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.

Grass carp Mre11A activates IFN 1 response by targeting STING to defend against GCRV infection.

Mre11A is considered as a cytosolic DNA receptor in mammals. However, it is rarely known about Mre11A in other vertebrates. Recently, a mammalian ortholog of Mre11A has been identified in grass carp (Ctenopharyngodon idellus) in our lab. Phylogenetic-tree analysis provided evidence for a close genetic relationship between C.idellus Mre11A and Carassius auratus Mre11A. The tissue expression profile of CiMre11A was detected, with a relatively higher level of expression in kidney, intestines, liver and spleen than that in other tissues after grass carp reovirus (GCRV) infection. Similarly, CiMre11A was also up-regulated in CIK cells after treatment with GCRV. Q-PCR and dual-luciferase assays indicated that the transcription levels of IFN1 and ISG15 were inhibited by CiMre11A knockdown, but were gradually augmented after CIK cells were transfected with increasing amounts of CiMre11A. Subcellular localization assays showed that a part of CiMre11A was translocated from the nucleus to the cytoplasm. Co-immunoprecipitation and co-localization assays demonstrated that CiMre11A interacts with CiSTING in response to GCRV infection. In CIK cells, the expressions of both IFN1 and ISG15 were acutely up-regulated by CiMre11A overexpression, as well as by co-overexpression of CiMre11A and CiSTING. CiMre11A and CiSTING induced the phosphorylation and cytoplasmic-to-nuclear translocation of IRF7 in CIK cells. The multiplication of GCRV in CIK cells was inhibited by the overexpression of CiMre11A and CiSTING.

Molecular characterization of an emerging reassortant mammalian orthoreovirus in China.

Mammalian orthoreoviruses (MRVs) infect almost all mammals, and there are some reports on MRVs in China. In this study, a novel strain was identified, which was designated as HLJYC2017. The results of genetic analysis showed that MRV HLJYC2017 is a reassortant strain. According to biological information analysis, different serotypes of MRV contain specific amino acid insertions and deletions in the σ1 protein. Neutralizing antibody epitope analysis revealed partial cross-protection among MRV1, MRV2, and MRV3 isolates from China. L3 gene recombination in MRV was identified for the first time in this study. The results of this study provide valuable information on MRV reassortment and evolution.

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.

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.

Multiple proteins differing between laboratory stocks of mammalian orthoreoviruses affect both virus sensitivity to interferon and induction of interferon production during infection.

In the course of previous works, it was observed that the virus laboratory stock (T3DS) differs in sequence from the virus encoded by the ten plasmids currently in use in many laboratories (T3DK), and derived from a different original virus stock. Seven proteins are affected by these sequence differences. In the present study, replication of T3DK was shown to be more sensitive to the antiviral effect of interferon. Infection by the T3DK virus was also shown to induce the production of higher amount of ß and α-interferons compared to T3DS. Two proteins, the µ2 and λ2 proteins, were found to be responsible for increased sensitivity to interferon while both µ2 and λ1 are responsible for increased interferon secretion. Altogether this supports the idea that multiple reovirus proteins are involved in the control of induction of interferon and virus sensitivity to the interferon-induced response. While interrelated, interferon induction and sensitivity can be separated by defined gene combinations. While both µ2 and λ2 were previously suspected of a role in the control of the interferon response, other proteins are also likely involved, as first shown here for λ1. This also further stresses that due caution should be exerted when comparing different virus isolates with different genetic background.

Distinct Effects of Type I and III Interferons on Enteric Viruses.

