Reovirus Replication Research Articles

Function, Architecture, and Biogenesis of Reovirus Replication Neoorganelles.

Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Reoviruses are common pathogens of mammals that have been linked to celiac disease and show promise for oncolytic applications. These viruses form nonenveloped, double-shelled virions that contain ten segments of double-stranded RNA. Replication organelles in reovirus-infected cells are nucleated by viral nonstructural proteins µNS and σNS. Both proteins partition the endoplasmic reticulum to form the matrix of these structures. The resultant membranous webs likely serve to anchor viral RNA–protein complexes for the replication of the reovirus genome and the assembly of progeny virions. Ongoing studies of reovirus replication organelles will advance our knowledge about the strategies used by viruses to commandeer host biosynthetic pathways and may expose new targets for therapeutic intervention against diverse families of pathogenic viruses.

Oncolytic immunotherapy and bortezomib synergy improves survival of refractory multiple myeloma in a preclinical model

AbstractThe oncolytic reovirus (RV) has demonstrated clinical efficacy and minimal toxicity in a variety of cancers, including multiple myeloma (MM). MM is a malignancy of plasma cells that is considered treatable but incurable because of the 90% relapse rate that is primarily from drug resistance. The systemic nature of MM and the antitumor immunosuppression by its tumor microenvironment presents an ongoing therapeutic challenge. In the present study, we demonstrate that RV synergizes with the standard-of-care MM drug bortezomib (BTZ) and, importantly, enhances its therapeutic potential in therapy-resistant human MM cell lines in vitro. Using the syngeneic Vk*MYC BTZ-resistant immunocompetent transplantable MM murine model, we also demonstrate that mice harboring BTZ-insensitive MM tumors respond to the RV/BTZ combination treatment in terms of decreased tumor burden and improved overall survival (P < .00001). We demonstrate that BTZ augments RV replication in tumor-associated endothelial cells and myeloma cells, leading to enhanced viral delivery and thereby stimulating cytokine release, immune activity, apoptosis, and reduction of the MM-associated immune suppression. We conclude that combined RV/BTZ is an attractive therapeutic strategy with no safety signals for the treatment of MM.

Oncolytics Virus Replication Using Pelareorep (Reolysin) and Carfilzomib in Relapsed Myeloma Patients Increases PD-L1 Expression with Clinical Responses

