p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and µNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Heterogeneous Nuclear Ribonucleoprotein A1 and Lamin A/C Modulate Nucleocytoplasmic Shuttling of Avian Reovirus p17.

Avian reovirus (ARV) p17 protein continuously shuttles between the nucleus and the cytoplasm via transcription-dependent and chromosome region maintenance 1 (CRM1)-independent mechanisms. Nevertheless, whether cellular proteins modulate nucleocytoplasmic shuttling of p17 remains unknown. This is the first report that heterogeneous nuclear ribonucleoprotein (hnRNP) A1 serves as a carrier protein to modulate nucleocytoplasmic shuttling of p17. Both in vitro and in vivo studies indicated that direct interaction of p17 with hnRNP A1 maps within the amino terminus (amino acids [aa] 19 to 40) of p17 and the Gly-rich region of the C terminus of hnRNP A1. Furthermore, our results reveal that the formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Utilizing sequence and mutagenesis analyses, we have identified nuclear export signal (NES) 19LSLRELAI26 of p17. Mutations of these residues causes a nuclear retention of p17. In this work, we uncovered that the N-terminal 21 amino acids (aa 19 to 40) of p17 that comprise the NES can modulate both p17 and hnRNP A1 interaction and nucleocytoplasmic shuttling of p17. In this work, the interaction site of p17 with lamin A/C was mapped within the amino terminus (aa 41 to 60) of p17 and p17 colocalized with lamin A/C at the nuclear envelope. Knockdown of hnRNP A1 or lamin A/C led to inhibition of nucleocytoplasmic shuttling of p17 and reduced virus yield. Collectively, the results of this study provide mechanistic insights into hnRNP A1 and lamin A/C-modulated nucleocytoplasmic shuttling of the ARV p17 protein.IMPORTANCE Avian reoviruses (ARVs) cause considerable economic losses in the poultry industry. The ARV p17 protein continuously shuttles between the nucleus and the cytoplasm to regulate several cellular signaling pathways and interacts with several cellular proteins to cause translation shutoff, cell cycle arrest, and autophagosome formation, all of which enhance virus replication. To date the mechanisms underlying nucleocytoplasmic shuttling of p17 remain largely unknown. Here we report that hnRNP A1 and lamin A/C serve as carrier and mediator proteins to modulate nucleocytoplasmic shuttling of p17. The formation of p17-hnRNP A1-transportin 1 carrier-cargo complex is required to modulate p17 nuclear import. Furthermore, we have identified an NES-containing nucleocytoplasmic shuttling domain (aa 19 to 40) of p17 that is critical for binding to hnRNP A1 and for nucleocytoplasmic shuttling of p17. This study provides novel insights into how hnRNP A1 and lamin A/C modulate nucleocytoplasmic shuttling of the ARV p17 protein.

Construction of polycistronic baculovirus surface display vectors to express the PCV2 Cap(d41) protein and analysis of its immunogenicity in mice and swine.

To increase expression levels of the PCV2 Cap(d41) protein, novel baculovirus surface display vectors with multiple expression cassettes were constructed to create recombinant baculoviruses BacSC-Cap(d41), BacDD-2Cap(d41), BacDD-3Cap(d41), and BacDD-4Cap(d41). Our results reveal that the recombinant baculovirus BacDD-4Cap(d41) was able to express the highest levels of Cap(d41) protein. Optimum conditions for expressing the PCV2 Cap(d41) protein were determined, and our results show that 107 of Sf-9 infected with the recombinant baculovirus BacDD-4Cap(d41) at an MOI of 5 for 3 days showed the highest level of protein expression. Mice immunized with the 4Cap(d41) vaccine which was prepared from the recombinant baculovirus-infected cells (107) elicited higher ELISA titers compared to the Cap (d41) vaccine. The 4Cap(d41) vaccine could elicit anti-PCV2 neutralizing antibodies and IFN-γ in mice, as confirmed by virus neutralization test and IFN-γ ELISA. Moreover, the swine lymphocyte proliferative responses indicated that the 4Cap(d41) vaccine was able to induce a clear cellular immune response. Flow cytometry analysis showed that the percentage of CD4+ T cells and CD4+/CD8+ ratio was increased significantly in SPF pigs immunized with the 4Cap(d41) vaccine. Importantly, the 4Cap(d41) vaccine induced an IFN-γ response, further confirming that its effect is through cellular immunity in SPF pigs. An in vivo challenge study revealed that the 4Cap(d41) and the commercial vaccine groups significantly reduce the viral load of vaccinated pigs as compared with the CE negative control group. Taken together, we have successfully developed a 4Cap(d41) vaccine that may be a potential subunit vaccine for preventing the disease associated with PCV2 infections.

