Mammalian reovirus µ1 protein attenuates RIG-I and MDA5-mediated signaling transduction by blocking IRF3 phosphorylation and nuclear translocation.

Mammalian reovirus (MRV) is a non-enveloped, gene segmented double-stranded RNA (dsRNA) virus. It is an important zoonotic pathogen that infects many mammals and vertebrates that act as natural hosts and causes respiratory and digestive tract diseases. Studies have reported that RIG-I and MDA5 in the innate immune cytoplasmic RNA-sensing RIG-like receptor (RLR) signaling pathway can recognize dsRNA from MRV and promote antiviral type I interferon (IFN) responses. However, the mechanism by which many MRV-encoded proteins evade the host innate immune response remains unclear. Here, we show that exogenous µ1 protein promoted the proliferation of MRV in vitro, while knockdown of MRV µ1 protein expression by shRNA could impair MRV proliferation. Specifically, µ1 protein inhibited MRV or poly(I:C)-induced IFN-ß expression, and attenuated RIG-I/MDA5-mediated signaling axis transduction during MRV infection. Importantly, we found that µ1 protein significantly decreased IFN-ß mRNA expression induced by MDA5, RIG-I, MAVS, TBK1, IRF3(5D), and degraded the protein expression of exogenous MDA5, RIG-I, MAVS, TBK1 and IRF3 via the proteasomal and lysosomal pathways. Additionally, we show that µ1 protein can physically interact with MDA5, RIG-I, MAVS, TBK1, and IRF3 and attenuate the RIG-I/MDA5-mediated signaling cascades by blocking the phosphorylation and nuclear translocation of IRF3. In conclusion, our findings reveal that MRV outer capsid protein µ1 is a key factor in antagonizing RLRs signaling cascades and provide new strategies for effective prevention and treatment of MRV infection.

Inhibition of HIF-1α accumulation in prostate cancer cells is initiated during early stages of mammalian orthoreovirus infection.

Mammalian orthoreovirus (MRV) is a safe and effective cancer killing virus that has completed Phase I-III clinical trials against numerous cancer types. While many patients experience benefit from MRV therapy, pre-defined set points necessary for FDA approval have not been reached. Therefore, additional research into MRV biology and the effect of viral therapy on different tumor genetic subtypes and microenvironments is necessary to identify tumors most amenable to MRV virotherapy. In this work we analyzed the stage of viral infection necessary to inhibit HIF-1α, an aggressive cancer activator induced by hypoxia. We demonstrated that two viral capsid proteins were not necessary and that a step parallel with virus core movement across the endosomal membrane was required for this inhibition. Altogether, this work clarifies the mechanisms of MRV-induced HIF-1α inhibition and provides biological relevance for using MRV to inhibit the devastating effects of tumor hypoxia.

Reovirus and the Host Integrated Stress Response: On the Frontlines of the Battle to Survive.

Cells are continually exposed to stressful events, which are overcome by the activation of a number of genetic pathways. The integrated stress response (ISR) is a large component of the overall cellular response to stress, which ultimately functions through the phosphorylation of the alpha subunit of eukaryotic initiation factor-2 (eIF2α) to inhibit the energy-taxing process of translation. This response is instrumental in the inhibition of viral infection and contributes to evolution in viruses. Mammalian orthoreovirus (MRV), an oncolytic virus that has shown promise in over 30 phase I-III clinical trials, has been shown to induce multiple arms within the ISR pathway, but it successfully evades, modulates, or subverts each cellular attempt to inhibit viral translation. MRV has not yet received Food and Drug Administration (FDA) approval for general use in the clinic; therefore, researchers continue to study virus interactions with host cells to identify circumstances where MRV effectiveness in tumor killing can be improved. In this review, we will discuss the ISR, MRV modulation of the ISR, and discuss ways in which MRV interaction with the ISR may increase the effectiveness of cancer therapeutics whose modes of action are altered by the ISR.

Closely related reovirus lab strains induce opposite expression of RIG-I/IFN-dependent versus -independent host genes, via mechanisms of slow replication versus polymorphisms in dsRNA binding σ3 respectively.

