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

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

IL-6/STAT3 axis is hijacked by GCRV to facilitate viral replication via suppressing type â IFN signaling.

Grass carp reovirus (GCRV) infections and hemorrhagic disease (GCHD) outbreaks are typically seasonally periodic and temperature-dependent, yet the molecular mechanism remains unclear. Herein, we depicted that temperature-dependent IL-6/STAT3 axis was exploited by GCRV to facilitate viral replication via suppressing type Ⅰ IFN signaling. Combined multi-omics analysis and qPCR identified IL-6, STAT3, and IRF3 as potential effector molecules mediating GCRV infection. Deploying GCRV challenge at 18 °C and 28 °C as models of resistant and permissive infections and switched to the corresponding temperatures as temperature stress models, we illustrated that IL-6 and STAT3 expression, genome level of GCRV, and phosphorylation of STAT3 were temperature dependent and regulated by temperature stress. Further research revealed that activating IL-6/STAT3 axis enhanced GCRV replication and suppressed the expression of IFNs, whereas blocking the axis impaired viral replication. Mechanistically, grass carp STAT3 inhibited IRF3 nuclear translocation via interacting with it, thus down-regulating IFNs expression, restraining transcriptional activation of the IFN promoter, and facilitating GCRV replication. Overall, our work sheds light on an immune evasion mechanism whereby GCRV facilitates viral replication by hijacking IL-6/STAT3 axis to down-regulate IFNs expression, thus providing a valuable reference for targeted prevention and therapy of GCRV.

Deep sequencing identified miR-193b-3p as a positive regulator of autophagy targeting Akt3 in Ctenopharyngodon idella CIK cells during GCRV infection.

Recent research has highlighted complex and close interaction between miRNAs, autophagy, and viral infection. In this study, we observed the autophagy status in CIK cells infected with GCRV at various time points. We found that GCRV consistently induced cellar autophagy from 0 h to 12 h post infection. Subsequently, we performed deep sequencing on CIK cells infected with GCRV at 0 h and 12 h respectively, identifying 38 DEMs and predicting 9581 target genes. With the functional enrichment analyses of GO and KEGG, we identified 35 autophagy-related target genes of these DEMs, among which akt3 was pinpointed as the most central hub gene using module assay of the PPI network. Then employing the miRanda and Targetscan programs for prediction, and verification through a double fluorescent enzyme system and qPCR method, we confirmed that miR-193 b-3p could target the 3'-UTR of grass carp akt3, reducing its gene expression. Ultimately, we illustrated that grass carp miR-193 b-3p could promote autophagy in CIK cells. Above results collectively indicated that miRNAs might play a critical role in autophagy of grass carp during GCRV infection and contributed significantly to antiviral immunity by targeting autophagy-related genes. This study may provide new insights into the intricate mechanisms involved in virus, autophagy, and miRNAs.

Bmp4 in Zebrafish Enhances Antiviral Innate Immunity through p38 MAPK (Mitogen-Activated Protein Kinases) Pathway.

Bone morphogenetic proteins (BMPs) are a group of structurally and functionally related signaling molecules that comprise a subfamily, belonging to the TGF-ß superfamily. Most BMPs play roles in the regulation of embryonic development, stem cell differentiation, tumor growth and some cardiovascular and cerebrovascular diseases. Although evidence is emerging for the antiviral immunity of a few BMPs, more BMPs are needed to determine whether this function is universal. Here, we identified the zebrafish bmp4 ortholog, whose expression is up-regulated through challenge with grass carp reovirus (GCRV) or its mimic poly(I:C). The overexpression of bmp4 in epithelioma papulosum cyprini (EPC) cells significantly decreased the viral titer of GCRV-infected cells. Moreover, compared to wild-type zebrafish, viral load and mortality were significantly increased in both larvae and adults of bmp4-/- mutant zebrafish infected with GCRV virus. We further demonstrated that Bmp4 promotes the phosphorylation of Tbk1 and Irf3 through the p38 MAPK pathway, thereby inducing the production of type I IFNs in response to virus infection. These data suggest that Bmp4 plays an important role in the host defense against virus infection. Our study expands the understanding of BMP protein functions and opens up new targets for the control of viral infection.