Rationale: Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are tempting in immunosuppressive cancers such as multiple myeloma (MM). PD-1 inhibition alone has not been effective (Lesokhin, JCO, 2016). Evidence suggests that tumor response is determined by tumor PD-L1 expression and presence of infiltrating cytotoxic T-cell lymphocytes (CTLs). Intravenous Pelareorep (Reolysin), the infusible form of reovirus (RV), has been shown to upregulate IFN-regulated gene expression, CTL infiltration, and the PD1/PD-L1 axis in myeloma cell lines (Kelly KR et al, Leukemia, 2018) and in patients with brain tumors (Samson A et al, Sci Trans Med, 2018).Introduction:Viral oncolytic therapy with Pelareorep is supported by preclinical data indicating that its antitumor activity results from direct cytolysis and immune responses against infected MM cells. Our phase 1 single agent Reolysin trial in patients with relapsed MM confirmed the tolerability of Pelareorep and demonstrated preferential infection into myeloma cells but not microenvironmental cells in the marrow by intracellular staining for RV genome. In PART ONE of our follow-up trial, Carfilzomib (Kyprolis)-sensitive patients were accrued. Correlative studies included bone marrow aspirate pretreatment on cycle 1 day 1 and day 9 to assess RV infection of myeloma cells, replication within myeloma cells, and PD-L1 expression on myeloma cell surface. Pelareorep, Carfilzomib, and 20 mg IV Dexamethasone were all infused per the standard Carfilzomib schedule on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle.Part ONE: Carfilzomib-sensitive patients were infused at dose level one with Pelareorep 3 x 1010TCID50/day and Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1 and 27 mg/m2 thereafter, on days 1, 2, 8, 9, 15 and 16 of a 28-day cycle. The MTD will be determined as the highest dose combination with fewer than 33% DLTs observed in cycle 1. There were 2 VGPRs, 2 PRs, 1 MR, and one patient with stable disease after cycle 1. All evaluable patients showed RV infection and replication in the post-treatment BM aspirates. In the 4 bortezomib-refractory patients in the first cohort, all have shown viral replication, and this correlated directly with activated caspase-3 in the MM cells and clinical response.Part TWO: Carfilzomib-refractory patients were treated with Carfilzomib 20 mg/m2 on days 1 and 2 of cycle 1, and 56 mg/m2 thereafter. Seven patients have been enrolled to date. In 3 patients processed to date with both pre- and post-treatment biopsies available, RV infection was detected in myeloma cells (2 patients) and endothelial cells (one patient). Replication was not seen. In these patients there was no strong evidence of increased activated caspase-3 expression in myeloma cells, nor was there a statistically significant increased CD8 cell infiltration or checkpoint protein expression after treatment.Conclusion: An increase in viral infection, viral replication, and PD-L1 expression on the surface of myeloma cells has been demonstrated in patients that had a clinical response to protocol therapy. A future study to assess the safety and tolerability of PD-1 inhibition in relapsed myeloma patients is now open to accrual (NCT03605719). Dose escalation is ongoing and the effects of the addition of Reolysin to Carfilzomib/Dexamethasone will be determined in this trial. DisclosuresHofmeister:Bristol-Myers Squibb: Research Funding; Adaptive biotechnologies: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Nuovo:Oncolytics Biotech Inc: Research Funding. Lonial:Amgen: Research Funding. Kaufman:Roche: Consultancy; Abbvie: Consultancy; Karyopharm: Other: data monitoring committee; Janssen: Consultancy; BMS: Consultancy. Nooka:GSK: Consultancy, Membership on an entity's Board of Directors or advisory committees; Adaptive technologies: Consultancy, Membership on an entity's Board of Directors or advisory committees; Spectrum Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Heffner:Pharmacyclics: Research Funding; ADC Therapeutics: Research Funding; Genentech: Research Funding; Kite Pharma: Research Funding.

Functional characterization of duck TBK1 in IFN-β induction

TRAF family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1) serves as hub molecule at the crossroad of multiple signaling pathways of type I interferon (IFN) induction. The importance of TBK1 in innate immunity has been demonstrated in mammalian, however the characterization and function of TBK1 in avian remains largely unknown. In this study, we cloned duck TBK1 (duTBK1) from duck embryo fibroblasts (DEFs) for the first time, which encoded 729 amino acids and had a high amino acid identity with goose and cormorant TBK1s. The duTBK1 showed a diffuse cytoplasmic localization in DEFs and was extensively expressed in all tested tissues. Overexpression of duTBK1 induced IFN-β production through the activation of IRF1 and NF-κB in DEFs. The N-terminal kinase domain and the ubiquitin-like domain in middle of duTBK1 played pivotal roles in IFN-β induction as well as in IRF1 and NF-κB activation. Furthermore, knockdown of duTBK1 by small interfering RNA significantly decreased poly(I:C)- or Sendai virus (SeV)-induced IFN-β expression. In addition, duTBK1 expression dramatically reduced the replication of both duck reovirus (DRV) and duck Tembusu virus (DTMUV) in DEFs. These results suggested that the duTBK1 played a pivotal role in mediating duck antiviral innate immunity.

Reovirus σNS and μNS Proteins Remodel the Endoplasmic Reticulum to Build Replication Neo-Organelles.

ABSTRACTLike most viruses that replicate in the cytoplasm, mammalian reoviruses assemble membranous neo-organelles called inclusions that serve as sites of viral genome replication and particle morphogenesis. Viral inclusion formation is essential for viral infection, but how these organelles form is not well understood. We investigated the biogenesis of reovirus inclusions. Correlative light and electron microscopy showed that endoplasmic reticulum (ER) membranes are in contact with nascent inclusions, which form by collections of membranous tubules and vesicles as revealed by electron tomography. ER markers and newly synthesized viral RNA are detected in inclusion internal membranes. Live-cell imaging showed that early in infection, the ER is transformed into thin cisternae that fragment into small tubules and vesicles. We discovered that ER tubulation and vesiculation are mediated by the reovirus σNS and μNS proteins, respectively. Our results enhance an understanding of how viruses remodel cellular compartments to build functional replication organelles.