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK-cyclin complexes and enhancement of p53 and cyclin H interaction.

The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

Avian reovirus σA-modulated suppression of lactate dehydrogenase and upregulation of glutaminolysis and the mTOC1/eIF4E/HIF-1α pathway to enhance glycolysis and the TCA cycle for virus replication.

Adenosine triphosphate (ATP) is an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. Three cellular factors, isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (vATPase) co-immunoprecipitated with ARV σA and were identified by 2D-LC/MS/MS. ARV enhances glycolytic flux through upregulation of glycolytic enzymes. Increased ATP levels in both ARV-infected and σA-transfected cells were observed by a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams. Furthermore, σA upregulates IDH3B and glutamate dehydrogenase (GDH) to promote glutaminolysis, activating HIF-1α. Both HIF-1α level and viral yield in IDH3B-depleted and glutamine-deprived cells, and inhibition of glutaminolysis was significantly reduced. The σAR155/273A mutant loses its ability to enter the nucleolus, impairing its ability to regulate glycolysis. In addition, we have identified the conserved untranslated regions (UTR) of the 5'- and 3'-termini of the ARV genome segments that are required for viral protein synthesis in an ATP-dependent manner. Deletion of either the 5'- or 3'-UTR impaired viral protein synthesis. Knockdown of σA reduced the ATP level and significantly decreased virus yield, suggesting that σA enhances ATP formation to promote virus replication. Collectively, this study provides novel insights into σA-modulated suppression of LDHA and activation of IDH3B and GDH to activate the mTORC1/eIF4E/HIF-1α pathways to upregulate glycolysis and the TCA cycle for virus replication.

Cell entry of bovine ephemeral fever virus requires activation of Src-JNK-AP1 and PI3K-Akt-NF-κB pathways as well as Cox-2-mediated PGE2 /EP receptor signalling to enhance clathrin-mediated virus endocytosis.

Although we have previously demonstrated that cell entry of bovine ephemeral fever virus (BEFV) follows a clathrin-mediated and dynamin 2-dependent endocytosis pathway, the cellular mechanism mediating virus entry remains unknown. Here, we report that BEFV triggers simultaneously Src-JNK-AP1 and PI3K-Akt-NF-κB signalling pathways in the stage of virus binding to induce clathrin and dynamin 2 expressions, while vesicular stomatitis virus only activates Src-JNK signalling to enhance its entry. Activation of these pathways by ultraviolet-inactivated BEFV suggests a role for virus binding but not viral internalization and gene expression. By blocking these signalling pathways with specific inhibitors, BEFV-induced expressions of clathrin and dynamin 2 were significantly diminished. By labelling BEFV with 3,3'-dilinoleyloxacarbocyanine perchlorate to track viral entry, we found that virus entry was hindered by both Src and Akt inhibitors, suggesting that these signalling pathways are crucial for efficient virus entry. In addition, BEFV also triggers Cox-2-catalysed prostaglandin E2 (PGE2) synthesis and induces expressions of G-protein-coupled E-prostanoid (EP) receptors 2 and 4, leading to amplify signal cascades of Src-JNK-AP1 and PI3K-Akt-NF-κB, which elevates both clathrin and dynamin 2 expressions. Furthermore, pretreatment of cells with adenylate cyclase (cAMP) inhibitor SQ22536 reduced BEFV-induced Src phosphorylation as well as clathrin and dynamin 2 expressions. Our findings reveal for the first time that BEFV activates the Cox-2-mediated PGE2/EP receptor signalling pathways, further enhancing Src-JNK-AP1 in a cAMP-dependent manner and PI3K-Akt-NF-κB in a cAMP-independent manner. Accordingly, BEFV stimulates PGE2/EP receptor signalling amplifying Src-JNK-AP1 and PI3K-Akt-NF-κB pathways in an autocrine or paracrine fashion to enhance virus entry.