Interferons (IFNs) are key host cytokines in the innate immune response to viral infection, and recent work has identified unique roles for IFN subtypes in regulating different aspects of infection. Currently emerging is a common theme that type III IFNs are critical in localized control of infection at mucosal barrier sites, while type I IFNs are important for broad systemic control of infections. The intestine is a particular site of interest for exploring these effects, as in addition to being the port of entry for a multitude of pathogens, it is a complex tissue with a variety of cell types as well as the presence of the intestinal microbiota. Here we focus on the roles of type I and III IFNs in control of enteric viruses, discussing what is known about signaling downstream from these cytokines, including induction of specific IFN-stimulated genes. We review viral strategies to evade IFN responses, effects of IFNs on the intestine, interactions between IFNs and the microbiota, and briefly discuss the role of IFNs in controlling viral infections at other barrier sites. Enhanced understanding of the coordinate roles of IFNs in control of viral infections may facilitate development of antiviral therapeutic strategies; here we highlight potential avenues for future exploration.

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.

Virus-Infected Human Mast Cells Enhance Natural Killer Cell Functions.

Mucosal surfaces are protected from infection by both structural and sentinel cells, such as mast cells. The mast cell's role in antiviral responses is poorly understood; however, they selectively recruit natural killer (NK) cells following infection. Here, the ability of virus-infected mast cells to enhance NK cell functions was examined. Cord blood-derived human mast cells infected with reovirus (Reo-CBMC) and subsequent mast cell products were used for the stimulation of human NK cells. NK cells upregulated the CD69 molecule and cytotoxicity-related genes, and demonstrated increased cytotoxic activity in response to Reo-CBMC soluble products. NK cell interferon (IFN)-γ production was also promoted in the presence of interleukin (IL)-18. In vivo, SCID mice injected with Reo-CBMC in a subcutaneous Matrigel model, could recruit and activate murine NK cells, a property not shared by normal human fibroblasts. Soluble products of Reo-CBMC included IL-10, TNF, type I and type III IFNs. Blockade of the type I IFN receptor abrogated NK cell activation. Furthermore, reovirus-infected mast cells expressed multiple IFN-α subtypes not observed in reovirus-infected fibroblasts or epithelial cells. Our data define an important mast cell IFN response, not shared by structural cells, and a subsequent novel mast cell-NK cell immune axis in human antiviral host defense.

Reovirus intermediate subviral particles constitute a strategy to infect intestinal epithelial cells by exploiting TGF-ß dependent pro-survival signaling.

Intestinal epithelial cells (IECs) constitute the primary barrier that separates us from the outside environment. These cells, lining the surface of the intestinal tract, represent a major challenge that enteric pathogens have to face. How IECs respond to viral infection and whether enteric viruses have developed strategies to subvert IECs innate immune response remains poorly characterized. Using mammalian reovirus (MRV) as a model enteric virus, we found that the intermediate subviral particles (ISVPs), which are formed in the gut during the natural course of infection by proteolytic digestion of the reovirus virion, trigger reduced innate antiviral immune response in IECs. On the contrary, infection of IECs by virions induces a strong antiviral immune response that leads to cellular death. Additionally, we determined that virions can be sensed by both TLR and RLR pathways while ISVPs are sensed by RLR pathways only. Interestingly, we found that ISVP infected cells secrete TGF-ß acting as a pro-survival factor that protects IECs against virion induced cellular death. We propose that ISVPs represent a reovirus strategy to initiate primary infection of the gut by subverting IECs innate immune system and by counteracting cellular-death pathways.

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.

A single amino acid substitution in the mRNA capping enzyme λ2 of a mammalian orthoreovirus mutant increases interferon sensitivity.

In the last few years, the development of a plasmid-based reverse genetics system for mammalian reovirus has allowed the production and characterization of mutant viruses. This could be especially significant in the optimization of reovirus strains for virotherapeutic applications, either as gene vectors or oncolytic viruses. The genome of a mutant virus exhibiting increased sensitivity to interferon was completely sequenced and compared with its parental virus. Viruses corresponding to either the parental or mutant viruses were then rescued by reverse genetics and shown to exhibit the expected phenotypes. Systematic rescue of different viruses harboring either of the four parental genes in a mutant virus backbone, or reciprocally, indicated that a single amino acid substitution in one of λ2 methyltransferase domains is the major determinant of the difference in interferon sensitivity between these two viruses.

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.

Mice immunogenicity after vaccination by DNA vaccines containing individual genes of a new type of reovirus.