Black carp RIOK3 suppresses MDA5-mediated IFN signaling in the antiviral innate immunity.

In mammals, right open reading frame kinase 3 (RIOK3) is related with cancer development and immune regulation. To explore the role of teleost RIOK3 in the antiviral innate immunity, the homolog of RIOK3 (bcRIOK3) from black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Sequence analysis revealed that bcRIOK3 is conserved in vertebrates. The transcription of bcRIOK3 varied in host cells in response to the stimulation of spring viremia of carp virus (SVCV), poly (I:C), and LPS. Immunoblotting (IB) and immunofluorescence (IF) assays identified bcRIOK3 as a cytoplasmic protein with a molecular weight of ∼60 kDa. It was interesting that bcRIOK3 knockdown led to the decreased basal mRNA levels of IFNa, IFNb and Viperin; however, triggered obviously higher mRNA levels of the above genes after viral infection and enhanced host resistance to SVCV. Like its mammalian counterpart, bcRIOK3 overexpression in EPC cells showed a significant inhibitory effect on black carp MDA5 (bcMDA5)-mediated transcription of interferon promoters and antiviral activity. Co-immunoprecipitation and immunofluorescent assays identified the association between bcRIOK3 and bcMDA5. Further analysis revealed that bcRIOK3 enhanced the K48-linked ubiquitination and proteasome-dependent degradation of bcMDA5, and it weakened the oligomerization of bcMDA5 under poly (I:C) stimulation. In summary, our data conclude that RIOK3 dampens MDA5-mediated IFN signaling by promoting its degradation in black carp, which provide new insights into the regulation of IFN signaling in teleost.

Modulation of Reoviral Cytolysis (I): Combination Therapeutics.

Patients with stage IV gastric cancer suffer from dismal outcomes, a challenge especially in many Asian populations and for which new therapeutic options are needed. To explore this issue, we used oncolytic reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel, and docetaxel) for the treatment of gastric and other gastrointestinal cancer cells in vitro and in a mouse model. Cell viability in vitro was quantified by WST-1 assays in human cancer cell lines treated with reovirus and/or chemotherapeutic agents. The expression of reovirus protein and caspase activity was determined by flow cytometry. For in vivo studies, athymic mice received intratumoral injections of reovirus in combination with irinotecan or paclitaxel, after which tumor size was monitored. In contrast to expectations, we found that reoviral oncolysis was only poorly correlated with Ras pathway activation. Even so, the combination of reovirus with chemotherapeutic agents showed synergistic cytopathic effects in vitro, plus enhanced reovirus replication and apoptosis. In vivo experiments showed that reovirus alone can reduce tumor size and that the combination of reovirus with chemotherapeutic agents enhances this effect. Thus, we find that oncolytic reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and chemotherapeutic agents synergistically enhanced cytotoxicity in human gastric cancer cell lines in vitro and in vivo. Our data support the use of reovirus in combination with chemotherapy in further clinical trials, and highlight the need for better biomarkers for reoviral oncolytic responsiveness.

Modulation of Reoviral Cytolysis (II): Cellular Stemness.

Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is capable of killing both murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells' reoviral-permissive state to a resistant one. In a breast cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming rendered the cells permissive to cytolysis. Bioinformatic analysis indicates that expression of the Yamanaka pluripotency factors may be associated with regulating reoviral selectivity. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.

Transcriptome Analysis of the Spleen Provides Insight into the Immune Regulation of GCRV Resistance in Grass Carp (Ctenopharyngodon idella).