The Dearing isolate of Mammalian orthoreovirus (T3D) is a prominent model of virus-host relationships and a candidate oncolytic virotherapy. Closely related laboratory strains of T3D, originating from the same ancestral T3D isolate, were recently found to exhibit significantly different oncolytic properties. Specifically, the T3DPL strain had faster replication kinetics in a panel of cancer cells and improved tumor regression in an in vivo melanoma model, relative to T3DTD. In this study, we discover that T3DPL and T3DTD also differentially activate host signalling pathways and downstream gene transcription. At equivalent infectious dose, T3DTD induces higher IRF3 phosphorylation and expression of type I IFNs and IFN-stimulated genes (ISGs) than T3DPL. Using mono-reassortants with intermediate replication kinetics and pharmacological inhibitors of reovirus replication, IFN responses were found to inversely correlate with kinetics of virus replication. In other words, slow-replicating T3D strains induce more IFN signalling than fast-replicating T3D strains. Paradoxically, during co-infections by T3DPL and T3DTD, there was still high IRF3 phosphorylation indicating a phenodominant effect by the slow-replicating T3DTD. Using silencing and knock-out of RIG-I to impede IFN, we found that IFN induction does not affect the first round of reovirus replication but does prevent cell-cell spread in a paracrine fashion. Accordingly, during co-infections, T3DPL continues to replicate robustly despite activation of IFN by T3DTD. Using gene expression analysis, we discovered that reovirus can also induce a subset of genes in a RIG-I and IFN-independent manner; these genes were induced more by T3DPL than T3DTD. Polymorphisms in reovirus σ3 viral protein were found to control activation of RIG-I/ IFN-independent genes. Altogether, the study reveals that single amino acid polymorphisms in reovirus genomes can have large impact on host gene expression, by both changing replication kinetics and by modifying viral protein activity, such that two closely related T3D strains can induce opposite cytokine landscapes.

Toll like receptor 3 as an immunotherapeutic target for KRAS mutated colorectal cancer.

New therapeutic interventions are essential for improved management of patients with metastatic colorectal cancer (mCRC). This is especially critical for those patients whose tumors harbor a mutation in the KRAS oncogene (40-45% of all patients). This patient cohort is excluded from receiving anti-EGFR monoclonal antibodies that have added a significant therapeutic benefit for KRAS wild type CRC patients. Reovirus, a double stranded (ds) RNA virus is in clinical development for patients with chemotherapy refractory KRAS mutated tumors. Toll Like Receptor (TLR) 3, a member of the toll like receptor family of the host innate immune system is the pattern recognition motif for dsRNA pathogens. Using TLR3 expressing commercial HEK-BlueTM-hTLR3 cells we confirm that TLR3 is the host pattern recognition motif responsible for the detection of reovirus. Further, our investigation with KRAS mutated HCT116 cell line showed that effective expression of host TLR3 dampens the infection potential of reovirus by mounting a robust innate immune response. Down regulation of TLR3 expression with siRNA improves the anticancer activity of reovirus. In vivo experiments using human CRC cells derived xenografts in athymic mice further demonstrate the beneficial effects of TLR3 knock down by improving tumor response rates to reovirus. Strategies to mitigate the TLR3 response pathway can be utilized as a tool towards improved reovirus efficacy to specifically target the dissemination of KRAS mutated CRC.

[Exogenous Bacillus pumilus RNase (binase) suppresses the reproduction of reovirus serotype 1].

The experimental study identified the antiviral activity of Bacillus pumilus RNase (binase) against the reovirus of serotype 1/strain Lang. For the first time, it has been found that 50 µg/mL of binase effectively reduced the hemagglutinin and cytocidal activity of reovirus in Vero cell line. The preincubation of the enzyme with reovirus before infection of the cells inhibited the viral replication. To determine the stagedependent effect of reovirus reproduction upon binase inhibition, the infected cells were treated with binase or RNase A at different phases of the infectious cycle. The treatment of virus-infected cells has revealed that both enzymes have a maximal antiviral effect on the reovirus propagation during early phases of the reovirus reproduction cycle, with binase being more effective than RNase A. It has been hypothesized that the combined action of the oncolytic reovirus and binase is promising for the elimination of tumor cells carrying mutated RAS gene.

African Swine Fever Virus NP868R Capping Enzyme Promotes Reovirus Rescue during Reverse Genetics by Promoting Reovirus Protein Expression, Virion Assembly, and RNA Incorporation into Infectious Virions.

