We checked hinokitiol activated the hypoxia-inducible factor-1α (HIF-1α) and identified that hinokitiol-induced HIF-1α regulated autophagy. Taken together, this study is the first report demonstrating that hinokitiol protected against prion protein-induced neurotoxicity via autophagy regulated by HIF-1α. We suggest that hinokitiol is a possible therapeutic strategy in neuronal disorders including prion disease.Prion diseases are fatal neurodegenerative disorders that are derived from structural changes of the native PrPc. Recent studies indicated that hinokitiol induced autophagy known to major function that keeps cells alive under stressful conditions. We investigated whether hinokitiol induces autophagy and attenuates PrP (106-126)-induced neurotoxicity. We observed increase of LC3-II protein level, GFP-LC3 puncta by hinokitiol in neuronal cells. Addition to, electron microscopy showed that hinokitiol enhanced autophagic vacuoles in neuronal cells. We demonstrated that hinokitiol protects against PrP (106-126)-induced neurotoxicity  Whole cell proteins were electrophoresed on a 10-15% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane. Immunoreactivity was detected through sequential incubation with primary antibodies, horseradish peroxidase-conjugated secondary antibodies, and an enhanced chemiluminescence reagent, i.e., West save gold detection kit (AbFrontier Inc.). The primary antibodies used for immunoblotting were anti-LC3B , anti-P62 , anti-ATG5  and anti-β-actin . Images were examined using a Fusion FX7 imaging system . Signal band densitometry was analyzed using the Bio-1D software .Primary neuronal cells and SK-N-SH cells were placed in lysis buffer [25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 100 mM NaCl, 1 mM EDTA (ethylene diamine tetra acetic acid), 5 mM MgClTotal ribonucleic acid (RNA) was extracted from primary neuronal cells using the easy-spin™ Total RNA Extraction Kit . The cDNA synthesis was carried out following the instructions in the TaKaRa PrimeScript TM 1st strand cDNA synthesis kit . For qRT-PCR, 1 μl of gene primers with SYBR Green  in 20 μl of reaction volume was applied. The primers were: p62/SQSTM1 , Hif-1α , and β-actin  . All reactions with iTaq SYBR Green Supermix  were performed on the CFX96 real-time PCR detection system .TEM samples were analyzed by Transmission Electron Microscope  installed in the Center for University-Wide Research Facilities (CURF) at Chonbuk National University. After fixing SK-N-SH cell samples in 2.5% glutaraldehyde  in PBS (pH: 7.2), specimens were post-fixed in 1% osmium tetroxide , dehydrated in graded ethanol and propylene oxide , and then embedded in epoxy resin , as used previously. Serial ultrathin sections were cut on an LKB-III ultratome . Ultrathin sections were stained with uranyl acetate  and lead citrate  and were examined using a Hitachi H7600 electron microscope  at an accelerating voltage of 100 kV.t-tests or Welch's corrections were used to compare differences between the two groups. One-way ANOVAs followed by the Dunnett's test were used for multiple comparisons. All statistical analyses were performed with GraphPad Prism software. Results were considered significant at * p < 0.05, ** p < 0.01 or *** p < 0.001.Unpaired


Recent evidence suggests that troxerutin, a trihydroxyethylated derivative of natural bioflavonoid rutin, exhibits beneficial effects on diabetes-related symptoms. Here we investigated the effects of troxerutin on the enhancement of hepatic gluconeogenesis in high-fat diet (HFD)-treated mice and the mechanisms underlying these effects. Mice were divided into four groups: Control group, HFD group, HFD + Troxerutin group, and Troxerutin group. Troxerutin was treated by daily oral administration at doses of 150 mg/kg/day for 20 weeks. Tauroursodeoxycholic acid (TUDCA) was used to inhibit endoplasmic reticulum stress (ER stress). Our results showed that troxerutin effectively improved obesity and related metabolic parameters, and liver injuries in HFD-treated mouse. Furthermore, troxerutin significantly attenuated enhancement of hepatic gluconeogenesis in HFD-fed mouse. Moreover, troxerutin notably suppressed nuclear factor-κB (NF-κB) p65 transcriptional activation and release of inflammatory cytokines in HFD-treated mouse livers. Mechanismly, troxerutin dramatically decreased Nucleotide oligomerization domain (NOD) expression, as well as interaction between NOD1/2 with interacting protein-2 (RIP2), by abating oxidative stress-induced ER stress in HFD-treated mouse livers, which was confirmed by TUDCA treatment. These improvement effects of troxerutin on hepatic glucose disorders might be mediated by its anti-obesity effect. In conclusion, troxerutin markedly diminished HFD-induced enhancement of hepatic gluconeogenesis via its inhibitory effects on ER stress-mediated NOD activation and consequent inflammation, which might be mediated by its anti-obesity effect. Over the past two decades, it has been increasingly appreciated that low-grade inflammation plays a crucial role in type 2 diabetes (T2D) etiology and progression . Excess Nucleotide oligomerization domain (NOD) proteins including NOD1 and NOD2, which belong to a class of intracellular pattern recognition receptors, sense invading bacteria by recognizing intracellular fragments of bacterial peptidoglycan , thereby activating innate immune response to clear those pathogens ,5. In adTroxerutin, known as vitamin P4, is a trihydroxyethylated derivative of natural bioflavonoid rutin, which is widely distributed in tea, coffee, cereal grains, and a variety of fruits and vegetable ,14,15. TAfter four weeks of treatment, HFD-fed mice developed significant obesity A. FurtheAn evident liver injury, as evidenced by significantly elevated serum alanine aminotransferase (ALT) level, was observed in HFD-treated mice G. HematoThese results indicate that troxerutin improves obesity and related metabolic parameters, and liver injuries in HFD-treated mouse.HFD feeding markedly impaired glucose intolerance in mice as evidenced by a diminished ability to lower their blood glucose A. A dramThese results suggest that troxerutin restores hepatic insulin signaling to diminish gluconeogenesis in HFD-treated mouse.interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1)  p65 subunit localization in the mouse livers A, indica (MCP-1) B. Intere (MCP-1) . No signIn combination with the abovementioned results of H&E staining G, these The levels of oxidative stress markers, including reactive oxygen species (ROS) and 4-hydroxynonenal , were markedly elevated in the livers of HFD-treated mice A–C. MoreThese results indicate that troxerutin blocks oxidative stress in the HFD-treated mouse livers.The unfolded protein response (UPR) was largely activated, characterized by the up-regulations of phosphorylated pancreatic endoplasmic reticulum resident kinase (p-PERK) (Thr980), phosphorylated eukaryotic translation initiation factor (p-eIF2α) (Ser51) and phosphorylated inositol-requiring 1 (p-IRE1) (Ser724) protein levels in the livers of the HFD-treated mice A. FurtheER stress is critical to the development of a variety of diseases, including diabetes ,29. In tInterestingly, troxerutin treatment markedly diminished the protein levels of p-PERK (Thr980), p-eIF2α (Ser51), p-IRE1 (Ser724) and TRAF2 in the livers of the HFD-treated mice A. No sigThese results reveal that troxerutin abates ER stress in the HFD-treated mouse livers, which may attenuate fasting hyperglycemia.NOD expression, including NOD1 and NOD2, in mouse livers . Therefore, our results suggested that troxerutin might ameliorate the enhancement of hepatic gluconeogenesis-mediated fasting hyperglycemia by abating ER stress in HFD-treated mouse. A recent study reveals that ER stress induces inflammation through triggering NOD signaling during tissue damage and microbial infection . It is wIn obesity, excessive accumulation of adipose tissue leads to aberrant adipokine secretion, which plays a critical role in the development of various obesity-related metabolic disorders such as insulin resistance and ectopic lipid accumulation . It is wO-(2-hydroxyethyl)] rutin; CAS NO. 7085-55-4, EINECS NO. 230-389-4; FORMULA: C33H42O19; MOL WT: 742.68) was obtained from Baoji Fangsheng Biotechnology Co., Ltd . Normal diet  and HFD diet  were purchased from Research Diets .Troxerutin , rabbit anti-total-Akt, rabbit anti-phospho-PERK (Thr980), rabbit anti-total-PERK, rabbit anti-total-eIF2α, rabbit anti-TRAF2, rabbit anti-NF-κB p65, rabbit anti-histone H3 antibodies and HRP-conjugated anti-rabbit antibodies from Cell Signaling Technology ; rabbit anti-phospho-eIF2α (Ser51), rabbit anti-phopho-IRE1 (Ser724), rabbit anti-total-IRE1 and rabbit anti-NOD1 antibodies from Abcam ; rat anti-NOD2 antibody from eBioscience ; rabbit anti-RIP2, HRP-conjugated anti-mouse and HRP-conjugated anti-rat antibodies from Santa Cruz Biotechnology ; mouse anti-β-actin antibody from Chemicon ; rabbit anti-4-HNE antibody from Alpha Diagnostics ; Texas Red-conjugated anti-rabbit antibody was from Vector Laboratories .