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 σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.

Proteasome activity is required for reovirus entry into cells.

IMPORTANCE: Due 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 that the proteasome system, which is critical for cellular protein turnover, affects 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 control reovirus entry.

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 reduced 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 previously unappreciated role for WDR81 in promoting the replication of viruses that transit through late endosomes.

Early Events in Reovirus Infection Influence Induction of Innate Immune Response.

Mammalian orthoreovirus (reovirus) is a double-stranded RNA (dsRNA) 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 endosomal proteases are required even late in infection, when disassembly is complete, to induce an immune response to reovirus. Additionally, counter to dogma, our data demonstrate that 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. IMPORTANCE Viruses 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 that 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.

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-κB, a key transcriptional regulator of the hosts' innate immune response. One mechanism by which reovirus infection results in inhibition of NF-κB is through a diminishment in levels of upstream activators, IKKß and NEMO. Here, we demonstrate a second, distinct mechanism by which reovirus blocks NF-κB. We report that expression of a single viral protein, σ3, is sufficient to inhibit expression of NF-κB target genes. Further, σ3-mediated blockade of NF-κB occurs without changes to IκB kinase (IKK) levels or activity. Among NF-κB targets, the expression of type I interferon is significantly diminished by σ3 expression. Expression of NF-κB 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 σ3. This work identifies a new role for reovirus σ3 as a viral antagonist of NF-κB-dependent antiviral pathways. IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that a single protein, σ3, produced by mammalian reovirus, impairs the function of NF-κB. We demonstrate that by blocking NF-κB, σ3 diminishes the hosts' response to infection to promote viral replication. This work identifies a second, previously unknown, mechanism by which reovirus blocks this aspect of the host cell response.

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 µ1, 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 nonlethal in wild-type mice, whereas more 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, as well as 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 than T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a nonlethal 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. IMPORTANCE Reovirus is a nonenveloped 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 nonlethal 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.

Curriculum Guidelines for Graduate and Undergraduate Virology Courses.

Curriculum guidelines for virology are needed to best guide student learning due to the continuous and ever-increasing volume of virology information, the need to ensure that undergraduate and graduate students have a foundational understanding of key virology concepts, and the importance in being able to communicate that understanding to both other virologists and nonvirologists. Such guidelines, developed by virology educators and the American Society for Virology Education and Career Development Committee, are described herein.

ICTV Virus Taxonomy Profile: Spinareoviridae 2022.

Spinareoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (9-12 linear segments) dsRNA genomes of 23-29 kbp. Spinareovirids have a broad host range, infecting animals, fungi and plants. Some have important pathogenic potential for humans (e.g. Colorado tick fever virus), livestock (e.g. avian orthoreoviruses), fish (e.g. aquareoviruses) and plants (e.g. rice ragged stunt virus and rice black streaked dwarf virus). This is a summary of the ICTV Report on the family Spinareoviridae, which is available at ictv.global/report/spinareoviridae.

ICTV Virus Taxonomy Profile: Sedoreoviridae 2022.

Sedoreoviridae is a large family of icosahedral viruses that are usually regarded as non-enveloped with segmented (10-12 linear segments) dsRNA genomes of 18-26 kbp. Sedoreovirids have a broad host range, infecting mammals, birds, crustaceans, arthropods, algae and plants. Some of them have important pathogenic potential for humans (e.g. rotavirus A), livestock (e.g. bluetongue virus) and plants (e.g. rice dwarf virus). This is a summary of the ICTV Report on the family Sedoreoviridae, which is available at ictv.global/report/sedoreoviridae.

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-κB), interferon regulatory factor (IRF), and the signal transducer and activator of transcription proteins (STAT) family of transcription factors. Such a strategy allows viruses to evade immune surveillance and maximize their replication. Using an unbiased transcriptome sequencing (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-κB family of transcription factors is lower in infected cells. Potent agonists of NF-κB such as tumor necrosis factor alpha (TNF-α) and vgRNA failed to induce NF-κB-dependent gene expression in infected cells. We demonstrate that NF-κB signaling is blocked due to loss of critical members of the inhibitor of kappa B kinase (IKK) complex, NF-κB essential modifier (NEMO), and IKKß. The loss of the IKK complex components prevents nuclear translocation and phosphorylation of NF-κB, thereby preventing gene expression. Our study demonstrates that reovirus infection selectively blocks NF-κB, likely to counteract its antiviral effects and promote efficient viral replication.IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-κB 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-κB is inactive. Further, we demonstrate that NF-κB is rendered inactive because virus infection results in reduced levels of upstream intermediaries (called IKKs) that are needed for NF-κB function. Based on previous evidence that active NF-κB limits reovirus infection, we conclude that inactivating NF-κB is a viral strategy to produce a cellular environment that is favorable for virus replication.