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid µ1 protein to µ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation.IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure.

Expression of Ifnlr1 on Intestinal Epithelial Cells Is Critical to the Antiviral Effects of Interferon Lambda against Norovirus and Reovirus.

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1 We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity.IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1-/- mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections.

PI3K/Akt/p53 pathway inhibits reovirus infection.

Viral infections activate many host signaling pathways, including the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which has recently attracted considerable interest due to its central role in modulating virus replication. This study demonstrated that the sero-type 3 reovirus strain Masked Palm Civet/China/2004 (MPC/04) could transiently activate the PI3K/Akt pathway in A549 cells at earlier time points of infection. The blockage of PI3K/Akt activation increased viral RNA synthesis and yield. The role of the downstream effectors MDM2/p53 of PI3K/Akt in regulating reovirus replication was further analyzed. We found that during reovirus infection, the level of phosphorylated MDM2 (p-MDM2) was increased and the expression of p53 was reduced. In addition, the blockage of PI3K/Akt by Ly294002 or knockdown of Akt by siRNA reduced the level of p-MDM2 and increased the level of p53. Both indicated that the downstream effectors MDM2/p53 of PI3K/Akt were activated. Pre-treatment with Nutlin, which can destroy MDM2 and p53 cross-talk and increase the expression of p53 RNA and protein, dose-dependently enhanced reovirus replication. Additionally, the overexpression of p53 alone also supported reovirus replication, and knockdown of p53 significantly inhibited viral replication. This study demonstrates that PI3K/Akt/p53 activated by mammalian reovirus can serve as a pathway for inhibiting virus replication/infection, yet the precise mechanism of this process remains under further investigation.

Activity levels of cathepsins B and L in tumor cells are a biomarker for efficacy of reovirus-mediated tumor cell killing.

Reovirus has gained much attention as an anticancer agent; however, the mechanism of the tumor cell-specific replication of reovirus is not fully understood. Although Ras activation is known to be crucial for tumor cell-specific replication of reovirus, it remains controversial which cellular factors are required for the reovirus-mediated tumor cell killing. In this study, we systematically investigated which cellular factors determined the efficiencies of reovirus-mediated tumor cell killing in various human cultured cell lines. The efficiency of reovirus-mediated cell killing varied widely among the cell lines. Junction adhesion molecule-A, a reovirus receptor, was highly expressed in almost all cell lines examined. Ras activation levels were largely different between the cell lines; however, there were no apparent correlations among the reovirus-mediated cell killing efficiencies and Ras activation status. On the other hand, activity levels of the cysteine proteases cathepsins B and L, which are crucial for proteolytic disassembly of the outer capsid proteins of reovirus, showed a tendency to be correlated with the efficiency of reovirus-mediated cell killing. These results indicate that the activity of cathepsins B and L is the most suitable as a biomarker for the efficacy of reovirus-mediated oncolysis among the factors examined in this study.

Amino acids substitutions in σ1 and µ1 outer capsid proteins of a Vero cell-adapted mammalian orthoreovirus are required for optimal virus binding and disassembly.

In a recent study, the serotype 3 Dearing strain of mammalian orthoreovirus was adapted to Vero cells; cells that exhibit a limited ability to support the early steps of reovirus uncoating and are unable to produce interferon as an antiviral response upon infection. The Vero cell-adapted virus (VeroAV) exhibits amino acids substitutions in both the σ1 and µ1 outer capsid proteins but no changes in the σ3 protein. Accordingly, the virus was shown not to behave as a classical uncoating mutant. In the present study, an increased ability of the virus to bind at the Vero cell surface was observed and is likely associated with an increased ability to bind onto cell-surface sialic acid residues. In addition, the kinetics of µ1 disassembly from the virions appears to be altered. The plasmid-based reverse genetics approach confirmed the importance of σ1 amino acids substitutions in VeroAV's ability to efficiently infect Vero cells, although µ1 co-adaptation appears necessary to optimize viral infection. This approach of combining in vitro selection of reoviruses with reverse genetics to identify pertinent amino acids substitutions appears promising in the context of eventual reovirus modification to increase its potential as an oncolytic virus.

Reovirus type-2-triggered autoimmune cholangitis in extrahepatic bile ducts of weanling DBA/1J mice.