® Reagent and 20× peroxide from Cell Signaling Technology ; Trizol reagent from Invitrogen ; Moloney murine leukemia virus reverse transcriptase, random primers, and SYBR premix Ex TaqII from Takara .Other reagents were obtained from the following sources: TUDCA from Calbiochem ; hematoxylin and eosin from Sigma-Aldrich ; nuclear/cytoplasmic isolation kit and bicinchoninic acid assay kit from Pierce Biotechnology ; ALT assay kit from the Jiancheng Institute of Biotechnology ; mouse insulin enzyme-linked immunosorbent assay (ELISA) kit from ALPCO Diagnostics ; adiponectin ELISA kit from Abcam ; GSH assay kit from Cayman Chemical ; 20× LumiGLOAll of the experimental protocols and euthanasia procedures were approved by the Institutional Animal Care and Use Committee of Jiangsu Normal University . Male ICR mice (eight weeks old) were purchased from a Branch of the National Rodent Breeder Center . Mice were maintained at constant temperature (23 ± 1 °C) and humidity (60%), with food and drinking water ad libitum, and were kept on a 12 h light/dark cycle. After one week of acclimatization to the laboratory conditions, mice were randomly divided into four groups: Control group, HFD group, HFD + Troxerutin group, and Troxerutin group, and received the following treatments for 20 weeks: Mice in the Control group and the Troxerutin group were fed a normal diet. Mice in the HFD group and the HFD + Troxerutin group were fed a HFD.The mice of HFD + Troxerutin group and Troxerutin group were administered troxerutin in distilled water orally at the dose of 150 mg/kg/day. An equal volume of distilled water was given to the mice in the Control group, and HFD group by daily oral gavage. The troxerutin dosage used in this study was according to our previous work .Eighteen weeks after HFD feeding, some mice of the HFD group were divided into two subgroups. TUDCA, an ER stress inhibitor, solubilized in phosphate buffered saline (PBS), was given to one subgroup (HFD + TUDCA group) during two weeks by daily intraperitoneal (i.p.) injections at the dose of 200 mg/kg/day, and another subgroup (HFD-control group) received daily i.p. injections of an equal volume of PBS.After 20 weeks of treatment, overnight fasted mice were deeply anaesthetized and sacrificed, and the blood and liver were immediately collected for experiments or stored at −70 °C for later use.After 19 weeks of HFD treatment, glucose tolerance tests were performed as described in our previous work . OvernigLiver slice collection and hematoxylin-eosin staining were performed as previously described ,18. The Immunofluorescence staining of cryo-fixed liver sections was performed as described previously . The livThe preparation of liver homogenates were performed as described in our previous work ,18. The The serum ALT activity was measured by spectrophotometric methods using a kit according to the manufacturer’s instructions. Fasting blood glucose levels were determined with an Ascensia Elite glucose meter. Serum insulin and adiponectin levels were determined with the enzyme-linked immunosorbent assay kits following the manufacturer’s instructions.ROS level was assayed as previously described, which was based on the oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (H2-DCF-DA) to 2′,7′-dichlorofluorescein (DCF) ,18. ROS The hepatic GSH contents were assayed using a commercially available GSH assay kit. After reaction with 5,5-dithiobes-(2-ni-trobenzoic acid) (DTNB), the GSH contents were measured by a spectrophotometer (Shimadzu UV-2501PC) at 405 nm. The GSH contents were calculated as the contents (μmol GSH) per mg protein.SOD1 activity was determined following the method previously described . SOD1 ac2O2 consumed/min/mg of tissue protein.CAT activity was determined as described in our previous work . CAT act2, 1 mM NaF, 1% NP-40, and 1 mM Na3VO4, immune complexes were analyzed by Western blot with anti-NOD1, anti-NOD2 and anti-RIP2 antibodies.Immunoprecipitations were conducted as previously described . After p® Reagent and 20× peroxide. The optical density (OD) values of the detected bands were quantified by Scion Image analysis software  and were normalized using appropriate internal controls .The Western blot analyses were performed as previously described ,18. BriePEPCK, Forward: 5′-CAGCCAGTGCCCCATTATT-3′, Reverse: 5′-CCACCAAAGATGATACCCTCA-3′; G6P, Forward: 5′-TGGCCTGGCTTATTGTACCT-3′, Reverse: 5′-GTGCTAAGAGGAAGACCCGA-3′; NOD1, Forward: 5′-TGACGTTCCTGGGTTTATACAACA-3′, Reverse: 5′-CCAGGATTTGGGCCACATAC-3′; NOD2, Forward: 5′-CCTGGTACGTGCCCAAAGTAG-3′, Reverse: 5′-GCCAAGTAGAAAGCGGCAAA-3′; IL-1β, Forward: 5′-AAATACCTGTGGCCTTGGGC-3′, Reverse: 5′-CTTGGGATCCACACTCTCCAG-3′; TNF-α, Forward: 5′-TCTCATTCCTGCTTGTGG-3′, Reverse: 5′-ACTTGGTGGTTTGCTACG-3′; MCP-1, Forward: 5′-AGGTCCCTGTCATGCTTCTG-3′, Reverse: 5′-GCTGCTGGTGATCCTCTTGT-3′; β-actin, Forward: 5′-TGCTGTCCCTGTATGCCTCTG-3′, Reverse: 5′-TTGATGTCACGCACGATTTCC-3′. The relative levels of target mRNAs, were normalized to β-actin mRNA, and were calculated by the comparative cycle threshold (Ct) method.The quantitative real time polymerase chain reaction was performed as described in our previous work . The prit-test. Statistical significance was set to p < 0.05.Data analysis was conducted using SPSS software version 11.5 . All of the data were expressed as the means ± standard deviation (SD) and were analyzed by one-way ANOVA followed by Tukey’s Honestly Significant Difference (HSD) post-hoc test and Student’s Troxerutin displayed significant inhibitory effects on the enhancement of hepatic gluconeogenesis by attenuating ER stress-mediated NOD activation and consequent inflammation in the livers of HFD-treated mice, which might be mediated by its anti-obesity effect. Our results showed that troxerutin abated oxidative stress to restrain ER stress, therefore blocking NOD signaling, consequently suppressing NF-κB p65 transcriptional activation and release of inflammatory cytokines, thereby diminishing hepatic gluconeogenesis, ultimately lowering fasting glucose levels in HFD-fed mice. Moreover, troxerutin ameliorated obesity and decline of circulating adiponectin in HFD-treated mouse, indicating these improvements of hepatic glucose disorders might be mediated by its anti-obesity effect. This study provides insight into a novel mechanism for T2D pathogenesis and indicates that troxerutin is a candidate for pharmacological treatment of fasting hyperglycemia via its inhibitory effects on elevated hepatic gluconeogenesis.


MicroRNAs regulate multiple aspects of the host response to viral infection. This study verified that the expression of cfa-miR-143 was upregulated in vivo and in vitro by canine influenza virus (CIV) H3N2 infection. To understand the role of cfa-miR-143 in CIV-infected cells, the target gene of cfa-miR-143 was identified and assessed for correlations with proteins involved in the apoptosis pathway. A dual luciferase reporter assay showed that cfa-miR-143 targets insulin-like growth factor binding protein 5 (Igfbp5). Furthermore, a miRNA agomir and antagomir of cfa-miR-143 caused the downregulation and upregulation of Igfbp5, respectively, in CIV-infected madin-darby canine kidney (MDCK) cells. This study demonstrated that cfa-miR-143 stimulated p53 and caspase3 activation and induced apoptosis via the p53 pathway in CIV H3N2-infected cells. In conclusion, CIV H3N2 induced the upregulation of cfa-miR-143, which contributes to apoptosis via indirectly activating the p53-caspase3 pathway. MicroRNAs (miRNAs) are small, non-coding RNAs 18–22 nt in length that suppress gene expression via perfect or near-perfect base-pairing with complementary sequences in mRNA and participate in a wide range of biological processes ,4,5,6,7 Recently, miRNA expression profiles of influenza-infected hosts have been generated through high-throughput sequencing (HTS) and microarray technologies ,17,18,19Since 2006, we have been committed to epidemic monitoring and exploring the pathogenesis of canine influenza virus (CIV). We have previously shown that cfa-miR-143 is upregulated in the lungs of CIV H3N2-infected dogs . miR-14350) according to the Reed-Muench calculation method in MDCK cells [Madin-Darby Canine Kidney (MDCK) cells  and 293T cells were cultured in Minimal Essential Medium (MEM)  and Dulbecco’s Modified Eagle Medium (DMEM) containing 5% fetal bovine serum (Gibco) and 200 IU penicillin-streptomycin (Gibco). Cells were harvested using 0.25% trypsin-EDTA (1×) (Gibco). A/Canine/Guangdong/01/2007(H3N2) was propagated in 11-day-old specific pathogen free (SPF) embryonated eggs. Viral titers were measured by determining the 50% tissue culture infectious dose (TCIDCK cells  and a plCK cells .7 EID50 (50% egg infectious dose)/animal of H3N2 CIV in a 1mL volume. For the uninfected controls, six additional beagles were inoculated with 1 mL of Phosphate Buffer Saline (PBS) and housed in a separate room. All animals, except for those euthanized for lung tissue collection, were monitored daily for body temperature and virus shedding for 14 days, and serum antibodies were detected at 14 days post-inoculation (dpi). At 3 dpi and 7 dpi, three animals in each inoculated group were euthanized. Lungs were collected to assess viral replication and miRNA expression. This study protocol was reviewed and approved by the Institutional Review Board of South China Agricultural University (identification code: 2014-07).One-month-old beagles were shown to be sero-negative by a hemagglutination-inhibition (HI) assay for seasonal influenza viruses  