Cell Entry of Avian Reovirus Modulated by Cell-Surface Annexin A2 and Adhesion G Protein-Coupled Receptor Latrophilin-2 Triggers Src and p38 MAPK Signaling Enhancing Caveolin-1- and Dynamin 2-Dependent Endocytosis.

The specifics of cell receptor-modulated avian reovirus (ARV) entry remain unknown. By using a viral overlay protein-binding assay (VOPBA) and an in-gel digestion coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we determined that cell-surface annexin A2 (AnxA2) and adhesion G protein-coupled receptor Latrophilin-2 (ADGRL2) modulate ARV entry. Direct interaction between the ARV σC protein and AnxA2 and ADGRL2 in Vero and DF-1 cells was demonstrated in situ by proximity ligation assays. By using short hairpin RNAs (shRNAs) to silence the endogenous AnxA2 and ADGRL2 genes, ARV entry could be efficiently blocked. A significant decrease in virus yields and the intracellular specific signal for σC protein was observed in Vero cells preincubated with the specific AnxA2 and ADGRL2 monoclonal antibodies, indicating that AnxA2 and ADGRL2 are involved in modulating ARV entry. Furthermore, we found that cells pretreated with the AnxA2/S100A10 heterotetramer (A2t) inhibitor A2ti-1 suppressed ARV-mediated activation of Src and p38 mitogen-activated protein kinase (MAPK), demonstrating that Src and p38 MAPK serve as downstream molecules of cell-surface AnxA2 signaling. Our results reveal that suppression of cell-surface AnxA2 with the A2ti-1 inhibitor increased Csk-Cbp interaction, suggesting that ARV entry suppresses Cbp-mediated relocation of Csk to the membrane, thereby activating Src. Furthermore, reciprocal coimmunoprecipitation assays revealed that σC can interact with signaling molecules, lipid raft, and vimentin. The current study provides novel insights into cell-surface AnxA2- and ADGRL2-modulated cell entry of ARV which triggers Src and p38 MAPK signaling to enhance caveolin-1-, dynamin 2-, and lipid raft-dependent endocytosis. IMPORTANCE By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1-mediated and dynamin 2-dependent endocytosis. In this work, we demonstrated that ARV triggers Src activation by impeding Cbp-mediated relocation of Csk to the membrane in the early stages of the life cycle. This work provides better insight into cell-surface AnxA2 and ADGRL2, which upregulate Src and p38MAPK signaling pathways to enhance ARV entry and productive infection.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication.

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

Development of Polycistronic Baculovirus Surface Display Vectors to Simultaneously Express Viral Proteins of Porcine Reproductive and Respiratory Syndrome and Analysis of Their Immunogenicity in Swine.