BACKGROUND: Reovirus is a proposed cause of infantile biliary atresia. However, mechanistic insight regarding Reo-2 as a potential cholangiotropic virus is lacking. Furthermore, it is unknown whether Reo-2 infection can induce autoimmune-mediated bile duct injury. METHODS: Lesions of bile ducts in newborn DBA/1J mice infected with Reo-2 were analyzed immunopathologically. RESULTS: Damage to biliary epithelia occurs after Reo-2 infection. In addition, nonsuppurative cholangitis with fibrosis in extrahepatic (especially septal) bile ducts developed following complete viral clearance from the liver. At the inflamed ducts, major histocompatibility complex class I expressing((+)) and FAS(+) cholangiocytes were associated with FAS ligand(+) lymphocytes and tumor necrosis factor-α(+) mononuclear cells (macrophages and lymphocytes). These cholangiocytes were apoptotic and necrotic. Moreover, affected ducts were infiltrated by CD3(+), CD4(+), CD8(+), IFN-γ(+), and FAS(+) lymphocytes. Analysis of blood from Reo-2-infected mice revealed that they developed anticholangiocyte cytoplasm antibodies and had high serum IFN-γ concentration. Notably, there was no increase in Foxp3(+) lymphocytes at inflamed ducts, lymph nodes, and thymi. CONCLUSION: Reo-2 infection induced T-helper cell type 1-dependent injury to bile ducts in weanling mice. The lesions observed in mice may be analogous to those associated with human infantile biliary atresia, which are caused by an autoimmune-mediated process.

Reolysin is a novel reovirus-based agent that induces endoplasmic reticular stress-mediated apoptosis in pancreatic cancer.

Activating mutation of KRas is a genetic alteration that occurs in the majority of pancreatic tumors and is therefore an ideal therapeutic target. The ability of reoviruses to preferentially replicate and induce cell death in transformed cells that express activated Ras prompted the development of a reovirus-based formulation for cancer therapy called Reolysin. We hypothesized that Reolysin exposure would trigger heavy production of viral products leading to endoplasmic reticular (ER) stress-mediated apoptosis. Here, we report that Reolysin treatment stimulated selective reovirus replication and decreased cell viability in KRas-transformed immortalized human pancreatic duct epithelial cells and pancreatic cancer cell lines. These effects were associated with increased expression of ER stress-related genes, ER swelling, cleavage of caspase-4, and splicing of XBP-1. Treatment with ER stress stimuli including tunicamycin, brefeldin A, and bortezomib (BZ) augmented the anticancer activity of Reolysin. Cotreatment with BZ and Reolysin induced the simultaneous accumulation of ubiquitinated and viral proteins, resulting in enhanced levels of ER stress and apoptosis in both in vitro and in vivo models of pancreatic cancer. Our collective results demonstrate that the abnormal protein accumulation induced by the combination of Reolysin and BZ promotes heightened ER stress and apoptosis in pancreatic cancer cells and provides the rationale for a phase I clinical trial further investigating the safety and efficacy of this novel strategy.

HeLa cell response proteome alterations induced by mammalian reovirus T3D infection.

BACKGROUND: Cells are exposed to multiple stressors that induce significant alterations in signaling pathways and in the cellular state. As obligate parasites, all viruses require host cell material and machinery for replication. Virus infection is a major stressor leading to numerous induced modifications. Previous gene array studies have measured infected cellular transcriptomes. More recently, mass spectrometry-based quantitative and comparative assays have been used to complement such studies by examining virus-induced alterations in the cellular proteome. METHODS: We used SILAC (stable isotope labeling with amino acids in cell culture), a non-biased quantitative proteomic labeling technique, combined with 2-D HPLC/mass spectrometry and reciprocal labeling to identify and measure relative quantitative differences in HeLa cell proteins in purified cytosolic and nuclear fractions after reovirus serotype 3 Dearing infection. Protein regulation was determined by z-score analysis of each protein's label distribution. RESULTS: A total of 2856 cellular proteins were identified in cytosolic fractions by 2 or more peptides at >99% confidence and 884 proteins were identified in nuclear fractions. Gene ontology analyses indicated up-regulated host proteins were associated with defense responses, immune responses, macromolecular binding, regulation of immune effector processes, and responses to virus, whereas down-regulated proteins were involved in cell death, macromolecular catabolic processes, and tissue development. CONCLUSIONS: These analyses identified numerous host proteins significantly affected by reovirus T3D infection. These proteins map to numerous inflammatory and innate immune pathways, and provide the starting point for more detailed kinetic studies and delineation of virus-modulated host signaling pathways.

