Mammalian orthoreovirus can exit cells in extracellular vesicles.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

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.

p38 Mitogen-Activated Protein Kinase Signaling Enhances Reovirus Replication by Facilitating Efficient Virus Entry, Capsid Uncoating, and Postuncoating Steps.

Mammalian orthoreovirus serotype 3 Dearing is an oncolytic virus currently undergoing multiple clinical trials as a potential cancer therapy. Previous clinical trials have emphasized the importance of prescreening patients for prognostic markers to improve therapeutic success. However, only generic cancer markers such as epidermal growth factor receptor (EGFR), Hras, Kras, Nras, Braf, and p53 are currently utilized, with limited benefit in predicting therapeutic efficacy. This study aimed to investigate the role of p38 mitogen-activated protein kinase (MAPK) signaling during reovirus infection. Using a panel of specific p38 MAPK inhibitors and an inactive inhibitor analogue, p38 MAPK signaling was found to be essential for establishment of reovirus infection by enhancing reovirus endocytosis, facilitating efficient reovirus uncoating at the endo-lysosomal stage, and augmenting postuncoating replication steps. Using a broad panel of human breast cancer cell lines, susceptibility to reovirus infection corresponded with virus binding and uncoating efficiency, which strongly correlated with status of the p38ß isoform. Together, results suggest p38ß isoform as a potential prognostic marker for early stages of reovirus infection that are crucial to successful reovirus infection. IMPORTANCE The use of Pelareorep (mammalian orthoreovirus) as a therapy for metastatic breast cancer has shown promising results in recent clinical trials. However, the selection of prognostic markers to stratify patients has had limited success due to the fact that these markers are upstream receptors and signaling pathways that are present in a high percentage of cancers. This study demonstrates that the mechanism of action of p38 MAPK signaling plays a key role in establishment of reovirus infection at both early entry and late replication steps. Using a panel of breast cancer cell lines, we found that the expression levels of the MAPK11 (p38ß) isoform are a strong determinant of reovirus uncoating and infection establishment. Our findings suggest that selecting prognostic markers that target key steps in reovirus replication may improve patient stratification during oncolytic reovirus therapy.

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.

Reovirus Type 3 Dearing Variants Do Not Induce Necroptosis in RIPK3-Expressing Human Tumor Cell Lines.

Reoviruses are used as oncolytic viruses to destroy tumor cells. The concomitant induction of anti-tumor immune responses enhances the efficacy of therapy in tumors with low amounts of immune infiltrates before treatment. The reoviruses should provoke immunogenic cell death (ICD) to stimulate a tumor cell-directed immune response. Necroptosis is considered a major form of ICD, and involves receptor-interacting protein kinase 1 (RIPK1), RIPK3 and phosphorylation of mixed-lineage kinase domain-like protein (MLKL). This leads to cell membrane disintegration and the release of damage-associated molecular patterns that can activate immune responses. Reovirus Type 3 Dearing (T3D) can induce necroptosis in mouse L929 fibroblast cells and mouse embryonic fibroblasts. Most human tumor cell lines have a defect in RIPK3 expression and consequently fail to induce necroptosis as measured by MLKL phosphorylation. We used the human colorectal adenocarcinoma HT29 cell line as a model to study necroptosis in human cells since this cell line has frequently been described in necroptosis-related studies. To stimulate MLKL phosphorylation and induce necroptosis, HT29 cells were treated with a cocktail consisting of TNFα, the SMAC mimetic BV6, and the caspase inhibitor Z-VAD-FMK. While this treatment induced necroptosis, three different reovirus T3D variants, i.e., the plasmid-based reverse genetics generated virus (T3DK), the wild-type reovirus T3D isolate R124, and the junction adhesion molecule-A-independent reovirus mutant (jin-1) failed to induce necroptosis in HT29 cells. In contrast, these viruses induced MLKL phosphorylation in murine L929 cells, albeit with varying efficiencies. Our study shows that while reoviruses efficiently induce necroptosis in L929 cells, this is not a common phenotype in human cell lines. This study emphasizes the difficulties of translating the results of ICD studies from murine cells to human cells.

First report on co-isolation and whole-genomic characterisation of mammalian orthorubulavirus 5 and mammalian orthoreovirus type 3 from domestic pigs in India.