Reovirus Nonstructural Protein σNS Acts as an RNA Stability Factor Promoting Viral Genome Replication.

Viral nonstructural proteins, which are not packaged into virions, are essential for the replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. The reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, the activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered, using in vitro and cell-based RNA degradation experiments, that σNS increases the RNA half-life. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases the viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication.IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the family Reoviridae encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different viruses in the family Reoviridae diverge in primary sequence, they are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the family Reoviridae assort and replicate their genomes.

PO-031 Sensitising P53 mutant HNSCC to oncolytic reovirus (RT3D) by targeting the unfolded protein response

Introduction Oncolytic viruses are able to enter and selectively replicate in cancer cells. These self-propagating agents kill through multiple mechanisms and have the potential to promote anti-tumour immune responses. Reovirus strain type 3 Dearing (RT3D) is an oncolytic virus actively under clinical investigation. RT3D has been shown to induce ER stress and trigger the downstream unfolded protein response (UPR) in a variety of tumour types. Recent evidence has indicated that proteasome inhibition in multiple myeloma, and BRAF inhibition in BRAF mutant melanoma, sensitise to RT3D through perturbation of the normal cellular response to ER stress. We sought to investigate if more direct modulation of UPR signalling could sensitise head and neck squamous cell carcinoma (HNSCC) to RT3D. Material and methods ER stress agents including novel agents targeting UPR signalling were screened for sensitisation to RT3D in a panel of HNSCC cell lines. Alterations to reovirus protein levels and viable virus particle production was assessed. A 3D tumour spheroid model was used as a more accurate method to study stress in vitro. Recombinant protein expression and shRNA techniques were utilised to study how the unfolded protein response impacts reovirus replication. ER stress measured by GRP78 expression was also assessed in HNSCC patient samples. Results and discussions The PERK inhibitor GSK2606414 and the canonical ER stress agent thapsigargin were shown to sensitise HNSCC to RT3D. PERK inhibition resulted in increased viral capsid protein levels and increased viable RT3D production from target cells. PERK inhibition enhanced the ability of RT3D to eradicate HNSCC 3D tumour spheroids. PERK inhibition increased RT3D spread in 3D spheroids while perturbing normal ER chaperone levels and inhibiting RT3D induced LC3A/B. In HNSCC patient samples, GRP78 was found to be elevated in tumour vs stroma with significant variation between patients. This highlights the importance in understanding the link between ER stress levels/UPR signalling and reovirus efficacy. Conclusion We demonstrate that modulators of the ER stress response represent a novel mechanism of sensitisation to RT3D in head and neck.

A Phase II Study of Pelareorep (REOLYSIN®) in Combination with Gemcitabine for Patients with Advanced Pancreatic Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with 1 and 5-year survival rates of ~18% and 7% respectively. FOLFIRINOX or gemcitabine in combination with nab-paclitaxel are standard treatment options for metastatic disease. However, both regimens are more toxic than gemcitabine alone. Pelareorep (REOLYSIN®), a proprietary isolate of reovirus Type 3 Dearing, has shown antitumor activity in clinical and preclinical models. In addition to direct cytotoxic effects, pelareorep can trigger antitumor immune responses. Due to the high frequency of RAS mutations in PDAC, we hypothesized that pelareorep would promote selective reovirus replication in pancreatic tumors and enhance the anticancer activity of gemcitabine. Chemotherapy-naïve patients with advanced PDAC were eligible for the study. The primary objective was Clinical Benefit Rate (complete response (CR) + partial response (PR) + stable disease (SD) ≥ 12 weeks) and secondary objectives include overall survival (OS), toxicity, and pharmacodynamics (PD) analysis. The study enrolled 34 patients; results included one partial response, 23 stable disease, and 5 progressive disease. The median OS was 10.2 months, with a 1- and 2-year survival rate of 45% and 24%, respectively. The treatment was well tolerated with manageable nonhematological toxicities. PD analysis revealed reovirus replication within pancreatic tumor and associated apoptosis. Upregulation of immune checkpoint marker PD-L1 suggests future consideration of combining oncolytic virus therapy with anti-PD-L1 inhibitors. We conclude that pelareorep complements single agent gemcitabine in PDAC.

