Reovirus infection induces transcriptome-wide unique A-to-I editing changes in the murine fibroblasts.

The conversion of Adenosine (A) to Inosine (I), by Adenosine Deaminases Acting on RNA or ADARs, is an essential post-transcriptional modification that contributes to proteome diversity and regulation in metazoans including humans. In addition to its transcriptome-regulating role, ADARs also play a major part in immune response to viral infection, where an interferon response activates interferon-stimulated genes, such as ADARp150, in turn dynamically regulating host-virus interactions. A previous report has shown that infection from reoviruses, despite strong activation of ADARp150, does not influence the editing of some of the major known editing targets, while likely editing others, suggesting a potentially nuanced editing pattern that may depend on different factors. However, the results were based on a handful of selected editing sites and did not cover the entire transcriptome. Thus, to determine whether and how reovirus infection specifically affects host ADAR editing patterns, we analyzed a publicly available deep-sequenced RNA-seq dataset, from murine fibroblasts infected with wild-type and mutant reovirus strains that allowed us to examine changes in editing patterns on a transcriptome-wide scale. To the best of our knowledge, this is the first transcriptome-wide report on host editing changes after reovirus infection. Our results demonstrate that reovirus infection induces unique nuanced editing changes in the host, including introducing sites uniquely edited in infected samples. Genes with edited sites are overrepresented in pathways related to immune regulation, cellular signaling, metabolism, and growth. Moreover, a shift in editing targets has also been observed, where the same genes are edited in infection and control conditions but at different sites, or where the editing rate is increased for some and decreased for other differential targets, supporting the hypothesis of dynamic and condition-specific editing by ADARs.

Nonstructural protein NS17 of grass carp reovirus Honghu strain promotes virus infection by mediating cell-cell fusion and apoptosis.

Fusion-associated small transmembrane (FAST) proteins can promote cell fusion, alter membrane permeability and trigger apoptosis to promote virus proliferation in orthoreoviruses. However, it is unknown whether FAST proteins perform these functions in aquareoviruses (AqRVs). Non-structural protein 17 (NS17) carried by grass carp reovirus Honghu strain (GCRV-HH196) belongs to the FAST protein family, and we preliminarily explored its relevance to virus infection. NS17 has similar domains to FAST protein NS16 of GCRV-873, comprising a transmembrane domain, a polybasic cluster, a hydrophobic patch and a polyproline motif. It was observed in the cytoplasm and the cell membrane. Overexpression of NS17 enhanced the efficiency of cell-cell fusion induced by GCRV-HH196 and promoted virus replication. Overexpression of NS17 also led to DNA fragmentation and reactive oxygen species (ROS) accumulation, and it triggered apoptosis. The findings illuminate the functions of NS17 in GCRV infection, and provide a reference for the development of novel antiviral strategies.

Oncolytic Reoviruses: Can These Emerging Zoonotic Reoviruses Be Tamed and Utilized?

Orthoreovirus is a nonenveloped double-stranded RNA virus under the Reoviridae family. This group of viruses, especially mammalian orthoreovirus (MRV), are reported with great therapeutic values due to their oncolytic effects. In this review, the life cycle and oncolytic effect of MRV and a few emerging reoviruses were summarized. This article also highlights the challenges and strategies of utilizing MRV and the emerging reoviruses, avian orthoreovirus (ARV) and pteropine orthoreovirus (PRV), as oncolytic viruses (OVs). Besides, the emergence of potential ARV and PRV as OVs were discussed in comparison to MRV. Finally, the risk of reovirus as zoonosis or reverse zoonosis (zooanthroponosis) were debated, and concerns were raised in this article, which warrant continue surveillance of reovirus (MRV, ARV, and PRV) in animals, humans, and the environment.

The µ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network.

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

Development of an oncolytic mammalian orthoreovirus expressing the near-infrared fluorescent protein iRFP720.

Fluorescence-guided surgery (FGS) is a useful method for removing invasive tumor tissues. For this, near-infrared (NIR) fluorescence probes are suitable for visualizing cancer cells due to their low autofluorescence, and an oncolytic mammalian orthoreovirus (MRV) expressing an NIR fluorescent protein is expected to be a novel tool for FGS. In this study, we identified the optimal insertion site of the NIR fluorescent protein gene iRFP720 (915 nt) in the MRV genome. We constructed genome plasmids for the L1, M1, and S2 segments, where a gene cassette comprising iRFP720 and T2A self-cleaving peptide was inserted in the 5' or 3' region of each segment. Through virus recovery, the recombinant MRV with the gene cassette at the M1 segment's 3' end, T3D-L(M1/3'iRFP720), was capable of replication and passaging with bright NIR fluorescence. However, the replication of T3D-L(M1/3'iRFP720) was approximately 1,000-fold lower than that of the wild-type virus. T3D-L(M1/3'iRFP720) production improved due to the transfection of a fusion-associated small transmembrane protein gene of fusogenic reovirus. Further, fluorescence signals were detected in T3D-L(M1/3'iRFP720)-infected human gastric and pancreatic cancer cells. Thus, the M1 segment's 3' end tolerates the expression of the long iRFP720 gene, which may propel the development of recombinant MRV vectors for FGS.