During a surveillance study to monitor porcine epidemic diarrohoea virus and transmissible gastroenteritis virus in India, a total of 1043 swine samples including faeces (n = 264) and clotted blood (n = 779) were collected and tested. Five samples (four faecal and one serum) showed cytopathic effects in Vero cells. Transmission electron microscopy of infectious cell supernatant revealed the presence of two types of virions. Next-generation sequencing (de novo) allowed the complete genome sequence of mammalian orthorubulavirus 5 (MRuV5; 15246 bp) and that of all 10 gene segments of mammalian orthoreovirus to be determined. Genetic analysis of MRuV5 revealed grouping of the Indian MRuV5 with isolates from various mammalian species in South Korea and China, sharing more than 99% nucleotide sequence identity. The deduced amino acid sequences of the HN, NP, and F genes of MRuV5 isolates showed three (92L, 111R, 447H), two (86S, 121S), and two (139T, 246T) amino acid substitutions, respectively, compared to previously reported virus strains. Phylogenic analysis based on S1 gene sequences showed the Indian MRV isolates to be clustered in lineage IV of MRV type 3, with the highest nucleotide sequence identity (97.73%) to MRV3 strain ZJ2013, isolated from pigs in China. The protein encoded by the MRV3 S1 gene was found to contain the amino acid residues 198-204NLAIRLP, 249I, 340D, and 419E, which are known to be involved in sialic acid binding and neurotropism. This is the first report of co-isolation and whole-genomic characterisation of MRuV5 and MRV3 in domestic pigs in India. The present study lays a foundation for further surveillance studies and continuous monitoring of the emergence and spread of evolving viruses that might have pathogenic potential in animal and human hosts.

Host-microbiota interactions and responses to grass carp reovirus infection in Ctenopharyngodon idellus.

Gut microbiota could facilitate host to defense diseases, but fish-microbiota interactions during viral infection and the underlying mechanism are poorly understood. We examined interactions and responses of gut microbiota to grass carp reovirus (GCRV) infection in Ctenopharyngodon idellus, which is the most important aquaculture fish worldwide. We found that GCRV infection group with serious haemorrhagic symptoms (G7s) showed considerably different gut microbiota, especially with an abnormally high abundance of gram-negative anaerobic Cetobacterium somerae. It also showed the lowest (p < 0.05) alpha-diversity but with much higher ecological process of homogenizing dispersal (28.8%), confirming a dysbiosis of the gut microbiota after viral infection. Interestingly, signaling pathways of NOD-like receptors (NLRs), toll-like receptors (TLRs), and lipopolysaccharide (LPS) stimulation genes were significantly (q-value < 0.01) enriched in G7s, which also significantly (p < 0.01) correlated with the core gut microbial genera of Cetobacterium and Acinetobacter. The results suggested that an expansion of C. somerae initiated by GCRV could aggravate host inflammatory reactions through the LPS-related NLRs and TLRs pathways. This study advances our understanding of the interplay between fish immunity and gut microbiota challenged by viruses; it also sheds new insights for ecological defense of fish diseases with the help of gut microbiota.

Polymorphisms in the Most Oncolytic Reovirus Strain Confer Enhanced Cell Attachment, Transcription, and Single-Step Replication Kinetics.

Reovirus serotype 3 Dearing (T3D) replicates preferentially in transformed cells and is in clinical trials as a cancer therapy. Laboratory strains of T3D, however, exhibit differences in plaque size on cancer cells and differences in oncolytic activity in vivo This study aimed to determine why the most oncolytic T3D reovirus lab strain, the Patrick Lee laboratory strain (T3DPL), replicates more efficiently in cancer cells than other commonly used laboratory strains, the Kevin Coombs laboratory strain (T3DKC) and Terence Dermody laboratory (T3DTD) strain. In single-step growth curves, T3DPL titers increased at higher rates and produced ∼9-fold higher burst size. Furthermore, the number of reovirus antigen-positive cells increased more rapidly for T3DPL than for T3DTD In conclusion, the most oncolytic T3DPL possesses replication advantages in a single round of infection. Two specific mechanisms for enhanced infection by T3DPL were identified. First, T3DPL exhibited higher cell attachment, which was attributed to a higher proportion of virus particles with insufficient (≤3) σ1 cell attachment proteins. Second, T3DPL transcribed RNA at rates superior to those of the less oncolytic T3D strains, which is attributed to polymorphisms in M1-encoding µ2 protein, as confirmed in an in vitro transcription assay, and which thus demonstrates that T3DPL has an inherent transcription advantage that is cell type independent. Accordingly, T3DPL established rapid onset of viral RNA and protein synthesis, leading to more rapid kinetics of progeny virus production, larger virus burst size, and higher levels of cell death. Together, these results emphasize the importance of paying close attention to genomic divergence between virus laboratory strains and, mechanistically, reveal the importance of the rapid onset of infection for reovirus oncolysis.IMPORTANCE Reovirus serotype 3 Dearing (T3D) is in clinical trials for cancer therapy. Recently, it was discovered that highly related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a murine model of melanoma. The current study reveals two mechanisms for the enhanced efficiency of T3DPL in cancer cells. Due to polymorphisms in two viral genes, within the first round of reovirus infection, T3DPL binds to cells more efficiency and more rapidly produces viral RNAs; this increased rate of infection relative to that of the less oncolytic strains gives T3DPL a strong inherent advantage that culminates in higher virus production, more cell death, and higher virus spread.