In this study, we investigated humoral and cellular immune responses in mice to DNA vaccines containing individual S or M genes of a new type of reovirus (nRV) isolate from a severe acute respiratory syndrome (SARS) patient in Beijing, China. Mice were immunized intramuscularly (i.m.) with 100 µg of S1, S2, S3, S4, M1, M2, and M3 DNA vaccine each 4 times in 2-week intervals and assayed for humoral IgG, IgG1, IgG2, and IgG2b antibodies by ELISA and for cellular immune response, particularly IFN-γ induction by ELISpot assay. Moreover, CD4+ and CD8+ T cell levels in peripheral blood mononuclear cells were assayed by flow cytometry. We found that all DNA vaccines induced IgG antibodies, predominantly of the IgG2a class and S3 DNA vaccine was the strongest inducer. M2 and S3 DNA vaccines elicited Th1- and Th2-based immune responses, respectively, while S1 and M3 DNA vaccines induced a mixed Th1/Th2 response. M1, S2, and S4 DNA vaccines were poorly immunogenic. To our knowledge, this is the first report characterizing mammalian reovirus DNA vaccines applied to a mouse model.

Respiratory infection of mice with mammalian reoviruses causes systemic infection with age and strain dependent pneumonia and encephalitis.

BACKGROUND: Because mammalian reoviruses are isolated from the respiratory tract we modeled the natural history of respiratory infection of adult and suckling mice with T1 Lang (T1L) and T3 Dearing (T3D) reoviruses. METHODS: Adult and suckling Balb/c mice were infected by the intranasal route and were assessed for dose response of disease as well as viral replication in the lung and other organs. Viral antigen was assessed by immunofluorescence and HRP staining of tissue sections and histopathology was assessed on formalin fixed, H + E stained tissue sections. RESULTS: Intranasal infection of adult mice resulted in fatal respiratory distress for high doses (10(7) pfu) of T1L but not T3D. In contrast both T1L and T3D killed suckling mice at moderate viral dosages (10(5) pfu) but differed in clinical symptoms where T1L induced respiratory failure and T3D caused encephalitis. Infections caused transient viremia that resulted in spread to peripheral tissues where disease correlated with virus replication, and pathology. Immunofluorescent staining of viral antigens in the lung showed reovirus infection was primarily associated with alveoli with lesser involvement of bronchiolar epithelium. Immunofluorescent and HRP staining of viral antigens in brain showed infection of neurons by T3D and glial cells by T1L. CONCLUSIONS: These mouse models of reovirus respiratory infection demonstrated age and strain dependent disease that are expected to be relevant to understanding and modulating natural and therapeutic reovirus infections in humans.

Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk.

Administration of interferon (IFN)-α exacerbates reovirus type-2-triggered autoimmune insulitis in DBA/1J mice.

The aim of this study is to clarify the effects of administrated interferon (IFN)-α on Reovirus type-2 (Reo-2)-triggered autoimmune insulitis. Newborn DBA/1J mice infected with Reo-2 (on day 0) showed the highest titre in the pancreas on day 5 and thereafter the titre declined. Similar viral growth curve with lower titre was found in the spleen and thymus. Insulitis with impaired glucose tolerance developed in infected mice on day 10, but not on day 5. IFN-α was produced in the blood on days 3 and 5, but not on days 7 and 10. During the virus growth phase, IFN-α positive((+)) cells were detected in some pancreatic islet cells and infiltrated dendritic cells (DCs) in interstitium. Virus antigen positive cells were detected in islet cells, but not in DCs. Administration with IFN-α (10(2) , 10(3) or 10(4) international unit) on day 7, which is the time of the disappearance of virus from the pancreas and IFN-α from the blood, exacerbated insulitis with increased glucose values compared to only infected mice. IFN-α administration at the same time of infection did not develop insulitis. In addition, IFN-α administration in uninfected mice on day 7 did not cause any damage to islet cells. The present study suggests that IFN-α may have a possibility to exacerbate Reo-2-tiggered mild insulitis.