To simultaneously express and improve expression levels of multiple viral proteins of a porcine reproductive and respiratory syndrome virus (PRRSV), polycistronic baculovirus surface display vectors were constructed and characterized. We engineered polycistronic baculovirus surface display vectors, namely, pBacDual Display EGFP(BacDD)-2GP2-2GP4 and pBacDD-4GP5N34A/N51A (mtGP5), which simultaneously express and display the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP4-gp64TM-CTD, and His-tagged mtGP5-gp64TM-CTD fusion proteins of PRRSV on cell membrane of Sf-9 cells. Specific pathogen-free (SPF) pigs were administered intramuscularly in 2 doses at 21 and 35 days of age with genetic recombinant baculoviruses-infected cells. Our results revealed a high level of ELISA-specific antibodies, neutralizing antibodies, IL-4, and IFN-γ in SPF pigs immunized with the developed PRRSV subunit vaccine. To further assess the co-expression efficiency of different gene combinations, pBacDD-GP2-GP3-2GP4 and pBacDD-2mtGP5-2M constructs were designed for the co-expression of the ectodomain of His-tagged GP2-gp64TM-CTD, His-tagged GP3-gp64TM-CTD, and His-tagged GP4-gp64TM-CTD proteins as well as the ectodomain of His-tagged mtGP5-gp64TM-CTD and His-tagged M-gp64TM-CTD fusion proteins of PRRSV. To develop an ELISA assay for detecting antibodies against PRRSV proteins, the sequences encoding the ectodomain of the GP2, GP3, GP4, mtGP5, and M of PRRSV were amplified and subcloned into the pET32a vector and expressed in E. coli. In this work, the optimum conditions for expressing PRRSV proteins were evaluated, and the results suggested that 4 × 105 of Sf-9 cells supplemented with 7% fetal bovine serum and infected with the recombinant baculoviruses at an MOI of 20 for three days showed a higher expression levels of the protein. Taken together, the polycistronic baculovirus surface display system is a useful tool to increase expression levels of viral proteins and to simultaneously express multiple viral proteins of PRRSV for the preparation of subunit vaccines.

Cordycepin enhances radiosensitivity to induce apoptosis through cell cycle arrest, caspase pathway and ER stress in MA-10 mouse Leydig tumor cells.

Radiotherapy is a localized treatment commonly used in various types of cancer. However, major limitation of radiotherapy is the development of resistance of tumor cells to radiosensitivity. Cordycepin, a predominant functional component of the Cordyceps sinensis, is considered to use in treating tumor cells. In the present study, we investigated the anticancer effect of the combination of radiation and cordycepin in the treatment of Leydig tumor cells. Results showed that the combination treatment has a synergistic effect significantly suppress cell viability and enhance the radiosensitivity in MA-10 mouse Leydig tumor cells. The combination treatment induced MA-10 cell apoptosis through increasing levels of cleaved caspase-3/-8/-9, poly ADP-ribose polymerase (PARP), and cytochrome c and decreasing levels of B-cell lymphoma 2 (Bcl-2). In addition, prolonged sub-G1 and G2/M arrest accompany with cell cycle-related protein regulation was observed in cells that received the combination treatment. The endoplasmic reticulum (ER) stress-related protein expressions were regulated after MA-10 cells treating with a combination of 100 µM cordycepin and 4 Gy radiation. Furthermore, the combination treatment also decreased the Leydig tumor mass by increasing cell apoptosis in tumor-bearing mice. In conclusion, cordycepin enhances radiosensitivity to induce mouse Leydig tumor cells toward apoptosis in vitro and in vivo. This study will provide a scientific basis for the development of therapeutic regimen of testicular cancer.

Oncolytic avian reovirus σA-modulated fatty acid metabolism through the PSMB6/Akt/SREBP1/acetyl-CoA carboxylase pathway to increase energy production for virus replication.

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase ß (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.

Molecular chaperone TRiC governs avian reovirus replication by protecting outer-capsid protein σC and inner core protein σA and non-structural protein σNS from ubiquitin- proteasome degradation.

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Aspirin and 5-Aminoimidazole-4-carboxamide Riboside Attenuate Bovine Ephemeral Fever Virus Replication by Inhibiting BEFV-Induced Autophagy.

Recent study in our laboratory has demonstrated that BEFV-induced autophagy via activation of the PI3K/Akt/NF-κB and Src/JNK pathways and suppression of the PI3K-AKt-mTORC1 pathway is beneficial for virus replication. In the current study, we found that both aspirin and 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR) siginificantly attenuated virus replication by inhibiting BEFV-induced autophagy via suppressing the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways as well as inducing reversion of the BEFV-suppressed PI3K-Akt-mTORC1 pathway. AICAR reversed the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways at the early to late stages of infection and induced reversion of the BEFV-suppressed PI3K-AKt-mTORC1 pathway at the late stage of infection. Our findings reveal that inhibition of BEFV-induced autophagy by AICAR is independent of AMPK. Furthermore, we found that AICAR transcriptionally downregulates the ATG related genes ULK1, Beclin 1, and LC3 and enhances Atg7 degradation by the proteasome pathway. Aspirin suppresses virus replication by inhibiting BEFV-induced autophagy. It directly suppressed the NF-κB pathway and reversed the BEFV-activated Src/JNK pathway at the early stage of infection and reversed the BEFV-suppressed PI3K/Akt/mTOR pathway at the late stage of infection. The current study provides mechanistic insights into the effects of aspirin and AICAR on BEFV replication through suppression of BEFV-induced autophagy.