Grass carp (Ctenopharyngodon idella) is one of the most economically important fish in China, and its production is commonly lost due to GCRV infection. To understand the molecular mechanism of GCRV resistance in grass carp, we compared the spleen transcriptome of the GCRV-resistant and susceptible individuals under GCRV infection (Res-Sus) and the GCRV-resistant individuals under different conditions of injection with GCRV and PBS (Res-Ctl). A total of 87.56 GB of clean data were obtained from 12 transcriptomic libraries of spleen tissues. A total of 379 DEGs (156 upregulated genes and 223 downregulated genes) were identified in the comparison group Res-Ctl. A total of 1207 DEGs (633 upregulated genes and 574 downregulated genes) were identified in the comparison group Res-Sus. And 54 DEGs were shared including immune-related genes of stc2 (stanniocalcin 2), plxna1 (plexin A1), ifnα (interferon alpha), cxcl 11 (C-X-C motif chemokine ligand 11), ngfr (nerve growth factor receptor), mx (MX dynamin-like GTPase), crim1 (cysteine-rich transmembrane BMP regulator 1), plxnb2 (plexin B2), and slit2 (slit guidance ligand 2). KEGG pathway analysis revealed significant differences in the expression of genes mainly involved in immune system and signal transduction, including antigen processing and presentation, Toll-like receptor signaling pathway, natural killer cell-mediated cytotoxicity, and Hippo signaling pathway. This study investigates the immune mechanism of the resistance to GCRV infection in grass carp and provides useful information for the development of methods to control the spread of the GCRV infection.

Metabolomics in rare minnow (Gobiocypris rarus) after infection by attenuated and virulent grass carp reovirus genotype â ¡.

Grass carp reovirus genotype Ⅱ (GCRV Ⅱ) causes hemorrhagic disease in a variety fish, seriously affecting the aquaculture industry in China. However, the pathogenesis of GCRV Ⅱ is unclear. Rare minnow is an ideal model organism to study the pathogenesis of GCRV Ⅱ. Herein, we applied liquid chromatography-tandem mass spectrometry metabolomics to investigate metabolic responses in the spleen and hepatopancreas of rare minnow injected with virulent GCRV Ⅱ isolate DY197 and attenuated isolate QJ205. Results indicated that marked metabolic changes were identified in both the spleen and hepatopancreas after GCRV Ⅱ infection, and the virulent DY197 strain induced more significantly different metabolites (SDMs) than the attenuated QJ205 strain. Moreover, most SDMs were downregulated in the spleen and tend to be upregulated in hepatopancreas. The Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis showed that tissue-specific metabolic responses were identified after viruses infection, and the virulent DY197 strain induced more SDMs involved in amino acid metabolism in the spleen, especially the tryptophan metabolism, cysteine and methionine metabolism, which were essential for immune regulation in host; Meanwhile, nucleotide metabolism, protein synthesis and metabolism related pathways were enriched in the hepatopancreas by both virulent and attenuated strains. Our findings revealed the large scale metabolic alterations in rare minnow in response to attenuated and virulent GCRV Ⅱ infection, which will lead to a better understanding of the pathogenesis of viruses and host-pathogens interactions.

Oncolytic viruses as emerging therapy against cancers including Oncovirus-induced cancers.

There are several human viruses with known potential for causing cancers including, Hepatitis B virus, Hepatitis C virus, Epstein-Barr virus, Kaposi's sarcoma herpesvirus, Human T-cell lymphotropic virus, Human papillomavirus, and Merkel cell polyomavirus. Cancer is the second leading cause of death that affects humans worldwide, especially in developing countries. Surgery, chemotherapy, and radiotherapy can cure about 60% of humans with cancer but recurrent and metastatic diseases remain a major reason for death. In recent years, understanding the molecular characteristics of cancer cells has led to the improvement of therapeutic strategies using novel emerging therapies. Oncolytic viruses with the potential of lysing cancer cells defined the field of oncolytic virology, hence becoming a biotechnology tool rather than just a cause of disease. This study mainly focused on targeting cell proliferation and death pathways in human tumor-inducing viruses by developing innovative therapies for cancer patients based on the natural oncolytic properties of reovirus. To kill tumor cells efficiently and reduce the chance of recurrence both the direct ability of reovirus infection to lyse the tumor cells and the stimulation of a potent host immune response are applied. Hence, bioengineered stem cells can be used as smart carriers to improve the efficacy of oncolytic reovirus and safety profiles.

Comparison of the Effect of Adipose Mesenchymal Stem Cells-Derived Secretome with and without Reovirus in CT26 Cells.