Distribution of Ifitm3 in Yellow-Feathered Broilers and Inhibition of Avian Reovirus Multiplication by Ifitm3

ABSTRACT This study was carried to express the interferon-induced transmembrane protein 3 (IFITM3) in vitro and examine its function in inhibition of avian reovirus (ARV) replication. The recombinant prokaryotic vector expressing yellow-feathered broiler IFITM3 was successfully constructed, and the recombinant protein was expressed in competent Escherichia coli BL21 cells. New Zealand white rabbits were immunized with the purified recombinant protein to prepare a polyclonal antibody, with a titer of 1:128,000. Immunohistochemistry, reverse transcription-PCR, and real-time fluorescence quantitative PCR showed that IFITM3 was distributed in the yellow-feathered broiler immune organs, and the expression of IFITM3 in bursa of Fabricius was more than in spleen and thymus. It was found that in the thymus, spleen and bursa of Fabricius the mRNA expression levels of IFNα and IFITM3 were significantly induced after ARV infection. And it was also certified in the chicken embryo fibroblasts (CEFs) which infected with ARV. Then the IFNα was added into the cell culture medium before CEFs were infected with ARV. The results indicated that the mRNA of IFITM3 expression was significantly increased and ARV multiplication was significantly inhibited. And when the expression of IFITM3 was knocked down by siRNA-IFITM3, the expression of IFITM3 was significantly reduced, but the ARV multiplication was significantly increased, which indicated that IFITM3 protein could inhibit the ARV replication.

Nonstructural Protein σ1s Is Required for Optimal Reovirus Protein Expression.

Reovirus nonstructural protein σ1s is required for the establishment of viremia and hematogenous viral dissemination. However, the function of σ1s during the reovirus replication cycle is not known. In this study, we found that σ1s was required for efficient reovirus replication in simian virus 40 (SV40)-immortalized endothelial cells (SVECs), mouse embryonic fibroblasts, human umbilical vein endothelial cells (HUVECs), and T84 human colonic epithelial cells. In each of these cell lines, wild-type reovirus produced substantially higher viral titers than a σ1s-deficient mutant. The σ1s protein was not required for early events in reovirus infection, as evidenced by the fact that no difference in infectivity between the wild-type and σ1s-null viruses was observed. However, the wild-type virus produced markedly higher viral protein levels than the σ1s-deficient strain. The disparity in viral replication did not result from differences in viral transcription or protein stability. We further found that the σ1s protein was dispensable for cell killing and the induction of type I interferon responses. In the absence of σ1s, viral factory (VF) maturation was impaired but sufficient to support low levels of reovirus replication. Together, our results indicate that σ1s is not absolutely essential for viral protein production but rather potentiates reovirus protein expression to facilitate reovirus replication. Our findings suggest that σ1s enables hematogenous reovirus dissemination by promoting efficient viral protein synthesis, and thereby reovirus replication, in cells that are required for reovirus spread to the blood.IMPORTANCE Hematogenous dissemination is a critical step in the pathogenesis of many viruses. For reovirus, nonstructural protein σ1s is required for viral spread via the blood. However, the mechanism by which σ1s promotes reovirus dissemination is unknown. In this study, we identified σ1s as a viral mediator of reovirus protein expression. We found several cultured cell lines in which σ1s is required for efficient reovirus replication. In these cells, wild-type virus produced substantially higher levels of viral protein than a σ1s-deficient mutant. The σ1s protein was not required for viral mRNA transcription or viral protein stability. Since reduced levels of viral protein were synthesized in the absence of σ1s, the maturation of viral factories was impaired, and significantly fewer viral progeny were produced. Taken together, our findings indicate that σ1s is required for optimal reovirus protein production, and thereby viral replication, in cells required for hematogenous reovirus dissemination.

The TRiC chaperonin controls reovirus replication through outer-capsid folding.