and for avian-origin CIV H3N2. All procedures and animal housing were carried out in the animal laboratory of South China Agricultural University. After the animals were lightly anesthetized using tiletamine–zolazepam , nine beagles were intranasally inoculated with 102 at 37 °C for 1 h. Then, the inoculum was discarded and replaced with MEM containing 2 μg/mL TPCK-trypsin and 0.2% bovine serum albumin (BSA), and the cells were incubated in 5% CO2 at 37 °C.MDCK cells were inoculated with CIV H3N2  and incubated in 5% COn = 3) and MDCK cells using a miRNA extraction kit . Total RNA was extracted from the same samples using Trizol reagent  according to manufacturer’s instructions. The total RNA concentration was measured by a spectrophotometer  and either used for subsequent experiments or stored at −80 °C until use.Small RNAs (<200 nt) were extracted from the lungs  and TaqMan Universal Master Mix-II  as previously described . A probeXhoI and PmeI sites. Igfbp5-3′UTR target site mutations were introduced using specific primers (® 2000 reagent (Invitrogen) with cfa-miR-143 mimic (50 nM) in 6-well plates. At 36 h post-transfection, luciferase activity was detected using a Dual-Glo Luciferase Assay Kit  according to the manufacturer’s protocol. Luminescence intensity was read using a GloMax® Discover System . The relative fluorescence value was calculated from the ratio of renal luciferase to firefly luciferase activity. Transfections were performed in duplicate and repeated thrice [The Igfbp5 gene (NCBI Reference Sequence: XM_847792.4) was predicted as the target gene of cfa-miR-143 based on biological information from the online software platforms miRDB and TargetScan-Vet. The genomic DNA of MDCK cells was extracted using a Genomic DNA Extraction Kit , and the 3′-untranslated region (UTR) of the target gene was amplified using specific primers . The PCR primers . cfa-miRd thrice .Total protein was extracted from MDCK cells infected with CIV H3N2 at specific time points using a Protein Assay Kit , and proteins including p53 (tumor suppressor protein), caspase3, cleaved caspase3, bax, bcl-2, PI3K (phosphatidylinositol-3 kinase), AKT, p-Akt and HA (hemagglutinin) were detected by Western blotting. The following primary antibodies were used: rabbit anti-Akt polyclonal antibody , rabbit anti-phosphorylated Akt  , mouse anti-p53 monoclonal antibody , rabbit anti-caspase3 monoclonal antibody (Beyotime), rabbit anti-cleaved caspase3 (Asp175) monoclonal antibody (Beyotime), anti-bcl-2 monoclonal antibody , rabbit anti-bax monoclonal antibody (Bioss), mouse anti-PI3K p85 monoclonal antibody , rabbit anti-phospho-PI3 Kinase p85 (Tyr458)/p55(Tyr199) antibody , and a mouse anti-HA (H3N2 CIV) polyclonal antibody from our lab. The secondary antibodies were an IRDye 680RD goat anti-rabbit IgG (H + L) polyclonal antibody (LICOR) and IRDye 800CW goat anti-rabbit IgG (H + L) polyclonal antibody (LICOR) . All antibody dilutions and the Western blotting procedure were performed per the manufacturer’s protocol. Relative expression levels were assessed relative to the reduced GAPDH protein expression detected using a mouse anti-GAPDH monoclonal antibody .For flow cytometry, cells were harvested by 0.25% trypsin (no EDTA), followed by washing with PBS. Cell death was assessed by two-color flow cytometric analysis using an Annexin-APC/PI apoptosis kit (Ebioscience) .t test. All assays were run in triplicate. The data are shown as the mean ± S.E., and a p value < 0.05 was considered statistically significant .Statistical significance was determined using the conventional Student’s 50/mL, respectively  due to the action of miRNA agomir but decreased to 9.04 ± 0.51% (p < 0.01) with siR-caspase3 (p < 0.01) with siR-TP53 .Manikandan et al. indicated that miR-143 likely indirectly upregulates p53 in oral squamous cell carcinoma . To undeCIV H3N2 induced the upregulation of cfa-miR-143, which promotes cell apoptosis by indirectly activating the p53-caspase3 pathway.


SLC6A4). In the present longitudinal study we assessed: (a) the effects of NICU-related stress and SLC6A4 methylation variations from birth to discharge on brain development at term equivalent age (TEA); (b) the association between brain volume at TEA and socio-emotional development  at 12 months corrected age (CA). Twenty-four infants had complete data at 12-month-age. SLC6A4 methylation was measured at a specific CpG previously associated with NICU-related stress and socio-emotional stress. Findings confirmed that higher NICU-related stress associated with greater increase of SLC6A4 methylation at NICU discharge. Moreover, higher SLC6A4 discharge methylation was associated with reduced anterior temporal lobe (ATL) volume at TEA, which in turn was significantly associated with less-than-optimal GMDS Personal-Social scale score at 12 months CA. The reduced ATL volume at TEA mediated the pathway linking stress-related increase in SLC6A4 methylation at NICU discharge and socio-emotional development at 12 months CA. These findings suggest that early adversity-related epigenetic changes might contribute to the long-lasting programming of socio-emotional development in VPT infants through epigenetic regulation and structural modifications of the developing brain.Very preterm (VPT) infants admitted to Neonatal Intensive Care Unit (NICU) are at risk for altered brain growth and less-than-optimal socio-emotional development. Recent research suggests that early NICU-related stress contributes to socio-emotional impairments in VPT infants at 3 months through epigenetic regulation  of the serotonin transporter gene ( SLC6A4) methylation ; c*d, β = -.28, p = .02, 95% C.I. ; e*f, β = -.29, p = .02, 95% C.I. .(TIF)Click here for additional data file.S1 TableNote. Chr17 = Chromosome 17; TSS = Transcriptional Start Site.(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


To address these questions, we have investigated neuromodulatory signaling in the context of Caenorhabditis elegans egg-laying. Egg-laying activity cycles between discrete states–short bursts of egg deposition (active phases) that alternate with prolonged quiescent periods (inactive phases). Here using genetic, pharmacological and optogenetic approaches for cell-specific activation and inhibition, we show that a group of neurosecretory cells (uv1) located in close spatial proximity to the egg-laying neuromusculature direct the temporal organization of egg-laying by prolonging the duration of inactive phases. We demonstrate that the modulatory effects of the uv1 cells are mediated by peptides encoded by the nlp-7 and flp-11 genes that act locally to inhibit circuit activity, primarily by inhibiting vesicular release of serotonin from HSN motor neurons. This peptidergic inhibition is achieved, at least in part, by reducing synaptic vesicle abundance in the HSN motor neurons. By linking the in vivo actions of specific neuropeptide signaling systems with the generation of stable behavioral outcomes, our study reveals how cycles of neuromodulation emanating from non-neuronal cells can fundamentally shape the organization of a behavioral program.Animal behaviors are often composed of distinct alternating behavioral states. Neuromodulatory signals are thought to be critical for establishing stable behavioral states and for orchestrating transitions between them. However, we have only a limited understanding of how neuromodulatory systems act  in vivo to elicit alternate behavioral outcomes. Egg-laying behavior in the model system C. elegans cycles between clusters of egg-laying and prolonged inactive periods. This temporal organization provides for spatial dispersal of eggs, presumably benefiting progeny by limiting local overcrowding and competition for food. Here we uncover a novel neuromodulatory mechanism that shapes the timing of egg-laying behavior. Specifically, we find that neuromodulatory signaling from a group of non-neuronal cells specifies transitions between active and inactive phases of egg-laying by regulating neurotransmitter release from the motor output neurons of the egg-laying circuit. Advancing our knowledge of how neuropeptides and other modulators act in the context of the circuits in which they are endogenously released will be critical in ongoing efforts to understand how alternate behavioral states, for example those underlying mood or arousal, are encoded.Animals have robust mechanisms in place to shape their behavior in a manner that is beneficial both for their survival and for the survival of their progeny. A class of signaling molecules known as neuropeptides have been implicated in driving transitions between behavioral states but we have only a limited understanding of how neuropeptide signaling modulates neural circuit activity  Animals have robust mechanisms in place to shape their behavior in a manner that is beneficial both for their survival and for the survival of their progeny. In many cases behaviors are composed from discrete, opposing behavioral states. Examples of behaviors that are organized in this manner range from the motor programs underlying feeding or locomotion to more complex behaviors such as mood or arousal –5. In eain vivo activation of specific neuromodulatory systems with changes in circuit performance and the generation of alternate behavioral outcomes