependent antiviral signaling

Viral antagonism of innate immune pathways is a common mechanism by which viruses evade immune surveillance. Infection of host cells with reovirus leads to the blockade of NF-{kappa}B, a key transcriptional regulator of the hosts innate immune response. One mechanism by which reovirus infection results in inhibition of NF-{kappa}B is through a diminishment in levels of upstream activators, IKK{beta} and NEMO. Here, we demonstrate a second, distinct mechanism by which reovirus blocks NF-{kappa}B. We report that expression of a single viral protein, {sigma}3, is sufficient to inhibit expression of NF-{kappa}B target genes. Further, {sigma}3-mediated blockade of NF-{kappa}B occurs without changes to IKK levels or activity. Expression of only a subset of NF-{kappa}B target genes is reduced. Among NF-{kappa}B targets, the expression of type I interferon is significantly diminished by {sigma}3 expression. Correspondingly, ectopic expression of {sigma}3 enhances viral replication. Expression of NF-{kappa}B target genes varies following infection with closely related reovirus strains. Our genetic analysis identifies that these differences are controlled by polymorphisms in the amino acid sequence of {sigma}3. This work identifies a new role for reovirus {sigma}3 as a viral antagonist of the NF-{kappa}B-dependent antiviral pathways. IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected cells. The NF-{kappa}B family of proteins is important for the cell to mediate this response. In this study, we show that a single protein, {sigma}3, produced by mammalian reovirus, impairs the function of NF-{kappa}B. We demonstrate that by blocking NF-{kappa}B, {sigma}3 diminishes the hosts response to infection and promotes viral replication. This work identifies a second, previously unknown mechanism by which reovirus blocks this aspect of the host cell response.

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↗

bioRxiv2020

Reovirus σ3 protein limits interferon expression and cell death induction

Induction of necroptosis by mammalian reovirus requires both type I interferon (IFN)-signaling and viral replication events that lead to production of progeny genomic dsRNA. The reovirus outer capsid protein {micro}1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein {sigma}3, which interacts with {micro}1, also functions in regulating cell death, we used siRNA-mediated knockdown. Similar to that observed by diminishment of {micro}1 expression, knockdown of newly synthesized {sigma}3 enhances necroptosis. {sigma}3 knockdown does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following {sigma}3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of {sigma}3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of {sigma}3 protein to bind dsRNA does not impact its capacity to diminish production of IFN. Consistent with this, infection with a virus harboring a mutation in the dsRNA binding domain of {sigma}3 does not result in enhanced production of IFN or cell death. Together, these data suggest that {sigma}3 limits the production of IFN to control innate immune signaling and cell death following infection through a mechanism that is independent of its dsRNA binding capacity. IMPORTANCEWe use mammalian reovirus as a model to study how virus infection modulates innate immune signaling and cell death induction. Here we sought to determine how viral factors regulate these processes. Our work highlights a previously unknown role for the reovirus outer capsid protein {sigma}3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. {sigma}3 limits the induction of necroptosis by preventing excessive production of interferon following infection.

microbiology↗

bioRxiv2020

Reovirus core proteins {lambda}1 and σ2 promote stability of disassembly intermediates and influence early replication events