Reovirus uses multiple endocytic pathways for cell entry.

Entry of reovirus virions has been well studied in several tissue culture systems. After attachment to junctional adhesion molecule A (JAM-A), virions undergo clathrin-mediated endocytosis followed by proteolytic disassembly of the capsid and penetration to the cytoplasm. However, during in vivo infection of the intestinal tract, and likely in the tumor microenvironment, capsid proteolysis (uncoating) is initiated extracellularly. We used multiple approaches to determine if uncoated reovirus particles, called intermediate subviral particles (ISVPs), enter cells by directly penetrating the limiting membrane or if they take advantage of endocytic pathways to establish productive infection. We found that entry and infection by reovirus ISVPs was inhibited by dynasore, an inhibitor of dynamin-dependent endocytosis, as well as by genistein and dominant-negative caveolin-1, which block caveolar endocytosis. Inhibition of caveolar endocytosis also reduced infection by reovirus virions. Extraction of membrane cholesterol with methyl-ß-cyclodextrin inhibited infection by virions but had no effect when infection was initiated with ISVPs. We found this pathway to be independent of both clathrin and caveolin. Together, these data suggest that reovirus virions can use both dynamin-dependent and dynamin-independent endocytic pathways during cell entry, and they reveal that reovirus ISVPs can take advantage of caveolar endocytosis to establish productive infection.

Reovirus-associated reduction of microRNA-let-7d is related to the increased apoptotic death of cancer cells in clinical samples.

We analyzed the in situ molecular correlates of infection from cancer patients treated with reovirus. Melanoma, colorectal, and ovarian cancer samples from such patients showed variable infection of the cancer cells but not the intermingled benign cells. RT in situ PCR showed most cancer cells contained the viral genome with threefold less having productive viral infection as documented by either tubulin or reoviral protein co-expression. Productive infection in the cancer cells was strongly correlated with co-expression of p38 and caspase-3 as well as apoptosis-related death (P<0.001). The cancer cell apoptotic death was due to a marked viral-induced inhibition of microRNA-let-7d that, in turn, upregulated caspase-3 activity. In summary, reovirus shows a striking tropism to cancer cells in clinical samples. A rate-limiting factor of reovirus-induced cancer cell death is productive viral infection that operates via the marked reduction of microRNA-let-7d and concomitant elevated caspase-3 expression.

Reverse genetics for mammalian reovirus.

Mammalian orthoreoviruses (reoviruses) are highly tractable models for studies of viral replication and pathogenesis. The versatility of reovirus as an experimental model has been enhanced by development of a plasmid-based reverse genetics system. Infectious reovirus can be recovered from cells transfected with plasmids encoding cDNAs of each reovirus gene segment using a strategy that does not require helper virus and is independent of selection. In this system, transcription of each gene segment is driven by bacteriophage T7 RNA polymerase, which can be supplied transiently by recombinant vaccinia virus (rDIs-T7pol) or by cells that constitutively express the enzyme. Reverse genetics systems have been developed for two prototype reovirus strains, type 1 Lang (T1L) and type 3 Dearing (T3D). Each reovirus cDNA was encoded on an independent plasmid for the first-generation rescue system. The efficiency of virus recovery was enhanced in a second-generation system by combining the cDNAs for multiple reovirus gene segments onto single plasmids to reduce the number of plasmids from 10 to 4. The reduction in plasmid number and the use of baby hamster kidney cells that express T7 RNA polymerase increased the efficiency of viral rescue, reduced the incubation time required to recover infectious virus, and eliminated potential biosafety concerns associated with the use of recombinant vaccinia virus. Reovirus reverse genetics has been used to introduce mutations into viral capsid and nonstructural components to study viral protein-structure activity relationships and can be exploited to engineer recombinant reoviruses for vaccine and oncolytic applications.

Avian and mammalian reoviruses use different molecular mechanisms to synthesize their {micro}NS isoforms.