remains a challenge. Uncovering basic principles by which neuromodulators influence circuit activity and behavior in more simple systems provides an important framework for tackling similar questions in the mammalian brain. In particular, the neural circuit controlling egg-laying behavior in the nematode Caenorhabditis elegans provides a relatively simple and experimentally tractable system to address these questions.How the nervous system establishes the duration of particular behavioral states and executes transitions between them, however, is largely unclear. Neuromodulators such as neuropeptides are attractive candidates for orchestrating these processes –11. PharC. elegans egg-laying behavior cycles probabilistically between two alternative states: active phases containing short bursts of egg-laying and prolonged periods of quiescence (inactive phases) , LX2047: lite-1(ce314);lin-15(n765ts);vsIs189[Pocr-2::ChR::YFP::ocr-2 3’UTR], IZ2634: nlp-7(tm2984);flp-11(tm2706);lite-1(ce314);lin-15(n765ts);vsIs189, NY2040: ynIs40[pflp-11::GFP], IZ1130: lin-15(n765ts);ufEx378 [pnlp-7::nlp-7::SL2::mCherry], IZ1135: nlp-7(tm2984), IZ2022: flp-11(tm2706), IZ1589: nlp-7(tm2984);flp-11(tm2706); IZ2263: nlp-7(tm2984);flp-11(tm2706);ufEx792, IZ2407: nlp-7(tm2984);flp-11(tm2706);ufEx876, IZ2834: nlp-7(tm2984);flp-11(tm2706);ufEx1099 , IZ1823: nlp-7(tm2984);flp-11(tm2706);ufEx582, LX1836: lite-1(ce314);lin-15(n765ts);wzIs30[pegl-6::ChR::YFP], IZ1587: nlp-7(tm2984); lite-1(ce314);lin-15(n765ts);wzIs30, IZ2008: flp-11(tm2706); lite-1(ce314);lin-15(n765ts);wzIs30, IZ2007: nlp-7(tm2984);flp-11(tm2706); lite-1(ce314);lin-15(n765ts);wzIs30, IZ1614: ufIs118;lite-1(ce314);lin-15(n765ts);wzIs30, IZ1236: ufIs118 , IZ2109: ufEx730[Phsp16.2::nlp-7], IZ1594: ufEx521, IZ2006: ufEx681, IZ1692: ufEx548, RB850: egl-47(ok677)V, IZ1235: egl-47(ok677);ufIs118, IZ1324: goa-1(n1134);ufIs118, IZ2096: egl-47(ok677), goa-1(n1134);ufIs118, MT2426: goa-1(n1134), IZ2683: egl-47(ok677), goa-1(n1134), IZ2474: egl-47(ok677), goa-1(n1134);ufIs118;lite-1(ce314);lin-15(n765ts);wzIs30, IZ236: ufIs6 , IZ1274: ufIs6;ufIs118, LX967: lin-15(n765ts);vsIs103, IZ1881: nlp-7(tm2984); lin-15(n765ts);vsIs103, IZ1880: flp-11(tm2706); lin-15(n765ts);vsIs103, IZ1884: nlp-7(tm2984);flp-11(tm2706); lin-15(n765ts);vsIs103, IZ1812: ufIs118; lin-15(n765ts);vsIs103, BL5752: inIs181(Pida-1::IDA-1::GFP);inIs182(Pida-1::IDA-1::GFP), IZ2681: inIs181;inIs182;ufEx378, TV38: wyIs12 , IZ2818: wyIs12;ufIs118, IZ2819: wyIs12;nlp-7(tm2984);flp-11(tm2706).All nematode strains were maintained at 20°C on agar nematode growth media plates seeded with HisCl: A fragment containing the coding sequence of HisCl SL2 trans-spliced with GFP was ligated into pKMC281 (a gift from the Koelle lab) between the ocr-2 promoter and the ocr-2 3’ UTR to generate pNB41 (pocr-2::HisCl::SL2::GFP::ocr-2 3’UTR). This was injected (100 ng/μl) into N2 animals along with the coinjection marker pBB107 . The resulting extrachromosomal array was stably inserted by X-ray integration to generate the transgene ufIs160 and outcrossed four times to wild type.nlp-7 expression pattern: pCL17  was injected into lin-15(n765ts) animals along with the coinjection marker pL15EK (lin-15(+), 50 ng/μl).nlp-7 and flp-11 rescue: For nlp-7 rescue, a 5104 bp PCR product containing the nlp-7 promoter, genomic locus and the 3’UTR  was injected at 50 ng/μl into strain IZ1589 along with the coinjection marker pHP6 . For flp-11 rescue, a 3240 bp PCR product containing about 2.5 kb of the flp-11 promoter, genomic locus and the 3’UTR  was injected (100 ng/μl) into strain IZ1589 along with pHP6 (30 ng/μl).NLP-7 overexpression: The NLP-7 OE strain (ufIs118) was generated by microinjection and subsequent X-ray integration of a 5104 bp PCR product (injected at 100 ng/μl) containing the nlp-7 promoter (about 3.5 kb), genomic locus and the 3’UTR  along with pHP6  as coinjection marker. The integrated strain was outcrossed five times with wild type.Heat shock overexpression of nlp-7: pNB35  was injected into N2 animals along with pHP6  as coinjection marker.uv1: The nlp-7 genomic sequence was ligated into pKMC281 (a gift from the Koelle lab) between the ocr-2 promoter and the ocr-2 3’ UTR to generate pNB28 (Pocr-2::nlp-7::ocr-2 3’UTR). A PCR product amplified from this plasmid was injected at 100 ng/μl into N2 animals together with pBB107 (30 ng/μl).VC: A fragment containing the lin-11 enhancer region fused to the pes-10 basal promoter was PCR amplified from pDM4 and ligated into pENTR/D-TOPO vector to generate a gateway entry vector. It was recombined with gateway destination vectors containing the nlp-7 genomic sequence and mCherry coding sequence to generate pNB14 and pNB15. pNB14  was coinjected with pNB15  into N2 animals together with pHP6 (30 ng/μl).Head and tail neurons: A 2537 bp fragment containing the nlp-7 promoter  was amplified from genomic DNA and ligated into pENTR/D-Topo vector to generate a Gateway entry vector. This was recombined to Gateway destination vectors containing the nlp-7 genomic sequence and mCherry coding sequence to generate pNB24 (Pnlp-7(2.5kb)::nlp-7) and pNB25 (Pnlp-7(2.5kb)::mCherry) respectively. pNB24 (100 ng/μl) and pNB25 (50 ng/μl) were coinjected into N2 animals together with pHP6 (30 ng/μl).Age-matched adults were obtained by collecting late fourth larval stage (L4) animals and culturing at 20°C for 30 hrs  were transferred to fresh nematode growth medium plates , allowed to lay eggs for 30 mins and removed. Eggs laid on the plates were examined by a high power dissecting microscope and categorized as described in Ringstad and Horvitz, 2008.Single one-day old (24–30 hrs after the L4 stage) adult animals were placed individually on NGM agar plates and videotaped at room temperature for 4–5 hours at a rate 0.5 frames/s. Intervals between clusters of egg-laying  and intervals between individual egg-laying events within a cluster  were determined by video analysis and manually scoring the timing of egg-laying events.in utero, age matched adults (24 hrs after the L4 stage) were placed on seeded NGM plates with or without histamine. After 6 hrs at room temperature, animals were individually bleached and the number of eggs retained in utero were counted. To analyze the timing of egg-laying events, one-day old adults were placed on histamine plates. After 1 hr of histamine exposure, animals were videotaped for 3–5 hrs while on the same histamine plates. The timing of individual egg-laying events was determined by manual video analysis. Control animals were treated similarly except that they were placed on plates lacking histamine.Histamine plates were made by diluting histamine dihydrochloride (Sigma Aldrich) into NGM agar media cooled to 55°C, to a final concentration of 50 mM. Plates were stored at 4°C and used within 2 weeks. To quantify the number of eggs Individual staged adult animals were placed in 50 μl of low salt M9 buffer, or M9 containing 7.5 mg/ml serotonin or 0.5 mg/ml fluoxetine. After 1 hour in each condition, the number of eggs laid by each animal was quantified.trans retinal to a final concentration of 100 μM and 150 μl of the mix was seeded onto individual NGM agar plates. Plates were stored at 4°C under dark conditions and used within one week. Photostimulation experiments were conducted using a fluorescent dissecting microscope (Zeiss steREO Discovery.V12) equipped with a GFP filter set. Light intensities were measured at the surface of assay plates using a light meter. For all photostimulation experiments, control and experimental animals were treated similarly except that controls did not receive retinal exposure.Synchronized young adult animals were transferred to retinal plates for roughly 18 hours prior to experiment. To prepare retinal plates, an overnight culture of OP50 was mixed with all-2) for 5 seconds and subsequent egg-laying events were monitored. Videotaping was continued for 5–6 mins after the last egg-laying event to ensure the end of an active phase. We noted that photostimulation elicited an escape-like response in a subset of animals expressing the Pocr-2::ChR2::ocr-2 3’UTR transgene ;flp-11(lf) double mutants where we did not observe appreciable inhibition of egg-laying  and assayed immediately for egg-laying response to blue light exposure.Groups of 10 age-matched adult animals (picked at L4 stage on the day prior to experiments) were transferred to NGM agar plates seeded with OP50, wrapped in parafilm and submerged into a water-bath at 33°C for 30 mins. After 30 mins, the animals were transferred to 20°C and the number of eggs laid were counted every hour.Images were acquired using a Zeiss Axioskop 2 microscope system and LSM Pascal 5 imaging software (Zeiss). Images were processed using ImageJ software. Worms were mounted on agarose pads and immobilized with 0.3 M sodium azide. All images were obtained from staged young adult animals (~24hrs after the L4 stage).Images were acquired using an Olympus BX51WI spinning disc confocal microscope. Age-matched adults (30 hrs after the L4 stage) were immobilized using 0.3 M sodium azide on slides with 2% agarose pads. z-stack images of HSN synapses and cell bodies were acquired from all animals. Images were analyzed using Volocity6.3 software. Total SNB-1::GFP volume, number of SNB-1::GFP varicosities and GFP::SYD-2 clusters in the HSN synaptic regions were determined as described in . Mean SNSNB-1::GFP and DsRed2 intensities in HSN cell bodies were determined by calculating their mean intensities within each cell body and subtracting the threshold value (1500 units above background).S1 Fignlp-7::nlp-7::SL2::mCherry. Scale bar, 100 μm. (C) Representative confocal images showing coexpression of Pnlp-7::SL2::mCherry with Pida-1::IDA-1::GFP in the uv1 cells, VC4 and VC5 motor neurons. Scale bar, 5 μm. (D) nlp-7 gene structure. Solid boxes represent exons. Sequences deleted in tm2984 allele are indicated. Predicted peptide products are shown to the right of corresponding gene models.