Reovirus Core Proteins λ1 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 composed of µ1-σ3 heterohexamers which surround the core. The core is composed of λ1 decamers held in place by σ2. After entry into the endosome, σ3 is proteolytically degraded and µ1 is cleaved and exposed to form infectious subvirion particles (ISVPs). ISVPs undergo further conformational changes to form ISVP*s, resulting in the release of µ1 peptides, which facilitate the penetration of the endosomal membrane to release transcriptionally active core particles into the cytoplasm. Previous work identified regions or specific residues within reovirus outer capsid proteins that impact the efficiency of cell entry. We examined the functions of the core proteins λ1 and σ2. We generated a reovirus T3D reassortant that carries strain T1L-derived σ2 and λ1 proteins (T3D/T1L L3S2). This virus displays lower ISVP stability and therefore converts to ISVP*s more readily. To identify the molecular basis for lability of T3D/T1L L3S2, we screened for hyperstable mutants of T3D/T1L L3S2 and identified three point mutations in µ1 that stabilize ISVPs. Two of these mutations are located in the C-terminal ϕ region of µ1, which has not previously been implicated in controlling ISVP stability. Independent of compromised ISVP stability, we also found that T3D/T1L L3S2 launches replication more efficiently and produces higher yields in infected cells than T3D. In addition to identifying a new role for the core proteins in disassembly events, these data highlight the possibility that core proteins may influence multiple stages of infection.IMPORTANCE Protein 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. In comparison to the parental T3D strain, 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 the 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.

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 double-stranded RNA (dsRNA). The reovirus outer capsid protein µ1 negatively regulates reovirus-induced necroptosis by limiting RNA synthesis. To determine if the outer capsid protein σ3, which interacts with µ1, also functions in regulating necroptosis, we used small interfering RNA (siRNA)-mediated knockdown. Similarly to what was observed in diminishment of µ1 expression, knockdown of newly synthesized σ3 enhances necroptosis. Knockdown of σ3 does not impact reovirus RNA synthesis. Instead, this increase in necroptosis following σ3 knockdown is accompanied by an increase in IFN production. Furthermore, ectopic expression of σ3 is sufficient to block IFN expression following infection. Surprisingly, the capacity of σ3 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 σ3 does not result in enhanced production of IFN or necroptosis. Together, these data suggest that σ3 limits the production of IFN to control innate immune signaling and necroptosis following infection through a mechanism that is independent of its dsRNA binding capacity.IMPORTANCE We 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 σ3 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires production of interferon. The σ3 protein limits the induction of necroptosis by preventing excessive production of interferon following infection.

Cell Killing by Reovirus: Mechanisms and Consequences.

Infection of host cells by mammalian reovirus in culture or in tissues of infected animals results in cell death. Cell death of infected neurons and myocytes contributes to the pathogenesis of reovirus-induced encephalitis and myocarditis in a newborn mouse model. Thus, reovirus-induced cell death has been used to investigate the basis of viral disease. Depending on the cell type, infection of host cells by reovirus results in one of two forms of cell death-apoptosis and necroptosis. In addition to the obvious differences in how these two forms of cell death are executed, the mechanisms by which reovirus infection initiates and transduces signals that lead to each of these types of cell death are distinct. In this review, we discuss how apoptosis and necroptosis are triggered by events at different stages of infection. We also describe how innate immune recognition of reovirus genomic material and type I interferon signaling pathways connect with the core components of the apoptosis and necroptosis machinery. The impact of different cell death mediators on viral pathogenesis and the potential of reovirus as an oncolytic vector are also outlined.

Cell Entry-Independent Role for the Reovirus µ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products.

The reovirus outer capsid protein µ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized µ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized µ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of µ1 also affects aspects of viral replication. We found that, while µ1 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 µ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when µ1 is knocked down. Following µ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of µ1 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 µ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCE We used 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 the reovirus outer capsid protein µ1 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; µ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

Selection and Characterization of a Reovirus Mutant with Increased Thermostability.

The environment represents a significant barrier to infection. Physical stressors (heat) or chemical agents (ethanol) 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) σ3 is degraded to generate the infectious subviral particle, and (ii) µ1 facilitates membrane penetration and subsequent core delivery. µ1-σ3 interactions also prevent inactivation; however, this activity is not fully characterized. Using forward and reverse genetic approaches, we identified two mutations (µ1 M258I and σ3 S344P) within heat-resistant strains. σ3 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.IMPORTANCE Nonenveloped 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, these findings show capsid function is balanced for optimal replication and for spread to a new host.