Baculovirus surface display of the HA protein of H5N2 avian influenza virus and its immunogenicity against a lethal challenge with H5N1 virus in chickens.

In the present study, we have generated several H5N2 HA recombinant baculoviruses for production of a HA subunit vaccine against the lethal H5N2 avian influenza virus (AIV). The effective display of functional HA on the cell membrane and baculoviral envelope was examined. Our results reveal that chickens immunized with the chimeric AIV HA protein fused with the baculovirus gp64 cytoplasmic domain (CTD) induced higher HI titer. To further increase the expression level of the H5N2 AIV HA protein, the HA gene of H5N2 AIV was amplified and cloned into three novel baculovirus surface display vectors BacDual DisplayEGFP-2HA, BacDual DisplayEGFP-3HA, BacDual DisplayEGFP-4HA which contains multiple expression cassettes for higher level display of HA proteins on the cell membrane and baculovirus envelope. To determine the optimum conditions for producing HA protein, various MOI, infection times, and shaker times for virus transfection were tested. Our results reveal that the conditions of an MOI of 5, 3 day post infection, and 15 min of shaker time have higher efficiency for HA protein production. Our results reveal that the baculovirus surface display vector pBacDual DisplayEGFP-4HA increases significantly the expression level of the H5N2 AIV HA protein. Chickens that received two doses of BacDual DisplayEGFP-4HA cell lysates formulated with Montanide ISA70 adjuvant elicited efficient immunogenicity and had an average HI titer of 7 log2 at 2 weeks post-vaccination. Challenge studies revealed that vaccinated chickens with HI titers 5 log2 were completely protected against the lethal H5N1 AIV challenge. Furthermore, HI titers could be maintained at 5 log2 for 20 weeks for laying hens. This study suggests that the HA protein expression from the baculovirus surface display system could be a safe and efficacious subunit vaccine for chickens.

Muscovy duck reovirus p10.8 protein induces ER stress and apoptosis through the Bip/IRE1/XBP1 pathway.

In the present study, the mechanisms underlying Muscovy duck reovirus (MDRV) p10.8 protein-induced ER stress and apoptosis in DF-1 cells and Muscovy duckling hepatic tissues were explored. On the fifth day post-infection, an increase in the mRNA levels of binding immunoglobulin protein (Bip) and X-box binding protein (XBP1), activation of XBP1/s, and an increase in percentage of apoptotic cells were observed in Muscovy duckling livers. The use of ER stress inducer Tunicamycin and ER stress inhibitor Tauroursodeoxycholic acid demonstrated that MDRV induces apoptosis via ER stress, leading to apoptosis. The use of Tunicamycin increased viral protein synthesis while Tauroursodeoxycholic acid reduced viral protein synthesis, suggesting that MDRV induces ER stress benefiting virus replication. The MDRV p10.8 is the major protein to induce ER stress and apoptosis. We found that p10.8 promotes the conversion of XBP1/u to XBP1/s and expands ER diameter, and increases the percentages of apoptotic cells in DF-1 and duckling liver tissues. To investigate the mechanism underlying the MDRV p10.8-induced ER stress and apoptosis, Western blot, siRNA, and co-immunoprecipitation (Co-IP) assays were performed. We found that the MDRV p10.8 protein up-regulates Bip, p-IRE1, XBP1s, and cleaved-caspase 3. Co-IP results reveal that the MDRV p10.8 protein disassociates the Bip/IRE1 complex. Inhibition of IRE1 by 4-methyl umbelliferone 8-carbaldehyde (4u8c) dramatically reversed the MDRV p10.8-modulated increase in levels of XBP1s and cleaved-caspase 3. Knockdown of XBP1 by siRNA reversed the increased level of p10.8-modulated cleaved-caspase 3. The present study provides mechanistic insights into the MDRV p10.8 protein induces ER stress, resulting in apoptosis via the Bip/IRE1/XBP1 pathway in DF-1 cells and duckling livers.