(A) Representative confocal images showing distribution of the dense core vesicle marker IDA-1::GFP (green) in a single uv1 cell. IDA-1::GFP shows a punctate distribution and is enriched near the cell periphery. Scale bar, 2 μm. (B) Fluorescent images of whole animals expressing P(TIF)Click here for additional data file.S2 Figflp-11::GFP. Scale bar, 100 μm. (B) flp-11 gene structure. Solid boxes represent exons. Sequences deleted in tm2706 allele are indicated. Predicted peptide products are shown to the right of corresponding gene models.(A) Fluorescent confocal images of whole animals expressing P(TIF)Click here for additional data file.S3 Fignlp-7;flp-11 double mutants (upper) or with uv1-specific expression of either flp-11 (middle) or nlp-7 (lower) in nlp-7;flp-11 double mutants. uv1-specific expression of either precursor produced a significant decrease in the proportion of eggs that were laid as 1–8 cell embryos. p<0.0001, Fisher’s exact test.Distribution of the developmental stages of eggs laid by either (TIF)Click here for additional data file.S4 FigQuantification of eggs laid in 1 hour for the genotypes indicated, in either control buffer (M9) or on NGM plates seeded with a thin bacterial lawn. Bars represent mean ± SEM for each condition. Numbers above bars indicate n for each condition. Data for M9 were duplicated from (TIF)Click here for additional data file.S5 Fig3 was considered as a varicosity [vsIs103, Ptph-1::SNB-1::GFP; Ptph-1::DsRed2) for the genotypes indicated. Scale bar, 3 μm.  Quantification of average SNB-1::GFP (D) or DsRed2 (E) intensity in cell bodies of the HSNs for the genotypes indicated. Bars represent mean ± SEM for each condition. Numbers in bars indicate the n for each condition. *p<0.05, ANOVA with Sidak’s test. Quantification of total SNB-1::GFP volume (A) and number of SNB-1::GFP varicosities (B) in HSN synaptic regions for the genotypes indicated. SNB-1::GFP volume greater then 1 μmricosity . (C) Rep(TIF)Click here for additional data file.S6 FigwyIs12, Punc-86::GFP::SYD-2; Podr-1::GFP) for the genotypes indicated. Scale bar, 3 μm.  Quantification of average GFP::SYD-2 intensity (B) or number of SYD-2 clusters (C) in the HSN synaptic region for the genotypes indicated. Bars represent mean ± SEM for each condition. Numbers in bars indicate the n for each condition.(A) Representative confocal images of HSN synapses in transgenic animals expressing the active zone marker GFP::SYD-2 in the HSNs ((TIF)Click here for additional data file.S7 Fig(A) During the uv1 “off” phase, spontaneous activity of the HSN neurons elicits serotonin release onto the Vm2 vulval muscles, triggering entry into an active phase of egg-laying. Reduced levels of uv1 activity promote “short” inactive phases (bottom). (B) During the uv1 “on” phase, activation of the uv1 cells triggers release of NLP-7 and FLP-11 peptides. Release of these peptides promotes the termination of an active phase and lengthens the duration of the inactive phase (bottom), at least in part, by reducing serotonergic activation of vulval muscles. Under normal (favorable) conditions, cycles of uv1 activity shape the timing of egg-laying events. Solid lines indicate synaptic connections. Dashed lines indicate volume transmission. Arrows indicate excitation. Circles indicate inhibition. In the lower panel of A and B, each tick mark represents a single egg-laying event.(TIF)Click here for additional data file.S1 MoviePocr-2::ChR2::YFP::ocr-2 3’UTR. Egg-laying events are indicated by numbers. Light stimulation is initiated immediately following the first egg-laying event. The timing and duration of blue light are indicated by blue text. The animal performs an avoidance response upon blue light exposure, likely due to ChR2 expression in 2–3 head sensory neurons Click here for additional data file.S2 Movienlp-7(lf);flp-11(lf) double mutant animal expressing channelrhodopsin in the uv1 cells continues egg-laying following light stimulation of the uv1 cells. Note that the avoidance response to light stimulation is unaffected by combined deletion of nlp-7 and flp-11, indicating that NLP-7 and FLP-11 peptides are required for inhibition of the egg-laying response but not for the execution of the avoidance behavior. Egg-laying events are indicated by numbers (black). Light stimulation is initiated immediately following the first egg-laying event. The timing and duration of stimulation are indicated by blue text. Video is played 6X faster than real time.(MP4)Click here for additional data file.S3 MoviePegl-6a::ChR2::YFP. Timing of light stimulation is indicated by white text. The animal initiates a burst of egg-laying a few seconds following the onset of light stimulation.Light stimulation of the HSNs in a control animal expressing (MOV)Click here for additional data file.


Poxviruses have evolved multiple strategies to subvert signaling by Nuclear Factor κB (NF-κB), a crucial regulator of host innate immune responses. Here, we describe an orf virus (ORFV) virion-associated protein, ORFV119, which inhibits NF-κB signaling very early in infection (≤ 30 min post infection). ORFV119 NF-κB inhibitory activity was found unimpaired upon translation inhibition, suggesting that virion ORFV119 alone is responsible for early interference in signaling. A C-terminal LxCxE motif in ORFV119 enabled the protein to interact with the retinoblastoma protein (pRb) a multifunctional protein best known for its tumor suppressor activity. Notably, experiments using a recombinant virus containing an ORFV119 mutation which abrogates its interaction with pRb together with experiments performed in cells lacking or with reduced pRb levels indicate that ORFV119 mediated inhibition of NF-κB signaling is largely pRb dependent. ORFV119 was shown to inhibit IKK complex activation early in infection. Consistent with IKK inhibition, ORFV119 also interacted with TNF receptor associated factor 2 (TRAF2), an adaptor protein recruited to signaling complexes upstream of IKK in infected cells. ORFV119-TRAF2 interaction was enhanced in the presence of pRb, suggesting that ORFV119-pRb complex is required for efficient interaction with TRAF2. Additionally, transient expression of ORFV119 in uninfected cells was sufficient to inhibit TNFα-induced IKK activation and NF-κB signaling, indicating that no other viral proteins are required for the effect. Infection of sheep with ORFV lacking the ORFV119 gene led to attenuated disease phenotype, indicating that ORFV119 contributes to virulence in the natural host. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb. Poxviruses have evolved multiple strategies to subvert signaling by NF-κB, a crucial regulator of host innate immune responses. Viruses often encode multiple inhibitory proteins, which largely target cytoplasmic activation events of NF-κB signaling. The retinoblastoma protein (pRb), a multifunctional protein best known for its tumor suppressor activity, has been suggested to affect NF-κB signaling during virus infection however, viral effectors and mechanisms of actions are unknown. Here, we identified a virion-associated orf virus NF-κB inhibitory protein, ORFV119, which interacts with pRb. ORFV119 was shown to inhibit IKK complex activation in a pRb-dependent manner early in infection. Results show that ORFV119 interacted with both pRb and TRAF2 and that a ORFV119-pRb complex likely is required for efficient interaction with TRAF2 and inhibition of NF-κB signaling. ORFV119 represents the first poxviral protein to interfere with NF-κB signaling through interaction with pRb. Orf virus (ORFV), the type member of the genus Parapoxvirus of the Poxviridae, is the causative agent of orf or contagious ecthyma, a ubiquitous disease of sheep and goats characterized by proliferative lesions affecting muco-cutaneous tissues of the mouth and muzzle ,2. Orf iin vivo [Like other parapoxviruses (PPV), ORFV is highly epitheliotropic, and keratinocytes and their ontogenetically related counterparts in the oral mucosa are the most important if not the only cell type to support ORFV replication in vivo . In addiin vivo ,8. Engagin vivo .The NF-κB family of transcription factors consists of five members, NF-κB-p65 (RelA), RelB, c-Rel, NF-κB-p50/p105, and NF-κB-p52/p100, which contain an N-terminal Rel homology domain (RHD) responsible for homo- and heterodimerization and for sequence specific DNA binding ,12. The Many viruses with dissimilar life styles are known to interfere with the NF-κB pathway. In particular, poxviruses have evolved multiple strategies to counteract NF-κB function, indicating that inhibition of NF-κB-mediated transcription is important for