Gastrointestinal cancer-associated fibroblasts expressing Junctional Adhesion Molecule-A are amenable to infection by oncolytic reovirus.

Gastrointestinal (GI) cancers are characterized by extensive tumor stroma that both promotes tumor progression and acts as a physical barrier for adjacent tumor cells, limiting the effect of current treatment modalities. Oncolytic virotherapy is currently investigated in clinical trials as a novel therapeutic agent for different malignancies of the GI tract, but it is largely unknown whether these viruses can also target the tumor stroma. Here, we investigated the tropism of two commonly studied OVs, adenovirus and reovirus, towards primary GI fibroblasts from human oesophageal, gastric, duodenal and pancreatic carcinomas (N = 36). GI fibroblasts were susceptible to type 3 Dearing (T3D) strain R124 and bioselected mutant reovirus (jin-3) infection but not oncolytic adenovirus (Ad5-Δ24). Efficient infection and apoptosis of human and mouse GI cancer-derived fibroblasts by these reoviruses was partially dependent on the expression of the reovirus entry receptor, Junctional Adhesion Molecule-A (JAM-A). Moreover, human GI cancer organoid-fibroblast co-cultures showed higher overall infectivity when containing JAM-A expressing fibroblasts as compared to JAM-A negative fibroblasts, indicating a potential role of JAM-A expressing fibroblasts for viral dissemination. We further show that JAM-A is not only necessary for efficient reovirus infection of fibroblasts but also partially mediates reovirus-induced apoptosis, dependent on signaling through the C-terminal PDZ-domain of JAM-A. Altogether, our data show the presence of JAM-A expressing fibroblasts in both human and murine GI cancers that are amenable to infection and induction of apoptosis by reovirus, extending the potential anti-cancer actions of reovirus with stromal targeting.

Reovirus µ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV's ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the µ2 protein, is the primary determinant of MRV's ability to alter AS, and that the amino acid at position 208 in µ2 is critical to induce these changes. Moreover, we showed that the expression of µ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of µ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and µ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS.

RNA Packaging in the Cystovirus Bacteriophages: Dynamic Interactions during Capsid Maturation.

The bacteriophage family Cystoviridae consists of a single genus, Cystovirus, that is lipid-containing with three double-stranded RNA (ds-RNA) genome segments. With regard to the segmented dsRNA genome, they resemble the family Reoviridae. Therefore, the Cystoviruses have long served as a simple model for reovirus assembly. This review focuses on important developments in the study of the RNA packaging and replication mechanisms, emphasizing the structural conformations and dynamic changes during maturation of the five proteins required for viral RNA synthesis, P1, P2, P4, P7, and P8. Together these proteins constitute the procapsid/polymerase complex (PC) and nucleocapsid (NC) of the Cystoviruses. During viral assembly and RNA packaging, the five proteins must function in a coordinated fashion as the PC and NC undergo expansion with significant position translation. The review emphasizes this facet of the viral assembly process and speculates on areas suggestive of additional research efforts.

Evidence for a non-fusogenic aquareovirus encoding a transmembrane protein.

Fusogenic aquareoviruses can induce host cell-cell fusion, forming syncytia via a fusion-associated transmembrane protein. However, there have been very few reports on non-fusogenic aquareoviruses encoding a membrane-associated protein. Previously, sequence-based analysis has indicated that grass carp reovirus strain 104 (GCRV-104), a non-fusogenic aquareovirus, encodes the proteins VP8 (nt 36-263) and VP15 (nt 400-822) in its genome segment S11. Here, we employed a liquid chromatography-tandem mass spectrometry assay to experimentally annotate small coding genes in the GCRV-104 genome and confirmed that segment S11 indeed functions as bicistronic mRNA. Notably, some additional polypeptides were identified that are encoded upstream of the VP15 open reading frame (ORF), which suggests that the virus uses a novel ORF with a non-AUG initiator codon, tentatively named VP15L (nt 274-822), which is longer than the previous putative VP15 ORF. Furthermore, a transmembrane domain was identified at the N-terminus of VP15L, but its function is unclear. Thus, the aquareovirus GCRV-104 potentially encodes a transmembrane protein, which opens a new perspective on the properties of viral proteins and the pathogenesis of this non-fusogenic reovirus.