Components of the Reovirus Capsid Differentially Contribute to Stability.

The mammalian orthoreovirus (reovirus) outer capsid is composed of 200 µ1-σ3 heterohexamers and a maximum of 12 σ1 trimers. During cell entry, σ3 is degraded by luminal or intracellular proteases to generate the infectious subviral particle (ISVP). When ISVP formation is prevented, reovirus fails to establish a productive infection, suggesting proteolytic priming is required for entry. ISVPs are then converted to ISVP*s, which is accompanied by µ1 rearrangements. The µ1 and σ3 proteins confer resistance to inactivating agents; however, neither the impact on capsid properties nor the mechanism (or basis) of inactivation is fully understood. Here, we utilized T1L/T3D M2 and T3D/T1L S4 to investigate the determinants of reovirus stability. Both reassortants encode mismatched subunits. When µ1-σ3 were derived from different strains, virions resembled wild-type particles in structure and protease sensitivity. T1L/T3D M2 and T3D/T1L S4 ISVPs were less thermostable than wild-type ISVPs. In contrast, virions were equally susceptible to heating. Virion associated µ1 adopted an ISVP*-like conformation concurrent with inactivation; σ3 preserves infectivity by preventing µ1 rearrangements. Moreover, thermostability was enhanced by a hyperstable variant of µ1. Unlike the outer capsid, the inner capsid (core) was highly resistant to elevated temperatures. The dual layered architecture allowed for differential sensitivity to inactivating agents.IMPORTANCE Nonenveloped and enveloped viruses are exposed to the environment during transmission to a new host. Protein-protein and/or protein-lipid interactions stabilize the particle and protect the viral genome. Mammalian orthoreovirus (reovirus) is composed of two concentric, protein shells. The µ1 and σ3 proteins form the outer capsid; contacts between neighboring subunits are thought to confer resistance to inactivating agents. We further investigated the determinants of reovirus stability. The outer capsid was disrupted concurrent with the loss of infectivity; virion associated µ1 rearranged into an altered conformation. Heat sensitivity was controlled by σ3; however, particle integrity was enhanced by a single µ1 mutation. In contrast, the inner capsid (core) displayed superior resistance to heating. These findings reveal structural components that differentially contribute to reovirus stability.

Cleavage of the C-Terminal Fragment of Reovirus µ1 Is Required for Optimal Infectivity.

The mammalian orthoreovirus (reovirus) outer capsid, which is composed of 200 µ1/σ3 heterohexamers and a maximum of 12 σ1 trimers, contains all of the proteins that are necessary for attaching to and entering host cells. Following attachment, reovirus is internalized by receptor-mediated endocytosis and acid-dependent cathepsin proteases degrade the σ3 protein. This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the µ1 membrane penetration protein is exposed. ISVPs undergo a second structural rearrangement to deposit the genome-containing core into the host cytoplasm. The conformationally altered particle is called ISVP*. ISVP-to-ISVP* conversion culminates in the release of µ1 N- and C-terminal fragments, µ1N and Φ, respectively. Released µ1N is thought to facilitate core delivery by generating size-selective pores within the endosomal membrane, whereas the precise role of Φ, particularly in the context of viral entry, is undefined. In this report, we characterize a recombinant reovirus that fails to cleave Φ from µ1 in vitro Φ cleavage, which is not required for ISVP-to-ISVP* conversion, enhances the disruption of liposomal membranes and facilitates the recruitment of ISVP*s to the site of pore formation. Moreover, the Φ cleavage-deficient strain initiates infection of host cells less efficiently than the parental strain. These results indicate that µ1N and Φ contribute to reovirus pore forming activity.IMPORTANCE Host membranes represent a physical barrier that prevents infection. To overcome this barrier, viruses utilize diverse strategies, such as membrane fusion or membrane disruption, to access internal components of the cell. These strategies are characterized by discrete protein-protein and protein-lipid interactions. The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of well-defined conformational changes, which conclude with pore formation and delivery of the viral genetic material. In this report, we characterize the role of the small, reovirus-derived Φ peptide in pore formation. Φ cleavage from the outer capsid enhances membrane disruption and facilitates the recruitment of virions to membrane-associated pores. Moreover, Φ cleavage promotes the initiation of infection. Together, these results reveal an additional component of the reovirus pore forming apparatus and highlight a strategy for penetrating host membranes.

Protein Mismatches Caused by Reassortment Influence Functions of the Reovirus Capsid.