successful infection of the host. This is not surprising as poxvirus infections are sensed by NF-κB-activating PRRs such as endosomal TLRs, RIG-I-like receptors, and the inflammasome . GeneralApart from VACV E3L (ORFV020), PPV lack homologues of poxviral NF-κB inhibitors present in other poxviral genera, suggesting that PPV have evolved novel mechanisms to counteract the NF-κB signaling pathway. Recently, we have described four NF-κB inhibitors encoded by ORFV, ORFV024, ORFV002, ORFV121 and ORFV073 –38. ORFVThe retinoblastoma tumor suppressor, pRb, is a multifunctional, predominantly nuclear protein encoded by the RB1 gene. pRb affects cell cycle, differentiation and metabolism, genome stability and apoptosis, mostly but not exclusively through transcription regulation . CentralHere we show that ORFV119, a LxCxE motif-containing virion protein unique to PPV, interferes with NF-κB signaling in a pRb-dependent manner early in infection by inhibiting IKK complex activation.Flag, and OV-IA82-RV119GxE-FlagLx, a revertant virus carrying a CxG substitution in ORFV119 LxCxE motif.Primary ovine fetal turbinate (OFTu) cells were obtained from Howard D. Lehmkuhl (USDA) and were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) , 2 mM L-glutamine, gentamicin (50 μg/ml), penicillin (100 IU/ml), and streptomycin (100 μg/ml). Human osteosarcoma cells , Human embryonic kidney (HEK 293T) and cervical cancer (HeLa) cells (obtained from American Type Culture Collection), were cultured in Dulbecco's modified essential medium (DMEM) supplemented as above. Cells were incubated at 37°C with 5% CO2. ORFV strain OV-IA82  was usedTo knockdown pRb expression in OFTu cells, siRNA directed against ovine RB1 was used. A pool of three RB1 sense (S) and anti-sense (AS) siRNA with following sequences 1-RB1-(S): CTACCTATAGCAGAAGTAT, RB1-(AS): ATACTTCTGCTATAGGTAG; 2-RB1-(S): GCTTATATATTTGACACAA, RB1-(AS): TTGTGTCAAATATATAAGC; 3-RB1-(S): CTCAGATTCACCTTTATTT, RB1-(AS): AAATAAAGGTGAATCTGAG (Custom Oligos: Sigma Aldrich) were used. Pooled RB1 siRNA (15 nM of each siRNA) were transfected in OFTu cells  using MISSION transfection reagent  following the manufactures’ protocol. At 48 h post transfection, pRb knockdown was examined by SDS-PAGE, using antibody against pRb . One MISSION siRNA Universal Negative Control  was included in all experiments.Rb- or OV-IA82-RV119GxE-Flag-Rb-Lx virus stocks , OFTu cells with reduced pRb levels (OFTuRb- cells) were infected with OV-IA82 or OV-IA82-RV119GxE-FlagLx virus and supernatants from infected cultures were collected at 24 h p.i and used for NF-κB-p65 nuclear translocation assays.To obtain OV-IA82Flag and pORFV119GxE-FlagLx, the ORFV119 or ORFV119GxELx coding sequences were PCR-amplified from the OV-IA82 genome and a plasmid containing synthetic ORFV119GxELx sequence (Genscript), respectively with primers 119-3xFlag-FW (EcoRI): 5’-TAAGGCCTCTGAATTCAATGGACTCTCGTAGGCTC GCTCTT-3’; 119-3xFlag-RV (BamHI): 5’-CAGAATACGTGGATCCTCAATCGCTGTCG CTGTCGCCGAG-3’ and cloned into p3xFlag-CMV-10 vector (pFlag) . Similarly, to obtain a ORFV119-green fluorescence protein (ORFV119GFP) expression vector, ORFV119 sequence was PCR amplified from OV-IA82 genome with primers 119-GFP-FW (EcoRI): 5’- TAAGGCCTCTGAATTCATGGACTCTCGTAGGCTCGCTCTT-3’ and 119-GFP-RV (BamHI): 5’- CAGAATACGTGGATCCAGATCGCTGTCGCTGTCGCCGA GCG-3’, and cloned into the vector pEGFP-N1 . DNA sequencing of plasmids confirmed the identity and integrity of the constructs.To construct expression plasmids pORFV119RFP) gene flanked by 528 bp sequences representing ORFV119 left and right flanking regions was synthesized and cloned in pUC57 vector . Similarly, to generate revertant viruses OV-IA82-RV119Flag and OV-IA82-RV119GxE-FlagLx, recombination cassettes pRV119Flag-GFP and pRV119GxE-Flag-GFPLx were synthesized containing N-terminally tagged ORFV119 or ORFV119GxELx sequences, respectively, and a GFP reporter gene, all flanked by approximately 600 bp of homologous sequence on either side to mediate recombination .To generate gene deletion mutant virus OV-IA82-Δ119, a recombination cassette (pΔ119-RFP) containing Vaccinia virus 7.5 promoter (VV7.5)-driven Red Fluorescent Protein  and transfected with recombination plasmids pRV119Flag-GFP or pRV119GxE-Flag-GFPLx. Recombinant viruses were isolated by limiting dilution and plaque assay using fluorescence microscopy as previously described [To obtain OV-IA82-Δ119, OFTu cells were infected with OV-IA82  and transfected with recombination plasmid pΔ119-RFP. To obtain OV-IA82-RV119escribed . IdentitFlag, OV-IA82-Δ119, or OV-IA82-RV119GxE-FlagLx  were disrupted by three cycles of freeze and thaw at three days p.i. Cellular debris were removed by centrifugation at 1500 rpm for 5 min, and supernatants pelleted by ultracentrifugation at 25,000 rpm for 1 h. Pellets were resuspended in MEM and aliquots frozen at -80°C. Viral titers were obtained by the Spearman-Karber’s 50% tissue culture infectious dose (TCID50) method.Semi-purified viruses were used in infection experiments. OFTu cells infected with OV-IA82, OV-IA82-RV119GxE-FlagLx or OV-IA82-RV119Flag  were harvested at three days p.i. and supernatant (EEV) and cellular (IMV) fractions collected following centrifugation at 1,500 rpm for 5 min. Virus in supernatants was pelleted by ultra-centrifugation , while cellular fractions were disrupted by three cycles of freeze and thaw and centrifuged to remove cell debris. Further purification steps were identical for EEV and IMV. Both preparations were sonicated, pelleted through a 36% sucrose cushion, and purified using double sucrose gradient centrifugation. Virus-containing bands were collected, and virus was recovered by centrifugation and resuspended in 10 mM TrisHcl. Whole cell extracts (60 μg) from OV-IA82-RV119Flag infected OFTu cells, and purified EEV and IMV virion proteins (10 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with primary antibodies against Flag , ORFV structural protein ORFV086 or Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH)  overnight at 4°C [Extracellular enveloped virus (EEV) and intracellular mature virus (IMV) were purified using double sucrose gradient purification protocol with modifications . OFTu cet at 4°C . Blots wFlag in presence or absence of CHX and harvested at 30 min, 1 h, 1.5 h and 2 h p.i. Total protein extracts (50 μg) were resolved by SDS-PAGE, blotted and transferred to nitrocellulose membranes, probed with antibody against Flag, p53  and GAPDH  as described above.To examine the effect of protein synthesis inhibitor cycloheximide (CHX) on expression of ORFV119 and control cellular protein p53. OFTu cells mock treated or treated with CHX (50 μg/ml)  for 30 min were infected with OV-IA82RV119Flag  for 2 h, 4 h, 6 h, 8 h, 12 h or 24 h p.i. Total protein extracts (50 μg) were resolved by SDS-PAGE, blotted and transferred to nitrocellulose membranes, and probed with primary antibody against Flag and GAPDH. Blots were then incubated with appropriate HRP-labeled secondary antibodies and developed using chemiluminescent reagents.To evaluate ORFV119 expression, OFTu cells were infected with OV-IA82 or OV-IA82-RV1195 cells/well) were cultured in four-well chamber slides  for 16 h and mock infected or infected with OV-IA82-Δ119 or OV-IA82-RV119Flag . Cells were fixed at 3 h, 6 h, 12 h, 16 h or 24 h p.i with 4% formaldehyde for 20 min, permeabilized with 0.2% Triton X 100 for 10 min, blocked with 1% bovine serum albumin  for 1 h, and incubated with primary antibody against Flag  overnight at 4°C. Cells were then stained with Alexa fluor 594 labelled secondary antibody , counterstained with DAPI (2 μg/ml) for 10 min and visualized by confocal microscopy .To assess the subcellular localization of ORFV119, OFTu cells . Virus yields were quantitated at 6 h, 12 h, 24 h, 36 h, and 48 h p.i. as described above.To assess the effect of ORFV119 in virus replication, one-step growth curves were determined in OFTu cells infected with OV-IA82, OV-IA82-Δ119 or OV-IA82-RV119Flag  and harvested at 2 h p.i. RNA was extracted using RNeasy Mini Kit  and reverse transcribed as previously described [To investigate the effect of OV-IA82-Δ119 infection on expression of NF-κB regulated genes, OFTu cells were mock infected or infected with OV-IA82, OV-IA82-Δ119 or OV-IA82-RV119escribed . mRNA foescribed . ExperimGFP) were treated with TNFα (20ng/ml) at 12 h post transfection for 30 min, fixed, permeabilized and blocked as described above, incubated with primary antibody against NF-κB-p65  for 2 h, stained with Alexa fluor 594-labeled secondary antibody  for 1 h, counterstained with DAPI, and examined by confocal microscopy. Numbers of GFP expressing cells (approximately 300 cells/sample) exhibiting nuclear NF-κB-p65 staining were determined in randomly selected fields and results were shown as mean percentage of GFP/ORFV119GFP expressing cells containing nuclear NF-κB-p65 over three independent experiments. Statistical analysis of data was performed by using the Student’s t test.The effect of ORFV119 in TNFα-induced activation of NF-κB was assessed by NF-κB-p65 nuclear translocation assay. HeLa cells transfected with control plasmid (pEGFP-N1) or a plasmid encoding ORFV119-GFP fusion protein . Cells were fixed at 30 min, 1 h, 2 h, 4 h, and 6 h p.i. and processed for NF-κB-p65 staining as