The capsids of mammalian reovirus contain two concentric protein shells, the core and the outer capsid. The outer capsid is comprised of {micro}1-{sigma}3 heterohexamers which surround the core. The core is comprised of {lambda}1 decamers held in place by {sigma}2. After entry into the endosome, {sigma}3 is proteolytically degraded and {micro}1 is cleaved and exposed to form ISVPs. ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of {micro}1 peptides which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work has identified regions or specific residues within reovirus outer capsid that impact the efficiency of cell entry. We examined the functions of the core proteins {lambda}1 and {sigma}2. We generated a reovirus T3D reassortant that carries strain T1L derived {sigma}2 and {lambda}1 proteins (T3D/T1L L3S2). This virus displays a lower ISVP stability and therefore converts to ISVP*s more readily. To identify the basis for lability of T3D/T1L L3S2, we screened for hyper-stable mutants of T3D/T1L L3S2 and identified three point mutations in {micro}1 that stabilize ISVPs. Two of these mutations are located in the C-terminal {phi} region of {micro}1, which has not previously been implicated in controlling ISVP stability. Independent from compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells. In addition to identifying a new role for the core proteins in disassembly events, these data highlight that core proteins may influence multiple stages of infection. IMPORTANCEProtein shells of viruses (capsids) have evolved to undergo specific changes to ensure the timely delivery of genetic material to host cells. The 2-layer capsid of reovirus provides a model system to study the interactions between capsid proteins and the changes they undergo during entry. We tested a virus in which the core proteins were derived from a different strain than the outer capsid. We found that this mismatched virus was less stable and completed conformational changes required for entry prematurely. Capsid stability was restored by introduction of specific changes to the outer capsid, indicating that an optimal fit between inner and outer shells maintains capsid function. Separate from this property, mismatch between these protein layers also impacted the capacity of virus to initiate infection and produce progeny. This study reveals new insights into the roles of capsid proteins and their multiple functions during viral replication.

microbiology↗

bioRxiv2019

Loss of IKK subunits limits NF-κB signaling in reovirus infected cells

Viruses commonly antagonize innate immune pathways that are primarily driven by Nuclear Factor-{kappa}B (NF-{kappa}B), Interferon Regulatory Factor (IRF) and Signal Transducer and Activator of Transcription (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased RNA-seq based approach to measure gene expression induced by transfected viral genomic RNA (vgRNA) and reovirus infection, we discovered that mammalian reovirus inhibits host cell innate immune signaling. We found that while vgRNA and reovirus infection both induce a similar IRF dependent gene expression program, gene expression driven by the NF-{kappa}B family of transcription factors is lower in infected cells. Potent agonists of NF-{kappa}B, such as Tumor Necrosis Factor alpha (TNF) and vgRNA, failed to induce NF-{kappa}B dependent gene expression in infected cells. We demonstrate that NF-{kappa}B signaling is blocked due to loss of critical members of the Inhibitor of KappaB Kinase (IKK) complex, NF-{kappa}B Essential MOdifier (NEMO) and IKK{beta}. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-{kappa}B, thereby preventing gene expression. Our studies demonstrate that reovirus infection selectively blocks NF-{kappa}B, likely to counteract its antiviral effects and promote efficient viral replication. IMPORTANCEHost cells mount a response to curb virus replication in infected cells and prevent infection of neighboring, as yet uninfected cells. The NF-{kappa}B family of proteins is important for the cell to mediate this response. In this study, we show that in cells infected with mammalian reovirus, NF-{kappa}B is inactive. Further, we demonstrate that NF-{kappa}B is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-{kappa}B function. Based on previous evidence that active NF-{kappa}B limits reovirus infection, we conclude that inactivating NF-{kappa}B is a viral strategy to produce a cellular environment that is favorable for virus replication.

microbiology↗

bioRxiv2019

Cell entry-independent role for the reovirus μ1 protein in regulating necroptosis and the accumulation of viral gene products

The reovirus outer capsid protein 1 regulates cell death in infected cells. To distinguish between the role of incoming, capsid-associated and newly synthesized 1, we used siRNA-mediated knockdown. Loss of newly synthesized 1 protein does not impact apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of 1 also impacts aspects of viral replication. We found that while 1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus strand RNA, plus strand RNA, and proteins that are not targeted by the 1 siRNA accumulate to a greater extent when compared to control siRNA-treated cells. Furthermore, we observe a decrease in sensitivity of these viral products to inhibition by GuHCl (which targets minus strand synthesis to produce dsRNA) when 1 is knocked down. Following 1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of 1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to a 1 knockdown potentiates RIP3 dependent cell death. IMPORTANCEWe use mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for reovirus outer capsid protein 1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection. 1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

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↗