Viruses are molecular machines sustained through a life cycle that requires replication within host cells. Throughout the infectious cycle, viral and cellular components interact to advance the multistep process required to produce progeny virions. Despite progress made in understanding the virus-host protein interactome, much remains to be discovered about the cellular factors that function during infection, especially those operating at terminal steps in replication. In an RNA interference screen, we identified the eukaryotic chaperonin TRiC (also called CCT) as a cellular factor required for late events in the replication of mammalian reovirus. We discovered that TRiC functions in reovirus replication through a mechanism that involves the folding of the viral σ3 outer-capsid protein into a form capable of assembling onto virus particles. TRiC also complexes with homologous capsid proteins of closely related viruses. Our data define a critical function for TRiC in the viral assembly process and raise the possibility that this mechanism is conserved in related nonenveloped viruses. These results also provide insight into TRiC protein substrates and establish a rationale for the development of small-molecule inhibitors of TRiC as potential antiviral therapeutics.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

Oncolytic Reovirus Infection Is Facilitated by the Autophagic Machinery.

Mammalian reovirus is a double-stranded RNA virus that selectively infects and lyses transformed cells, making it an attractive oncolytic agent. Despite clinical evidence for anti-tumor activity, its efficacy as a stand-alone therapy remains to be improved. The success of future trials can be greatly influenced by the identification and the regulation of the cellular pathways that are important for reovirus replication and oncolysis. Here, we demonstrate that reovirus induces autophagy in several cell lines, evident from the formation of Atg5-Atg12 complexes, microtubule-associated protein 1 light chain 3 (LC3) lipidation, p62 degradation, the appearance of acidic vesicular organelles, and LC3 puncta. Furthermore, in electron microscopic images of reovirus-infected cells, autophagosomes were observed without evident association with viral factories. Using UV-inactivated reovirus, we demonstrate that a productive reovirus infection facilitates the induction of autophagy. Importantly, knock-out cell lines for specific autophagy-related genes revealed that the expression of Atg3 and Atg5 but not Atg13 facilitates reovirus replication. These findings highlight a central and Atg13-independent role for the autophagy machinery in facilitating reovirus infection and contribute to a better understanding of reovirus-host interactions.

Genome packaging of reovirus is mediated by the scaffolding property of the microtubule network.

Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.

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.

A Cytoplasmic RNA Virus Alters the Function of the Cell Splicing Protein SRSF2.

To replicate efficiently, viruses must create favorable cell conditions and overcome cell antiviral responses. We previously reported that the reovirus protein μ2 from strain T1L, but not strain T3D, represses one antiviral response: alpha/beta interferon signaling. We report here that T1L, but not T3D, μ2 localizes to nuclear speckles, where it forms a complex with the mRNA splicing factor SRSF2 and alters its subnuclear localization. Reovirus replicates in cytoplasmic viral factories, and there is no evidence that reovirus genomic or messenger RNAs are spliced, suggesting that T1L μ2 might target splicing of cell RNAs. Indeed, RNA sequencing revealed that reovirus T1L, but not T3D, infection alters the splicing of transcripts for host genes involved in mRNA posttranscriptional modifications. Moreover, depletion of SRSF2 enhanced reovirus replication and cytopathic effect, suggesting that T1L μ2 modulation of splicing benefits the virus. This provides the first report of viral antagonism of the splicing factor SRSF2 and identifies the viral protein that determines strain-specific differences in cell RNA splicing.IMPORTANCE Efficient viral replication requires that the virus create favorable cell conditions. Many viruses accomplish this by repressing specific antiviral responses. We demonstrate here that some mammalian reoviruses, RNA viruses that replicate strictly in the cytoplasm, express a protein variant that localizes to nuclear speckles, where it targets a cell mRNA splicing factor. Infection with a reovirus strain that targets this splicing factor alters splicing of cell mRNAs involved in the maturation of many other cell mRNAs. Depletion of this cell splicing factor enhances reovirus replication and cytopathic effect. Our results provide the first evidence of viral antagonism of this splicing factor and suggest that downstream consequences to the cell are global and benefit the virus.

Response of Three Different Viruses to Interferon Priming and Dithiothreitol Treatment of Avian Cells.