described above. Cells (approximately 300/sample) were randomly selected and scored as mean percentage of cells containing nuclear NF-κB-p65 over three independent experiments. Statistical analysis of data was performed by using Student’s t test.To examine the role of ORFV119 on NF-κB-p65 nuclear translocation during ORFV infection, OFTu cells were mock infected or infected with OV-IA82, OV-IA82-RV119To investigate the effects of CHX on NF-κB-p65 nuclear translocation during ORFV infection, OFTu cells were pre-treated with CHX for 30 min, infected with OV-IA82 or OV-IA82-Δ119 in presence or absence of CHX, and fixed at 30 min or 1 h p.i. NF-κB-p65 nuclear translocation assay, scoring, quantification and analysis were performed as described above.Rb-, OV-IA82-RV119GxE-FlagLx and OV-IA82-RV119GxE-Flag-Rb-Lx virus infection on NF-κB-p65 nuclear translocation, OFTu or OFTuRb- cells were infected with OV-IA82, OV-IA82Rb-, OV-IA82-RV119GxE-FlagLx or OV-IA82-RV119GxE-Flag-Rb-Lx virus . Cells were fixed at 1 h p.i. and NF-κB-p65 nuclear translocation assay was performed as described above. Cells (approximately 300/sample) were randomly selected and scored for mean percentage of cells containing nuclear NF-κB-p65 over two independent experiments. Statistical analysis of data was performed by using Student’s t test.To investigate the effect of OV-IA82, OV-IA82Additionally, to assess the effect of pRb on NF-κB-p65 nuclear translocation, Saos-2 cells, a pRb negative cell line, were mock infected or infected with OV-IA82 or OV-IA82-Δ119  and fixed at 1 h, 1.5 h, 2 h, 4 h, and 6 h p.i. NF-κB-p65 nuclear translocation assay, quantification and analysis were performed as described above.Flag) were treated with TNFα (20ng/ml), and harvested 10 and 20 min post treatment. OFTu cells mock infected or infected with OV-IA82, OV-IA82-RV119Flag, OV-IA82-Δ119 or OV-IA82-RV119GxE-FlagLx  were harvested at 30 min and 1 h p.i. Total protein extracts (50 μg) were resolved by SDS-PAGE, blotted and transferred to nitrocellulose membranes and probed with specific antibody against phospho-IKKα/β (Ser176/180) , phospho-IκBα (Ser32/36) , phospho-NF-κB-p65 (Ser536) , IKKα/β , IκBα , NF-κB-p65  and GAPDH  or Flag. Blots were processed as described above. Protein bands were quantified for densitometric analysis using ImageJ software, Version 1.6.0  and fold changes calculated. Statistical analysis was performed by using Student’s t test.HeLa cells transfected with control plasmid (pFlag) or a plasmid encoding ORFV119-3xFlag fusion protein  and harvested at 30 min, 1 h, 2 h, 4 h, and 6 h p.i. Total protein extract (50 μg) were resolved by SDS-PAGE, blotted and transferred to nitrocellulose membranes, probed with phospho-NF-κB-p65 and NF-κB-p65 antibodies, and developed as described above.Flag. At 24 h after transfection, cells were induced with poly(I:C)  (500ng), poly(A:T)  (750ng) or ORFV DNA (1μg). ORFV DNA was extracted from OV-IA82 virus stock using QIAamp DNA Blood Mini Kit . Luciferase activities were determined at 20 h post- induction using the Dual Luciferase Reporter Assay (Promega) and a luminometer. Data were analyzed as previously described [The effect of ORFV119 on poly(I:C), poly(A:T) or ORFV DNA induced NF-κB-mediated transcriptional activity was investigated using a NF-κB promoter luciferase assay. Firefly luciferase gene under the control of NF-κB promoter (pNF-κB-Luc) and with a plasmid encoding sea pansy (Renilla reniformis) luciferase under the control of herpesvirus TK promoter (pRL-TK) were used in studies. HeLa cells cultured in 12-well plates were co-transfected with the vectors pNF-κB-Luc , and pRL-TK  and pFlag or pORFV119escribed . StatistGxE-FlagLx (MOI = 10). Firefly and sea pansy luciferase activities were measured at 1, 2, 4 and 6 h p.i. and expressed as relative fold changes in luciferase activity as described above.To investigate the effect of ORFV119 on E2F transcriptional activity in ORFV infected cells, OFTu cells were co-transfected with a pE2F-Luc  and pRL-TK (50 ng) plasmids. At 24 h post transfection, cells were mock infected or infected with OV-IA82, OV-IA82Δ119, or OV-IA82-RV119GxELx with pRb and ORFV119 with TRAF2 was assessed in virus-infected cells by co-immunoprecipitation. OFTu cells infected with OV-IA82-RV119Flag or OV-IA82-RV119GxE-FlagLx  or mock infected were harvested at 12 h p.i. Total protein extraction and co-immunoprecipitation was performed using nuclear complex Co-IP Kit  following the manufacturer’s instructions. For co-immunoprecipitation, total protein extracts were incubated with antibodies against Flag, TRAF2 and pRb in the high stringency buffer  overnight at 4°C, and then incubated with pre-washed 50 μl slurry of protein G agarose beads  at 4°C for 2 h. Beads were washed four times with high stringency buffer (described above) and bound proteins were eluted in Laemmli buffer. For immunoblot analysis, eluted proteins and control total protein cell lysates were resolved by SDS-PAGE, blotted and transferred to nitrocellulose membranes, probed with antibodies against Flag, TRAF2 and pRb and developed as described above. Light chain specific antibody against Rabbit IgG  was used for TRAF2 blots. A flag-expressing protein ORFV113Flag was used as a control for specificity of ORFV119 and TRAF2 interaction.The interaction of ORFV119 or ORFV119Flag also was assessed by co-immunoprecipitation. Antibodies for co-immunoprecipitation included anti-pRb , anti-TRAF2  and anti-Flag. Co-immunoprecipitations of total proteins extracts were performed as described above. The interaction of ORFV119 or ORFV119GxELx with putative cellular binding partners was assessed by co-immunoprecipitation in pORFV119Flag or pORFV119GxE-FlagLx transfected HEK 293T or HeLa cells at 12 h post transfection. Antibodies for co-immunoprecipitation included pRb  were harvested at 12h post transfection. Co-immunoprecipitations of total proteins extracts were performed as described above. Antibodies for co-immunoprecipitation included anti-TRAF2, anti-Flag and anti-pRb.The interaction of ORFV119 with TRAF2 also was assessed in Saos-2 cells, which do not express pRb. Saos-2 cells transfected with control plasmid (pFlag) and pORFV119Co-immunoprecipitation efficiency was calculated by normalizing the band intensities of co-immunoprecipitated proteins to those corresponding immunoprecipitated proteins and to the expression of corresponding input lysates as previously described .Flag (n = 4), or mock-infected (n = 3). Following anesthesia, the mucocutaneous junction of the right lower lip near the labial commissure was scarified along a two-centimeter line, and virus inoculum (0.5 ml) containing 107.5 TCID50/ml was applied topically using cotton swabs. In addition, the inner sides of hind limbs were scarified in a five-centimeter line and inoculated as above. Animals were monitored for 21 days for the presence of characteristic orf lesions. Pictures were taken of the labial inoculation sites at days 3, 5, 9, 12, 16 and 21 p.i. and the lesion sizes measured with a ruler. Skin biopsy specimens from hind limb inoculation sites were collected at days 2, 5, 8, 12 and 21 p.i. fixed in 10% buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin using standard methods.Five-month-old lambs randomly allocated to three experimental groups were inoculated with either OV-IA82-Δ119 (n = 4) or OV-IA82-RV119All animal procedures were approved by University of Nebraska-Lincoln Institutional Animal Care and Use Committee  and were performed in accordance with the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching.PPV ORFV119 amino acid sequences were aligned using Clustal Omega (EMBL-EBI). ORFV GeneBank accession numbers are (virus strains in parentheses) AY386263 (OV-IA82), AAP89015 (Orf11), ABA00637 (NZ2), 9AHH34303 (B029), ADY76823 (D1701), NP957896 (OV-SA00), AKU76741 (OV-GO), AKU76609 (OV-YX), AHZ33817 (NA1/11) and KP010356 (OV-SJ1). PCPV and BPSV GeneBank accession numbers are AEL20654 (PCPV F00-120R), AEO18268 (PCPV It1303/05), NP958027 (BPSV BV-AR02) and KM875471 (BPSV BV-TX09c5).ORFV119 encodes for proteins of 170 to 206 amino acids, with predicted molecular weights of 18.6 to 22.2 kDa and percentage amino acid identities ranging from 77% to 100% . HomologFlag). A protein of approximately 32 kDa was detected at 2 h p.i. with increasing protein levels observed at later time points. At 24 h p.i., the last time point investigated, an additional slightly higher molecular weight species of ORFV119 (approximately 35 kDa) was detected  were observed in the cytoplasm and especially in close proximity to the cell membrane. Remarkably, at late times p.i. (24 h), OV-IA82-RV119Flag infected cells exhibited abundant ORFV119 nuclear staining , pORFV119GxE-FlagLx (ORFV119GxELx) or control plasmid (pFlag), and protein extracts were prepared at 12 h post-transfection. Reciprocal co-immunoprecipitation assays with either anti-Flag or anti-pRb antibodies demonstrated that ORFV119 co-immunoprecipitates with pRb in both cell types. Co-immunoprecipitation of ORFV119 and pRb, however, was not observed with pORFV119GxE-FlagLx, a plasmid encoding ORFV119 in which C in the LxCxE motif was replaced by G, a change shown to abrogate interaction with pRb , and cell lysates prepared at 12 h p.i. Reciprocal co-immunoprecipitation with either