Multiple conformations of trimeric spikes visualized on a non-enveloped virus.

Many viruses utilize trimeric spikes to gain entry into host cells. However, without in situ structures of these trimeric spikes, a full understanding of this dynamic and essential process of viral infections is not possible. Here we present four in situ and one isolated cryoEM structures of the trimeric spike of the cytoplasmic polyhedrosis virus, a member of the non-enveloped Reoviridae family and a virus historically used as a model in the discoveries of RNA transcription and capping. These structures adopt two drastically different conformations, closed spike and opened spike, which respectively represent the penetration-inactive and penetration-active states. Each spike monomer has four domains: N-terminal, body, claw, and C-terminal. From closed to opened state, the RGD motif-containing C-terminal domain is freed to bind integrins, and the claw domain rotates to expose and project its membrane insertion loops into the cellular membrane. Comparison between turret vertices before and after detachment of the trimeric spike shows that the trimeric spike anchors its N-terminal domain in the iris of the pentameric RNA-capping turret. Sensing of cytosolic S-adenosylmethionine (SAM) and adenosine triphosphate (ATP) by the turret triggers a cascade of events: opening of the iris, detachment of the spike, and initiation of endogenous transcription.

Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

Genomic characterization of viruses associated with the parasitoid Anagyrus vladimiri (Hymenoptera: Encyrtidae).

Knowledge on symbiotic microorganisms of insects has increased dramatically in recent years, yet relatively little data are available regarding non-pathogenic viruses. Here we studied the virome of the parasitoid wasp Anagyrus vladimiri Triapitsyn (Hymenoptera: Encyrtidae), a biocontrol agent of mealybugs. By high-throughput sequencing of viral nucleic acids, we revealed three novel viruses, belonging to the families Reoviridae [provisionally termed AnvRV (Anagyrus vladimiri reovirus)], Iflaviridae (AnvIFV) and Dicistroviridae (AnvDV). Phylogenetic analysis further classified AnvRV in the genus Idnoreovirus, and AnvDV in the genus Triatovirus. The genome of AnvRV comprises 10 distinct genomic segments ranging in length from 1.5 to 4.2 kb, but only two out of the 10 ORFs have a known function. AnvIFV and AnvDV each have one polypeptide ORF, which is typical of iflaviruses but very un-common among dicistroviruses. Five conserved domains were found along both the ORFs of those two viruses. AnvRV was found to be fixed in an A. vladimiri population that was obtained from a mass rearing facility, whereas its prevalence in field-collected A. vladimiri was ~15 %. Similarly, the prevalence of AnvIFV and AnvDV was much higher in the mass rearing population than in the field population. The presence of AnvDV was positively correlated with the presence of Wolbachia in the same individuals. Transmission electron micrographs of females' ovaries revealed clusters and viroplasms of reovirus-like particles in follicle cells, suggesting that AnvRV is vertically transmitted from mother to offspring. AnvRV was not detected in the mealybugs, supporting the assumption that this virus is truly associated with the wasps. The possible effects of these viruses on A. vladimiri's biology, and on biocontrol agents in general, are discussed. Our findings identify RNA viruses as potentially involved in the multitrophic system of mealybugs, their parasitoids and other members of the holobiont.

Mesenchymal stem cells support delivery and boost the efficacy of oncolytic reoviruses in TC-1 tumor cells.

Cancer has remained a major health problem around the world. Mesenchymal stem cells (MSCs)-based therapy exhibits a therapeutic effect via different mechanisms. By using MSCs as carrier cells, the major problem of clearance of oncolytic viruses is resolved by neutralizing antibodies before they react with cancer cells. The aim of this study was to characterize the effect of infected MSCs by reovirus type-3 Dearing (T3D) for in vitro cancer therapy. Adipose-derived MSCs (AD-MSCs) were infected with reovirus T3D and its biological properties were evaluated. Then, the effects of reovirus-infected AD-MSCs on cytokine profile, nitric oxide (NO) production, and apoptosis induction in TC-1 cells were assessed. Our results indicated that the differentiation potential of AD-MSCs was affected by reovirus. However, phenotypes were not affected after infection. Then, the effects of reovirus-infected AD-MSCs in TC-1 cells showed an increased amount of tumor necrosis factor-alpha (TNF-α) and NO production and a decreased amount of transforming growth factor-beta 1 (TGF-ß1) and interleukin-10 (IL-10). Moreover, apoptosis significantly increased via coculturing of TC-1 cells with infected AD-MSCs, compared with control, and both internal and external apoptosis pathways are activated in experimental groups. In conclusion, the data showed that with increasing TNF-α and NO production and reducing IL-10 and TGF-ß production, AD-MSCs can enhance the oncolytic effect of reovirus in cancer cells. Furthermore, the results suggested that AD-MSCs can be used as effective carrier cells candidate for reovirus T3D to maximize their anticancer cell activity.