Previous reports revealed that the M3 gene of both avian and mammalian reoviruses express two isoforms of the non-structural protein µNS in infected cells. The larger isoforms initiate translation at the AUG codon closest to the 5' end of their respective m3 mRNAs, and were therefore designated µNS. In this study we have performed experiments to identify the molecular mechanisms by which the smaller µNS isoforms are generated. The results of this study confirmed the previous findings indicating that the smaller mammalian reovirus µNS isoform is a primary translation product, the translation of which is initiated at the internal AUG-41 codon of mammalian reovirus m3 mRNA. Our results further revealed that the smaller avian reovirus µNS isoform originates from a specific post-translational cleavage site near the amino terminus of µNS. This cleavage produces a 55 kDa carboxy-terminal protein, termed µNSC, and a 17 kDa amino-terminal polypeptide, designated µNSN. These results allowed us to extend the known avian reovirus protein-encoding capacity to 18 proteins, 12 of which are structural proteins and six of which are non-structural proteins. Our finding that avian and mammalian reoviruses use different mechanisms to express their µNSC isoforms suggests that these isoforms are important for reovirus replication.

A proapoptotic peptide derived from reovirus outer capsid protein {micro}1 has membrane-destabilizing activity.

The reovirus outer capsid protein µ1 is responsible for cell membrane penetration during virus entry and contains determinants necessary for virus-induced apoptosis. Residues 582 to 611 of µ1 are necessary and sufficient for reovirus-induced apoptosis, and residues 594 and 595 independently regulate the efficiency of viral entry and reovirus-induced cell apoptosis, respectively. Two of three α-helices within this region, helix 1 (residues 582 to 611) and helix 3 (residues 644 to 675), play a role in reovirus-induced apoptosis. Here, we chemically synthesized peptides representing helix 1 (H1), H1:K594D, H1:I595K, and helix 3 (H3) and examined their biological properties. We found that H1, but not H3, was able to cause concentration- and size-dependent leakage of molecules from small unilamellar liposomes. We further found that direct application of H1, but not H1:K594D, H1:I595K, or H3, to cells resulted in cytotoxicity. Application of the H1 peptide to L929 cells caused rapid elevations in intracellular calcium concentration that were independent of phospholipase C activation. Cytotoxicity of H1 was not restricted to eukaryotic cells, as the H1 peptide also had bactericidal activity. Based on these findings, we propose that the proapoptotic function of the H1 region of µ1 is dependent on its capacity to destabilize cellular membranes and cause release of molecules from intracellular organelles that ultimately induces cell necrosis or apoptosis, depending on the dose.

Inhibition of GSK-3beta enhances reovirus-induced apoptosis in colon cancer cells.

Reovirus functions as an oncolytic agent for many types of cancer including colon cancer. Although most studies have emphasized the role of activated Ras signaling in enhancing reoviral oncolysis in susceptible cells, we note that many colon cancers also display elevated beta-catenin. Thus, it is possible that enhanced beta-catenin may augment reoviral susceptibility in colon cancer cells. To explore this hypothesis, HEK293 cells were treated with the glycogen synthase kinase (GSK)-3beta inhibitor LiCl, thereby inducing beta-catenin, followed by reoviral infection. Co-administration with LiCl indeed enhanced cell death compared to reovirus infection alone, but this was not associated with elevated reoviral replication. Similarly, HEK293 cells expressing the Frizzled-1 receptor in Wnt3a-conditioned medium also showed reovirus replication equivalent to that in cells in control medium, further suggesting that up-regulation of beta-catenin does not enhance the replication of reovirus. Instead, we observed that inhibition of GSK-3beta with LiCl decreased reovirus-induced NF-kappaB activation, leading to accelerated apoptosis via caspase 8 activation. We further found that colon cancer HCT116 cells were sensitized to apoptosis by co-treatment with reovirus and a GSK-3beta inhibitor, AR-A014418. Finally, we identified that inhibition of NF-kappaB sensitized apoptosis of HEK293 or HCT 116 cells during reovirus infection. Taken together, we propose that inhibition of GSK-3beta sensitizes reovirus-induced apoptosis of colon cancer cells by down-regulation of NF-kappaB activity, offering a potentially improved therapeutic strategy for the treatment of colon cancer.