Single Amino Acid Differences between Closely Related Reovirus T3D Lab Strains Alter Oncolytic Potency In Vitro and In Vivo.

Little is known about how genetic variations in viruses affect their success as therapeutic agents. The type 3 Dearing strain of Mammalian orthoreovirus (T3D) is undergoing clinical trials as an oncolytic virotherapy. Worldwide, studies on reovirus oncolysis use T3D stocks propagated in different laboratories. Here, we report that genetic diversification among T3D stocks from various sources extensively impacts oncolytic activity. The T3D strain from the Patrick Lee laboratory strain (TD3PL) showed significantly stronger oncolytic activities in a murine model of melanoma than the strain from the Terence Dermody laboratory (T3DTD). Overall in vitro replication and cytolytic properties of T3D laboratory strains were assessed by measuring virus plaque size on a panel of human and mouse tumor cells, and results were found to correlate with in vivo oncolytic potency in a melanoma model. T3DPL produced larger plaques than T3DTD and than the T3D strain from the ATCC (T3DATCC) and from the Kevin Coombs laboratory (T3DKC). Reassortant and reverse genetics analyses were used to decipher key genes and polymorphisms that govern enhanced plaque size of T3DPL Five single amino acid changes in the S4, M1, and L3 genome segments of reovirus were each partially correlated with plaque size and when combined were able to fully account for differences between T3DPL and T3DTD Moreover, polymorphisms were discovered in T3DTD that promoted virus replication and spread in tumors, and a new T3DPL/T3DTD hybrid was generated with enhanced plaque size compared to that of T3DPL Altogether, single amino acid changes acquired during laboratory virus propagation can have a large impact on reovirus therapeutic potency and warrant consideration as possible confounding variables between studies.IMPORTANCE The reovirus serotype 3 Dearing (T3D) strain is in clinical trials for cancer therapy. We find that closely related laboratory strains of T3D exhibit large differences in their abilities to replicate in cancer cells in vitro, which correlates with oncolytic activity in a in a murine model of melanoma. The study reveals that five single amino acid changes among three reovirus genes strongly impact reovirus therapeutic potency. In general, the findings suggest that attention should be given to genomic divergence of virus strains during research and optimization for cancer therapy.

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.

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.

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.

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection.

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.

[Molecular Mechanisms of Apoptosis of Leukemia Cell Induced by Reovirus].

OBJECTIVE: To investigate the molecular mechanism of apoptosis of HL60 cells induced by oncolytic virus Reovirus type 3 (Reo3). METHODS: HL60 cells were infected with Reo3 at different multiplicity of infection (MOI) with the uninfected HL60 cells as control group. After 48 h of infection, the activity of HL60 cells infected with virus at different MOI was detected by CCK8 method to investigate the influence of MOI to cell activity. Simultaneously, the apoptotic rate of HL60 cells was detected by flow cytometry, and the activation level of double-stranded RNA-dependent protein kinase (PKR) and the expression of apoptotic-related protein in HL60 cells were detected by Western blot. Before infection with Reo3 for 48 h, HL60 cells were treated with 2-aminopurine (2-AP), a specific inhibitor of PKR, for 24 h. Afterward, the apoptotic level and expression of apoptotic related proteins were detected. RESULTS: Activity of HL60 cells was obviously inhibited after infected with Reo3 with a MOI of 1 for 48 h. The cell survival rate was (24.333±3.396)% and the apoptotic rate was (29.96±2.06)%. Both rates were all higher than those in the control group (P < 0.05). Western blot results showed that the expression levels of PKR, p-PKR, Bax, Caspase3 and cleaved Caspase3 in HL60 cells infected with Reo3 were higher than those in the control group (P < 0.05), while the expression level of Bcl-2 was lower (P < 0.05). Compared with the group without inhibitor, the apoptotic rate of HL60 cells pretreated with 2-AP decreased (P < 0.05), the phosphorylation level of PKR and the expression level of apoptotic-related protein also decreased (P < 0.05). CONCLUSION: Oncolytic virus Reo3 could activate PKR in HL60 cells and thus induce apoptosis of HL60 cells.