Colorectal cancer is the fourth leading cause of cancer-related deaths that has significantly increased over the past three decades. New therapeutic approaches, such as oncolytic viruses, have become very imperative recently to destroy cancer cells. The use of mesenchymal stem cells (MSCs) secretome that is produced in response to variant conditions involves different paracrine molecules secretion that has therapeutic potential in several chronic diseases. Mesenchymal stem cells and their derivatives are employed as regenerative medicine; nevertheless, there is ambiguity in the function of these cells in the control of malignancy. This study aimed to examine the apoptotic effect of secretomes derived from MSCs affected by encompassing oncolytic reoviruses. Mesenchymal stem cells were cultured after separation from abdominal adipose tissue of BALB/c mice. After three passages, the cells were infected by reovirus at the multiplicity of infection of 1 plaque-forming unit per cell. Uninfected and infected secretomes with reovirus were collected separately. The colorectal cancer CT26 cells were confronted with uninfected secretome, infected secretions, reovirus as a positive control, and Dulbecco's Modified Eagle Medium/High Glucose as negative control separately. Finally, apoptosis and necrosis were evaluated by flow cytometry. The infected secretome with reovirus was capable to induce apoptosis more than the uninfected secretome in CT26. However, the supernatant of reovirus infected cells was more capable to induce cell death, in comparison to the infected secretome. Infected MSCs with oncolytic reovirus produced a type of condition media that enhanced apoptosis induction and could have a therapeutic effect on cancer cells. Nonetheless, tumoral cells confronted with the oncolytic reovirus showed more capability in inducing apoptosis in CT26 cells. As a result, the use of oncolytic virus and infected secretome are more effective than uninfected secretome in inducing apoptosis.

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53.

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

Identification and immune responses of thrombocytes in bacterial and viral infections in grass carp (Ctenopharyngodon idella).

Thrombocytes are an important component in peripheral blood cells and play a crucial role in immune regulation. CD41 is one of the biomarkers of thrombocytes. In this study, grass carp (Ctenopharyngodon idella) CD41 protein was expressed in Escherichia coli and purified by affinity chromatography. Subsequently, New Zealand rabbits were immunized with this protein via subcutaneous injection. The antibody titer examined by enzyme linked immunosorbent assay was 1:12800. The concentration of rabbit polyclonal antibody purified by HiTrap-rprotein-AFF affinity chromatography column was 1.9 mg/mL. The specificity was identified by SDS-PAGE, Western blot, flow cytometry, and indirect immunofluorescence assays. The purified antibody was used to screen grass carp thrombocytes, and CD41+ cells were 14.13%. CD41+ cells were further verified by Giemsa staining, transmission electron microscopy and RT-PCR. mRNA expression of CD41 in thrombocytes was not affected by viral or bacterial challenge in vitro, while CD41 transcripts were remarkably induced post pathogenic infections in vivo, which results from the immature hematopoietic stem cells and thrombocytes. Indirect immunofluorescence assay revealed that grass carp reovirus (GCRV) could not invade thrombocytes; however, mRNA expressions of some representative innate immune genes (IFN1, IL-1ß, TNFα and Mx2) were significantly up-regulated post GCRV challenge. Meanwhile, the transcripts of some innate immune genes (IL-6 and TNFα) were swiftly increased post bacterial infection. These results indicated that the rabbit anti-CD41 polyclonal antibody possesses good specificity and can effectively bind to the CD41 protein on the surface of grass carp thrombocytes. Grass carp thrombocytes participate in immune regulation in viral and bacterial infections.

DAK inhibits MDA5-mediated signaling in the antiviral innate immunity of black carp.

Dihydroxyacetone kinase (DAK) functions as a negative regulator of melanoma differentiation-associated gene 5 (MDA5)-mediated interferon (IFN) production in human. To explore its role in teleost fish, DAK homologue of black carp (Mylopharyngodon piceus) has been cloned and characterized in this paper. The transcription of black carp DAK (bcDAK) variated in host cells in response to LPS, poly (I:C) and virus stimulation, and bcDAK was majorly distributed in the cytoplasm. Overexpressed bcDAK in EPC cells showed little IFN promoter-inducing ability in the reporter assay and no antiviral activity in plaque assay. When co-expressed with black carp MDA5 (bcMDA5) in EPC cells, bcDAK obviously inhibited bcMDA5-mediated IFN promoter transcription in reporter assay and the antiviral activity in plaque assay. The knockdown of bcDAK enhanced the antiviral activity of the host cells. The association between bcDAK and bcMDA5 has been identified through immunofluorescent staining and co-immunoprecipitation (co-IP) assay. Thus, the data generated in this study support the conclusion that black carp DAK interacts with MDA5 and negatively regulates MDA5-mediated antiviral signaling.