Cdc20 and molecular chaperone CCT2 and CCT5 are required for the Muscovy duck reovirus p10.8-induced cell cycle arrest and apoptosis.

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.

Avian reovirus p17 and σA act cooperatively to downregulate Akt by suppressing mTORC2 and CDK2/cyclin A2 and upregulating proteasome PSMB6.

Although we have shown that avian reovirus (ARV) p17-mediated inhibition of Akt leads to induction of autophagy, the precise mechanisms remain largely unknown. This study has identified a specific mechanism by which ARV coordinately regulates the degradation of ribosomal proteins by p17-mediated activation of E3 ligase MDM2 that targets ribosomal proteins and by σA-mediated upregulation of proteasome PSMB6. In addition to downregulating ribosomal proteins, p17 reduces mTORC2 assembly and disrupts mTORC2-robosome association, both of which inactivate mTORC2 leading to inhibition of Akt phosphorylation at S473. Furthermore, we discovered that p17 binds to and inhibits the CDK2/cyclin A2 complex, further inhibiting phosphorylation of Akt S473. The negative effect of p17 on mTORC2 assembly and Akt phosphorylation at S473 is reversed in cells treated with insulin or overexpression of CDK2. The carboxyl terminus of p17 is necessary for interaction with CDK2 and for induction of autophagy. Furthermore, p17-mediated upregulation of LC3-II could be partially reversed by overexpression of CDK2. The present study provides mechanistic insights into cooperation between p17 and σA proteins of ARV to negatively regulate Akt by downregulating complexes of mTORC2 and CDK2/cyclin A2 and upregulating PSMB6, which together induces autophagy and cell cycle arrest and benefits virus replication.

Suppression of Vimentin Phosphorylation by the Avian Reovirus p17 through Inhibition of CDK1 and Plk1 Impacting the G2/M Phase of the Cell Cycle.

The p17 protein of avian reovirus (ARV) causes cell cycle retardation in a variety of cell lines; however, the underlying mechanism(s) by which p17 regulates the cell cycle remains largely unknown. We demonstrate for the first time that p17 interacts with CDK1 and vimentin as revealed by reciprocal co-immunoprecipitation and GST pull-down assays. Both in vitro and in vivo studies indicated that direct interaction of p17 and CDK1/vimentin was mapped within the amino terminus (aa 1-60) of p17 and central region (aa 27-118) of CDK1/vimentin. Furthermore, p17 was found to occupy the Plk1-binding site within the vimentin, thereby blocking Plk1 recruitment to CDK1-induced vimentin phosphorylation at Ser 56. Interaction of p17 to CDK1 or vimentin interferes with CDK1-catalyzed phosphorylation of vimentin at Ser 56 and subsequently vimentin phosphorylation at Ser 82 by Plk1. Furthermore, we have identified upstream signaling pathways and cellular factor(s) targeted by p17 and found that p17 regulates inhibitory phosphorylation of CDK1 and blocks vimentin phosphorylation at Ser 56 and Ser 82. The p17-mediated inactivation of CDK1 is dependent on several mechanisms, which include direct interaction with CDK1, p17-mediated suppression of Plk1 by activating the Tpr/p53 and ATM/Chk1/PP2A pathways, and p17-mediated cdc25C degradation via an ubiquitin- proteasome pathway. Additionally, depletion of p53 with a shRNA as well as inhibition of ATM and vimentin by inhibitors diminished virus yield while Tpr and CDK1 knockdown increased virus yield. Taken together, results demonstrate that p17 suppresses both CDK1 and Plk1functions, disrupts vimentin phosphorylation, causes G2/M cell cycle arrest and thus benefits virus replication.