We have previously shown that the replication of avian reovirus (ARV) in chicken cells is much more resistant to interferon (IFN) than the replication of vesicular stomatitis virus (VSV) or vaccinia virus (VV). In this study, we have investigated the role that the double-stranded RNA (dsRNA)-activated protein kinase (PKR) plays in the sensitivity of these three viruses toward the antiviral action of chicken interferon. Our data suggest that while interferon priming of avian cells blocks vaccinia virus replication by promoting PKR activation, the replication of vesicular stomatitis virus appears to be blocked at a pretranslational step. Our data further suggest that the replication of avian reovirus in chicken cells is quite resistant to interferon priming because this virus uses strategies to downregulate PKR activation and also because translation of avian reovirus mRNAs is more resistant to phosphorylation of the alpha subunit of initiation factor eIF2 than translation of their cellular counterparts. Our results further reveal that the avian reovirus protein sigmaA is able to prevent PKR activation and that this function is dependent on its double-stranded RNA-binding activity. Finally, this study demonstrates that vaccinia virus and avian reovirus, but not vesicular stomatitis virus, express/induce factors that counteract the ability of dithiothreitol to promote eIF2 phosphorylation. Our data demonstrate that each of the three different viruses used in this study elicits distinct responses to interferon and to dithiothreitol-induced eIF2 phosphorylation when infecting avian cells. Type I interferons constitute the first barrier of defense against viral infections, and one of the best characterized antiviral strategies is mediated by the double-stranded RNA-activated protein kinase R (PKR). The results of this study revealed that IFN priming of avian cells has little effect on avian reovirus (ARV) replication but drastically diminishes the replication of vaccinia virus (VV) and vesicular stomatitis virus (VSV) by PKR-dependent and -independent mechanisms, respectively. Our data also demonstrate that the dsRNA-binding ability of ARV protein sigmaA plays a key role in the resistance of ARV toward IFN by preventing PKR activation. Our findings will contribute to improve the current understanding of the interaction of viruses with the host's innate immune system. Finally, it would be of interest to uncover the mechanisms that allow avian reovirus transcripts to be efficiently translated under conditions (moderate eIF2 phosphorylation) that block the synthesis of cellular proteins.

Moroxydine hydrochloride inhibits grass carp reovirus replication and suppresses apoptosis in Ctenopharyngodon idella kidney cells

Moroxydine hydrochloride (Mor) is known to have multi-antiviral activities against DNA and RNA viruses but very little information exists on its pharmacology. The paper was undertaken to explore the antiviral response and antiapoptotic mechanism of Mor against grass carp reovirus (GCRV) in Ctenopharyngodon idella kidney (CIK) cells. The results showed that exposing GCRV-infected cell to 6.3 μg mL−1 of Mor for 96 h avoid ca. 50% apoptosis. Meanwhile, Mor had lower cytotoxicity than ribavirin (Rib) as the value of safe concentration was threefold higher than effective concentration and the compound could ensure sufficient into and out of cells within 4 h when tested at the maximal safe concentration. Mor blocked the GCRV-induced cytopathic effects and eliminated nucleocapsids in CIK cells to keep the normal morphological structure. Moreover, the expressions of viral protein genes were significantly inhibited especially the guanylyl transferase and RNA-dependent RNA polymerase related expression. Furthermore, GCRV caused Bcl-2 down-regulation and Bax mitochondrial translocation was prevented by treatment of CIK cells with Mor. The downstream effector, caspase activity was also significantly inhibited in Mor treated cells. The potential mechanism might be that mitochondrial apoptotic signals were not activated by the intervention of Mor for targeting viral gene expression. Taken together, Mor showed high anti-GCRV activity and had been proved as a secure and promising agent in viral controlling in aquaculture industry.

The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication.

Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1–471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection.

Novel antiviral effect of lithium chloride on mammalian orthoreoviruses in vitro

Reovirus not only causes considerable economic loss in the swine industry of the United States and other countries, but also threatens the public health due to its zoonotic potential. According to previous reports, LiCl has antiviral activity against a number of viruses. The inhibitory effects of LiCl on reovirus life cycle in Vero cells were evaluated. The unpaired t-test and one-way ANOVA were used to analyze the differences between experimental groups. We first found that LiCl treatment significantly inhibited reovirus replication in a dose-dependent manner. Furthermore, we found that this antiviral activity of LiCl targets the early stage of viral replication. LiCl could be a potential drug against reovirus infection.