Bombyx mori Akirin hijacks a viral peptide vSP27 encoded by BmCPV circRNA and activates the ROS-NF-κB pathway against viral infection.

Bombyx mori cypovirus (BmCPV), a member of the family Reoviridae, is a model of Cypovirus, has a 10 segmented double-stranded RNA genome. However, so far, only one viral small peptide vSP27 with negative regulation on viral infection was identified; the mechanisms underlying host-BmCPV interaction are still unknown. Here, we identified that vSP27 was translated from a BmCPV derived circular RNA (circRNA-vSP27). Subsequently, results showed that vSP27 induced generation of ROS activated the NF-κB signaling pathway, induced the expression of antimicrobial peptides, and suppressed BmCPV infection. On the other hand, we identified a nuclear protein Akirin that could hijack vSP27, positively regulate the NF-κB pathway, and lead to inhibiting the viral infection. Altogether, our data suggested that BmCPV derived circRNA-vSP27 with small peptide translation activity may be employed by the host immunity in defense against the BmCPV infection.

Heat shock protein 70, glutamate dehydrogenase, and angiotensin-converting enzyme of Bombyx mori mediate the cell attachment of Cypovirus 1.

Dendrolimus punctatus causes great damage to pine forests worldwide. Dendrolimus punctatus cypovirus 1 (DpCPV-1) is an important pathogen of D. punctatus. However, the mechanism of DpCPV-1 cell entry has not been elucidated. In this study, we revealed that both GTase and MTase domains of VP3 (B-spike) and VP4 (A-spike) of DpCPV-1 interacted with the midgut proteins of Bombyx mori. Binding and competition assays revealed that GTase, MTase and VP4 played roles as viral attachment proteins. Far-Western blotting and LC-MS/MS analyses identified that heat shock protein 70 (BmHSP70), glutamate dehydrogenase (BmGDH), and angiotensin-converting enzyme (BmACE) in the midgut proteins as ligand candidates of the viral attachment proteins, and this was further verified by co-immunoprecipitation and fluorescence co-localization assays. Viral binding to the host midgut in vitro was inhibited by pre-treating B. mori midgut proteins with anti-BmHSP70, anti-BmGDH, anti-BmACE antibodies singly and in combination. Incubating DpCPV-1 virions with prokaryotically expressed BmHSP70, BmGDH, and BmACE also decreased viral attachment to the host midgut. In vivo bioassays revealed that viral infection in Helicoverpa armigera was partially neutralized by BmHSP70, BmGDH, and BmACE. Taking together, we concluded that HSP70, GDH, and ACE mediate DpCPV attachment and entry via binding to the viral attachment proteins, VP3 and VP4. The findings provide foundation for further understanding the entry mechanisms of cypoviruses.

Targeting JAK/STAT Signaling Antagonizes Resistance to Oncolytic Reovirus Therapy Driven by Prior Infection with HTLV-1 in Models of T-Cell Lymphoma.