Oncolytic reovirus induces ovarian cancer cell apoptosis in a TLR3-dependent manner.

Globally, ovarian cancer is the seventh most common cancer and the eighth-most common cause of cancer death among women with a five-year survival rate of less than 45%. Although reovirus is known to be effective for treating ovarian cancer, some types of tumor cells still exhibit resistance to reovirus. In order to solve this resistance problem in the treatment of ovarian cancer, we selected the reovirus-resistant OV-90 ovarian cancer cells to study reovirus oncolytic effects. We found that the viability of OV-90 cells decreased after reovirus double-stranded RNA (dsRNA) genome transfection. Interestingly, we observed that chemical blockage of the Toll-like receptor 3 (TLR3)-dsRNA binding complex in OV-90 cells and the inhibition of downstream TLR3 signaling disrupted OV-90 apoptosis triggered by reovirus dsRNA. Together, these results demonstrate that reovirus dsRNA induces reovirus-resistant tumor cell apoptosis through the TLR3 signaling pathway.

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Cytidine Monophosphate N-Acetylneuraminic Acid Synthetase and Solute Carrier Family 35 Member A1 Are Required for Reovirus Binding and Infection.

Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.

Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells.

Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.

Reovirus σ1 Conformational Flexibility Modulates the Efficiency of Host Cell Attachment.

Reovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can cross-link σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that cross-link the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride, which blocks virion-to-ISVP conversion. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment.IMPORTANCE Nonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that cross-link σ1 monomers in nonreducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that cross-linking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell binding.

Noncanonical Cell Death Induction by Reassortant Reovirus.

Triple-negative breast cancer (TNBC) constitutes 10 to 15% of all breast cancer and is associated with worse prognosis than other subtypes of breast cancer. Current therapies are limited to cytotoxic chemotherapy, radiation, and surgery, leaving a need for targeted therapeutics to improve outcomes for TNBC patients. Mammalian orthoreovirus (reovirus) is a nonenveloped, segmented, double-stranded RNA virus in the Reoviridae family. Reovirus preferentially kills transformed cells and is in clinical trials to assess its efficacy against several types of cancer. We previously engineered a reassortant reovirus, r2Reovirus, that infects TNBC cells more efficiently and induces cell death with faster kinetics than parental reoviruses. In this study, we sought to understand the mechanisms by which r2Reovirus induces cell death in TNBC cells. We show that r2Reovirus infection of TNBC cells of a mesenchymal stem-like (MSL) lineage downregulates the mitogen-activated protein kinase/extracellular signal-related kinase pathway and induces nonconventional cell death that is caspase-dependent but caspase 3-independent. Infection of different MSL lineage TNBC cells with r2Reovirus results in caspase 3-dependent cell death. We map the enhanced oncolytic properties of r2Reovirus in TNBC to epistatic interactions between the type 3 Dearing M2 gene segment and type 1 Lang genes. These findings suggest that the genetic composition of the host cell impacts the mechanism of reovirus-induced cell death in TNBC. Together, our data show that understanding host and virus determinants of cell death can identify novel properties and interactions between host and viral gene products that can be exploited for the development of improved viral oncolytics.IMPORTANCE TNBC is unresponsive to hormone therapies, leaving patients afflicted with this disease with limited treatment options. We previously engineered an oncolytic reovirus (r2Reovirus) with enhanced infective and cytotoxic properties in TNBC cells. However, how r2Reovirus promotes TNBC cell death is not known. In this study, we show that reassortant r2Reovirus can promote nonconventional caspase-dependent but caspase 3-independent cell death and that the mechanism of cell death depends on the genetic composition of the host cell. We also map the enhanced oncolytic properties of r2Reovirus in TNBC to interactions between a type 3 M2 gene segment and type 1 genes. Our data show that understanding the interplay between the host cell environment and the genetic composition of oncolytic viruses is crucial for the development of efficacious viral oncolytics.

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.