Characterization of a replicating expanded tropism oncolytic reovirus carrying the adenovirus E4orf4 gene.

While the mammalian orthoreovirus type 3 dearing (reovirus T3D) infects many different tumour cells, various cell lines resist the induction of reovirus-mediated cell death. In an effort to increase the oncolytic potency, we introduced transgenes into the S1 segment of reovirus T3D. The adenovirus E4orf4 gene was selected as transgene since the encoded E4orf4 protein induces cell death in transformed cells. The induction of cell death by E4orf4 depends in part on its binding to phosphatase 2A (PP2A). In addition to the S1-E4orf4 reovirus, two other reoviruses were employed in our studies. The reovirus rS1-RFA encodes an E4orf4 double-mutant protein that cannot interact with PP2A and the rS1-iLOV virus encoding the fluorescent marker iLOV as a reporter. The replacement of the codons for the junction adhesion molecule-A (JAM-A) binding head domain of the truncated spike protein blocks the entry of these recombinant viruses via the reovirus receptor JAM-A. Instead these viruses rely on internalization via binding to sialic acids on the cell surface. This expands their tropism and allows infection of JAM-A-deficient tumour cells. Here we not only demonstrate the feasibility of this approach but also established that the cytolytic activity of these recombinant viruses is largely transgene independent.

A randomized phase II study of weekly paclitaxel with or without pelareorep in patients with metastatic breast cancer: final analysis of Canadian Cancer Trials Group IND.213.

BACKGROUND: Pelareorep, a serotype 3 reovirus, has demonstrated preclinical and early clinical activity in breast cancer and synergistic cytotoxic activity with microtubule targeting agents. This multicentre, randomized, phase II trial was undertaken to evaluate the efficacy and safety of adding pelareorep to paclitaxel for patients with metastatic breast cancer (mBC). METHODS: Following a safety run-in of 7 patients, 74 women with previously treated mBC were randomized either to paclitaxel 80 mg/m2 intravenously on days 1, 8, and 15 every 4 weeks plus pelareorep 3 × 1010 TCID50 intravenously on days 1, 2, 8, 9, 15, and 16 every 4 weeks (Arm A) or to paclitaxel alone (Arm B). Primary endpoint was progression-free survival (PFS). Secondary endpoints were objective response rate, overall survival (OS), circulating tumour cell counts, safety, and exploratory correlative analyses. All comparisons used a two-sided test at an alpha level of 20%. Survival analyses were adjusted for prior paclitaxel. RESULTS: Final analysis was performed after a median follow-up of 29.5 months. Pelareorep was well tolerated. Patients in Arm A had more favourable baseline prognostic variables. Median adjusted PFS (Arm A vs B) was 3.78 mo vs 3.38 mo (HR 1.04, 80% CI 0.76-1.43, P = 0.87). There was no difference in response rate between arms (P = 0.87). Median OS (Arm A vs B) was 17.4 mo vs 10.4 mo (HR 0.65, 80% CI 0.46-0.91, P = 0.1). CONCLUSIONS: This first, phase II, randomized study of pelareorep and paclitaxel in previously treated mBC did not show a difference in PFS (the primary endpoint) or RR. However, there was a significantly longer OS for the combination. Further exploration of this regimen in mBC may be of interest.

NKp46 Recognizes the Sigma1 Protein of Reovirus: Implications for Reovirus-Based Cancer Therapy.