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that infects at least 10 million people worldwide and is associated with the development of T-cell lymphoma (TCL). The treatment of TCL remains challenging and new treatment options are urgently needed. With the goal of developing a novel therapeutic approach for TCL, we investigated the activity of the clinical formulation of oncolytic reovirus (Reolysin, Pelareorep) in TCL models. Our studies revealed that HTLV-1-negative TCL cells were highly sensitive to Reolysin-induced cell death, but HTLV-1-positive TCL cells were resistant. Consistent with these data, reovirus displayed significant viral accumulation in HTLV-1-negative cells, but failed to efficiently replicate in HTLV-1-positive cells. Transcriptome analyses of HTLV-1-positive vs. negative cells revealed a significant increase in genes associated with retroviral infection including interleukin-13 and signal transducer and activator of transcription 5 (STAT5). To investigate the relationship between HTLV-1 status and sensitivity to Reolysin, we infected HTLV-1-negative cells with HTLV-1. The presence of HTLV-1 resulted in significantly decreased sensitivity to Reolysin. Treatment with the JAK inhibitor ruxolitinib suppressed STAT5 phosphorylation and expression of the key anti-viral response protein MX1 and enhanced the anti-TCL activity of Reolysin in both HTLV-1-positive and negative cells. Our data demonstrate that the inhibition of the JAK/STAT pathway can be used as a novel approach to antagonize the resistance of HTLV-1-positive cells to oncolytic virus therapy.

Mesenchymal stem cells loaded with oncolytic reovirus enhances antitumor activity in mice models of colorectal cancer.

Oncolytic viruses (OVs) are promising alternative biological agents for treating cancer. However, triggered immune responses against viruses and their delivery to tumor sites are their primary limitations in cancer therapy. To address these challenges, mesenchymal stem cells (MSCs) can serve as permissive tools for OVs loading and delivery to tumor sites. Here, we evaluated the in vitro and in vivo antitumor capability of adipose-derived mesenchymal stem cells (AD-MSCs) as a new vehicle for Dearing strain of reovirus (ReoT3D) loading. We first isolated and confirmed the purity of MSCs, and the optimized dose of ReoT3D for MSCs loading was computed by a standard assay. Next, we used murine CT26 cell line to establish the colorectal cancer model in BALB/c mice and demonstrated the antitumor effects of MSCs loaded with reovirus. Our results demonstrated that multiplicity of infection (MOI) 1 pfu/cells of reovirus was the safe dose for loading into purified MSCs. Moreover, our anticancer experiments exhibited that treatment with MSCs loaded with ReoT3D was more effective than ReoT3D and MSCs alone. Higher anticancer impact of MSCs loaded with OV was associated with induction of apoptosis, cell cycle arrests, P53 expression in tumor sections, and reduced tumor growth and size. The present results suggest that MSCs as a permissive shuttle for oncolytic virus (OV) delivery increased the anticancer activity of ReoT3D in mice models of colorectal cancer and these findings should be supported by more preclinical and clinical studies.

Reovirus directly engages integrin to recruit clathrin for entry into host cells.

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, ß1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct ß1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and ß1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and ß1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for ß1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and ß1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Grass Carp Reovirus VP35 Degrades MAVS Through the Autophagy Pathway to Inhibit Fish Interferon Production.

Fish interferon (IFN) is a crucial cytokine for a host to resist external pathogens, conferring cells with antiviral capacity. Meanwhile, grass carp reovirus (GCRV) is a strong pathogen that causes high mortality in grass carp. Therefore, it is necessary to study the strategy used by GCRV to evade the cellular IFN response. In this study, we found that GCRV 35-kDa protein (VP35) inhibited the host IFN production by degrading mitochondrial antiviral signaling (MAVS) protein through the autophagy pathway. First, the overexpression of VP35 inhibited the IFN activation induced by polyinosinic-polycytidylic acid (poly I:C) and MAVS, and the expression of downstream IFN-stimulated genes (ISGs) was also decreased by using VP35 under the stimulation. Second, VP35 interacted with MAVS; the experiments of truncated mutants of MAVS demonstrated that the caspase recruitment domain (CARD) and proline-rich (PRO) domains of MAVS were not necessary for this binding. Then, MAVS was degraded by using VP35 in a dose-dependent manner, and 3-MA (the autophagy pathway inhibitor) significantly blocked the degradation, meaning that MAVS was degraded by using VP35 in the autophagy pathway. The result of MAVS degradation suggested that the antiviral capacity of MAVS was remarkably depressed when interrupted by VP35. Finally, in the host cells, VP35 reduced ifn transcription and made the cells vulnerable to virus infection. In conclusion, our results reveal that GCRV VP35 impairs the host IFN response by degrading MAVS through the autophagy pathway, supplying evidence of a fish virus immune evasion strategy.