Following attachment to host receptors via σ1, reovirus particles are endocytosed and disassembled to generate infectious subvirion particles (ISVPs). ISVPs undergo conformational changes to form ISVP*, releasing σ1 and membrane-targeting peptides from the viral µ1 protein. ISVP* formation is required for delivery of the viral core into the cytoplasm for replication. We characterized the properties of T3DF/T3DCS1, an S1 gene monoreassortant between two laboratory isolates of prototype reovirus strain T3D: T3DF and T3DC T3DF/T3DCS1 is poorly infectious. This deficiency is a consequence of inefficient encapsidation of S1-encoded σ1 on T3DF/T3DCS1 virions. Additionally, compared to T3DF, T3DF/T3DCS1 undergoes ISVP-to-ISVP* conversion more readily, revealing an unexpected role for σ1 in regulating ISVP* formation. The σ1 protein is held within turrets formed by the λ2 protein. To test if the altered properties of T3DF/T3DCS1 are due to a mismatch between σ1 and λ2 proteins from T3DF and T3DC, properties of T3DF/T3DCL2 and T3DF/T3DCS1L2, which express a T3DC-derived λ2, were compared. The presence of T3DC λ2 allowed more efficient σ1 incorporation, producing particles that exhibit T3DF-like infectivity. Compared to T3DF, T3DF/T3DCL2 prematurely converts to ISVP*, uncovering a role for λ2 in regulating ISVP* formation. Importantly, a virus with matching σ1 and λ2 displayed a more regulated conversion to ISVP* than either T3DF/T3DCS1 or T3DF/T3DCL2. In addition to identifying new regulators of ISVP* formation, our results highlight that protein mismatches produced by reassortment can alter virus assembly and thereby influence subsequent functions of the virus capsid.IMPORTANCE Cells coinfected with viruses that possess a multipartite or segmented genome reassort to produce progeny viruses that contain a combination of gene segments from each parent. Reassortment places new pairs of genes together, generating viruses in which mismatched proteins must function together. To test if such forced pairing of proteins that form the virus shell or capsid alters the function of the particle, we investigated properties of reovirus variants in which the σ1 attachment protein and the λ2 protein that anchors σ1 on the particle are mismatched. Our studies demonstrate that a σ1-λ2 mismatch produces particles with lower levels of encapsidated σ1, consequently decreasing virus attachment and infectivity. The mismatch between σ1 and λ2 also altered the capacity of the viral capsid to undergo conformational changes required for cell entry. These studies reveal new functions of reovirus capsid proteins and illuminate both predictable and novel implications of reassortment.

The Loop Formed by Residues 340 to 343 of Reovirus µ1 Controls Entry-Related Conformational Changes.

Reovirus particles are covered with 200 µ1/σ3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of stepwise disassembly events. First, the σ3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the µ1 protein rearranges into a protease-sensitive conformation to generate ISVP*s and releases two virus-encoded peptides, µ1N and Φ. The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by µ1 intratrimer, intertrimer, and trimer-core interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring µ1 trimers unwind and separate. Located within the µ1 jelly roll ß barrel domain, which is a known regulator of ISVP* formation, residues 340 to 343 form a loop and have been proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (Δ341 and Δ342). Both deletions destabilized the outer capsid. Notably, Δ342 impaired the viral life cycle; however, replicative fitness was restored by an additional change (V403A) within the µ1 jelly roll ß barrel domain. In the Δ341 and Δ342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also sufficiently flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.

Viral RNA at Two Stages of Reovirus Infection Is Required for the Induction of Necroptosis.

Necroptosis, a regulated form of necrotic cell death, requires the activation of the RIP3 kinase. Here, we identify that infection of host cells with reovirus can result in necroptosis. We find that necroptosis requires sensing of the genomic RNA within incoming virus particles via cytoplasmic RNA sensors to produce type I interferon (IFN). While these events that occur prior to the de novo synthesis of viral RNA are required for the induction of necroptosis, they are not sufficient. The induction of necroptosis also requires late stages of reovirus infection. Specifically, efficient synthesis of double-stranded RNA (dsRNA) within infected cells is required for necroptosis. These data indicate that viral RNA interfaces with host components at two different stages of infection to induce necroptosis. This work provides new molecular details about events in the viral replication cycle that contribute to the induction of necroptosis following infection with an RNA virus.IMPORTANCE An appreciation of how cell death pathways are regulated following viral infection may reveal strategies to limit tissue destruction and prevent the onset of disease. Cell death following virus infection can occur by apoptosis or a regulated form of necrosis known as necroptosis. Apoptotic cells are typically disposed of without activating the immune system. In contrast, necroptotic cells alert the immune system, resulting in inflammation and tissue damage. While apoptosis following virus infection has been extensively investigated, how necroptosis is unleashed following virus infection is understood for only a small group of viruses. Here, using mammalian reovirus, we highlight the molecular mechanism by which infection with a dsRNA virus results in necroptosis.