The recent approval of oncolytic virus for therapy of melanoma patients has increased the need for precise evaluation of the mechanisms by which oncolytic viruses affect tumor growth. Here we show that the human NK cell-activating receptor NKp46 and the orthologous mouse protein NCR1 recognize the reovirus sigma1 protein in a sialic-acid-dependent manner. We identify sites of NKp46/NCR1 binding to sigma1 and show that sigma1 binding by NKp46/NCR1 leads to NK cell activation in vitro Finally, we demonstrate that NCR1 activation is essential for reovirus-based therapy in vivo Collectively, we have identified sigma1 as a novel ligand for NKp46/NCR1 and demonstrated that NKp46/NCR1 is needed both for clearance of reovirus infection and for reovirus-based tumor therapy.IMPORTANCE Reovirus infects much of the population during childhood, causing mild disease, and hence is considered to be efficiently controlled by the immune system. Reovirus also specifically infects tumor cells, leading to tumor death, and is currently being tested in human clinical trials for cancer therapy. The mechanisms by which our immune system controls reovirus infection and tumor killing are not well understood. We report here that natural killer (NK) cells recognize a viral protein named sigma1 through the NK cell-activating receptor NKp46. Using several mouse tumor models, we demonstrate the importance of NK cells in protection from reovirus infection and in reovirus killing of tumors in vivo Collectively, we identify a new ligand for the NKp46 receptor and provide evidence for the importance of NKp46 in the control of reovirus infections and in reovirus-based cancer therapy.

Detection of mammalian orthoreovirus type-3 (Reo-3) infections in mice based on serotype-specific hemagglutination protein sigma-1.

BACKGROUND: Reovirus type-3 infections cause severe pathologies in young mice and thus influence animal experiments in many ways. Therefore, the Federation of Laboratory Animal Science Associations (FELASA) recommends an annual screening in laboratory mice as part of a thorough health monitoring program. Based on the high protein sequence homology among the different reovirus serotypes, immunofluorescence antibody assay and other indirect methods relying on the whole virus are presumably cross-reactive to antibodies triggered by mammalian orthoreovirus infections independent of the serotype. METHODS: The serotype-specific protein σ-1 was expressed in Escherichia coli with an N-terminal Strep-tag and a C-terminal His-tag. The purified Strep-rσ-1-His-construct was used to develop an indirect ELISA by testing defined positive and negative sera obtained by experimental infection of mice as well as field sera. RESULTS: The Strep-rσ-1-His-ELISA provided high sensitivity and specificity during validation. Notably, a high selectivity was also observed for sera positively tested for other relevant FELASA-listed pathogens. Screening of field samples indicated that a commercial reovirus type-3-based ELISA might be cross-reactive to other murine reovirus serotypes and thus produces false-positive results. CONCLUSIONS: The prevalence of reovirus type-3 might be overestimated in German animal facilities and most likely in other countries as well. The occurrence of other reovirus serotypes, however, raises the question if murine health monitoring programs should be extended to these pathogens.

Spontaneous activation of a MAVS-dependent antiviral signaling pathway determines high basal interferon-ß expression in cardiac myocytes.

Viral myocarditis is a leading cause of sudden death in young adults as the limited turnover of cardiac myocytes renders the heart particularly vulnerable to viral damage. Viruses induce an antiviral type I interferon (IFN-α/ß) response in essentially all cell types, providing an immediate innate protection. Cardiac myocytes express high basal levels of IFN-ß to help pre-arm them against viral infections, however the mechanism underlying this expression remains unclear. Using primary cultures of murine cardiac and skeletal muscle cells, we demonstrate here that the mitochondrial antiviral signaling (MAVS) pathway is spontaneously activated in unstimulated cardiac myocytes but not cardiac fibroblasts or skeletal muscle cells. Results suggest that MAVS association with the mitochondrial-associated ER membranes (MAM) is a determinant of high basal IFN-ß expression, and demonstrate that MAVS is essential for spontaneous high basal expression of IFN-ß in cardiac myocytes and the heart. Together, results provide the first mechanism for spontaneous high expression of the antiviral cytokine IFN-ß in a poorly replenished and essential cell type.

Reovirus µ1 Protein Affects Infectivity by Altering Virus-Receptor Interactions.

Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the σ1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor junctional adhesion molecule A (JAM-A). The µ1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded µ1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of µ1 in altering the cell attachment property of reovirus. Our data indicate that the T3D µ1-mediated enhancement in infectivity of T1L is dependent on the function of σ1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies revealed a previously unknown relationship between two nonadjacent reovirus outer capsid proteins, σ1 and µ1. IMPORTANCE: How reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the σ1 protein, and properties of σ1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of σ1 are influenced by the nature of µ1, a capsid protein that does not physically interact with σ1. These studies could explain the previously described role for µ1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example of how genetic reassortment between virus strains could produce phenotypes that are distinct from those of either parent.