anti-Flag or anti pRb antibodies showed that ORFV119 but not ORFV119GxE-FlagLx co-immunoprecipitates with pRb , TLR2 (6.48-fold), NF-κB1 (3.26-fold) and IL36α (3.7-fold) in cells infected with OV-IA82-Δ119 compared to OV-IA82 at 2 h p.i .GxE-FlagLx or OV-IA82-RV119Flag and NF-κB-p65 localization was examined by immunofluorescence. Infection with OV-IA82-Δ119 and OV-IA82-RV119GxE-FlagLx but not OV-IA82 or OV-IA82-RV119Flag led to rapid nuclear translocation of NF-κB-p65 as early as 30 min p.i. (P<0.05). Notably, levels of NF-κB-p65 nuclear translocation observed for OV-IA82-RV119GxE-FlagLx were significantly reduced compared to those observed with OV-IA82-Δ119 , IκBα (Ser32/36) and NF-κB-p65 (Ser536) compared to OV-IA82 infected cells , IκBα (19.35 and 21.24 fold) and NF-κB-p65 (13 and 13.24 fold) were observed at 30 min and 1 h p.i., respectively  and OV-IA82-Δ119 virions LxGxE-Flade novo viral protein synthesis in the infected cells, OFTu cells were pre-treated with the protein synthesis inhibitor cycloheximide (CHX) for 30 min followed by infection with OV-IA82 or OV-IA82-Δ119 for 30 min and 1 h in presence of the drug. If de novo synthesis of ORFV119 is required for inhibiting NF-κB signaling, then increased levels of NF-κB activation should be observed in OV-IA82 infected CHX treated cells. Low levels of ORFV119 proteins were detected at 30 min and 1 h p.i. likely representing input virion-associated protein; however, ORFV119 protein levels declined in CHX-treated OV-IA82-RV119Flag infected OFTu cells at subsequent times (To assess whether early inhibition of NF-κB-p65 nuclear translocation by ORFV119 involves nt times . Under tnt times . Immunofnt times . TogetheP<0.05).To determine if ORFV119 alone is sufficient for inhibiting TNFα-induced nuclear translocation of NF-κB-p65, immunofluorescence assays were performed in HeLa cells transiently expressing GFP or ORFV119-GFP fusion protein (119GFP). Following TNFα induction (30 min) ORFV119-GFP-expressing cells exhibited significantly reduced nuclear translocation of NF-κB-p65 (12.7%) compared to control cells expressing GFP alone (68%) , P<0.05.Flag plasmids. ORFV119 expression markedly reduced the TNFα induced phosphorylation of IKKα/β (20 and 50%), IκBα (25 and 68%) and NF-κB-p65 (40 and 60%) in cells expressing ORFV119Flag compared to control pFlag expressing cells at 10 and 20 min after TNFα induction. (P<0.05 and P<0.01). Together, results indicate that ORFV119 inhibits TNFα-induced NF-κB signaling by preventing activation of the IKK complex in the absence of any other viral protein.ORFV119 effect on TNFα induced activation of NF-κB-p65 was further investigated by examining phosphorylation of IKKα/β (Ser176/180), IκBα (Ser32/36) and NF-κB-p65 (Ser536) in HeLa cells transfected with pFlag or pORFV119Flag together with a plasmid encoding for a firefly luciferase reporter gene under the control of a NF-κB-responsive promoter. Cells were induced with poly(I:C), poly(A:T) or ORFV DNA at 24 h post-transfection and luciferase activities were determined at 20 h post-induction. No significant effect of ORFV119 expression on poly(I:C), poly(A:T) or ORFV DNA-induced NF-κB-mediated transcription was observed , poly(A:T) or ORFV DNA-induced NF-κB transcriptional activity, HeLa cells were co-transfected with pFlag or pORFV119observed . Thus, dGxE-FlagLx—a virus containing a mutation in the ORFV119 LxCxE motif that abrogates pRb binding—was unable to efficiently inhibit NF-κB signaling (Given that: 1) ORFV119 interacts with pRb , 2) the ignaling , we examRb-) were prepared from OFTu cells using siRNAs targeting ovine RB1 , OV-IA82-RV119GxE-FlagLx or OV-IA82-RV119GxE-Flag-Rb-Lx virus (OV-IA82-RV119GxE-FlagLx virus propagated in cells with reduced pRb levels) virus stocks. As expected from data described above for OV-IA82  or OV-IA82Rb- virus (OFTu cells/ OV-IA82Rb- virus) were used . Notably, using both OFTuRb- cells and OV-IA82Rb- virus resulted in significantly increased levels of NF-κB-p65 nuclear translocation in infected cells (17.2% NF-κB-p65 positive nuclei)   . As expectively) . Thus, pAdditional experiments examining involvement of pRb in ORFV119-mediated inhibition of NF-κB signaling were conducted in human osteosarcoma Saos-2 cells, a pRb-deficient cell line . pRb wasFlag  and harvested at 12 h p.i. Total cell protein extracts were immunoprecipitated with anti-Flag or anti-target cellular protein antibodies. Reciprocal co-immunoprecipitation demonstrated that ORFV119 co-immunoprecipitates with TRAF2  or PBS  in the right labial commissure and the inner side of the thighs, and disease course was monitored for 21 days. All virus-inoculated animals developed clinical orf as evidenced by erythema, papules, pustules, and scabby tissue deposition on the lips  still exhibited well-defined lesions at day 16 p.i. and regressing lesions still were present in two of the animals (#79 and #101) at day 21 p.i. (end point of the experiment) , OV-IA82-RV119the lips . Howevernt virus . By day eriment) . No lesiThe NF-κB signaling pathway plays key roles in the skin by regulating innate immune responses, inflammation, cell survival and cell proliferation –58. CrucORFV119 was shown to interact with pRb in infected and uninfected cells, indicating that other ORFV proteins are not required for the interaction. Similar to oncoproteins of small DNA viruses, binding to pRb was dependent on the ORFV119 LxCxE motif since a CxG substitution in the motif abrogated the interaction completely . This supRb has not been previously implicated in antiviral responses against poxviruses; however, it has been associated with NF-κB modulation by other viruses. For example, transiently expressed adenovirus E1A, a pRb-binding oncoprotein, was shown to inhibit NF-κB-dependent transcription induced by TNFα, the effect being dependent on E1A/pRb interaction . And pRbThrough association with pRb, ORFV119 inhibits NF-κB signaling by preventing IKK complex activation , with ORORFV119 is a virion protein  functionNotably, at late times post-infection (≥24 h p.i) ORFV119 also is observed in the nucleus of infected cells , suggestOther ORFV NF-κB inhibitors have been detected in the nucleus. ORFV002 localized to the nucleus and inhibited nuclear phosphorylation of NF-κB-p65 by interacting with mitogen stimulated stress kinase 1 (MSK1) ,39. And,119 homolog have also been reported [Examination of published parapoxviral genomes shows that a genomic region encompassing ORFV119 is affected sporadically by gaps or deletions of as much as 5.7 kbp –69. Notareported . An explORFV119 resulted in restricted lesion development and reduced time to resolution compared with revertant virus infection in the natural host. The attenuated phenotype of infection likely indicates improved host control of ORFV infection in the absence of ORFV119  to moderate , or marked attenuation (ORFV121) –38. The S1 FigFlag viruses , and titers were determined at 6 h, 12 h, 36 h, and 48 h p.i. and expressed as TCID50/ml. Results are mean values from two independent experiments.OFTu cells were infected with wild type OV-IA82, deletion mutant OV-IA82-Δ119, or revertant OV-IA82-RV119(TIF)Click here for additional data file.S2 FigFlag and harvested at 12h post transfection. Total cell lysate and proteins extracts immunoprecipitated with antibodies against anti-Flag (A) or anti-pRb (B), were examined by Western blot (WB) using anti-pRb or anti-Flag antibodies. Results are representative of two independent experiments. Percentage of pRb co-immunoprecipitated by ORFV119Flag: 21.5±2.34%; Percentage of ORFV119Flag co-immunoprecipitated by pRb: 38.7±2.61%.(A and B) HeLa cells were transfected with control plasmid (pFlag) or pORFV119(TIF)Click here for additional data file.S3 FigP < 0.05).(A) OFTu cells infected with OV-IA82 or OV-IA82-Δ119  were harvested at indicated times p.i. Total cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted and probed with antibodies against total or Phospho (Ser536) NF-κB-p65. (B) Densitometry of Phospho NF-κB-p65 bands were normalized to the total NF-κB-p65 bands. Fold changes are shown relative to OV-IA82 treatment and results are mean values of two independent experiments (*(TIF)Click here for additional data file.S4 FigGxE-FlagLx. Firefly and sea pansy luciferase activities were measured at 1, 2, 4 and 6 h p.i. and expressed as relative fold changes in luciferase activity compared to mock treatment . Results are mean values of three independent experiments. No significant difference was observed between the viruses.OFTu cells were co-transfected with a pE2F-Luc and pRL-TK plasmids. At 24 h post transfection cells were mock infected or infected with OV-IA82, OV-IA82Δ119, or OV-IA82-RV119(TIF)Click here for additional data file.S5 FigFlag. At 24 h after transfection, cells were induced with poly(I:C) (500 ng), poly(A:T) (750 ng) or ORFV DNA (1 μg). Cells were harvested at 20 h p.i., and firefly and sea pansy luciferase activities were measured and expressed as fold changes in luciferase activity compared to uninduced (UN) cells (*P<0.05). Results are mean values of two independent experiments.HeLa cells were co-transfected with pNF-κB-Luc, pRL-TK and pFlag or pORFV119(TIF)Click here for additional data file.S6 FigRb- cells were infected with OV-IA82-RV119GxE-FlagLx (LxGxE virus) or OV-IA82-RV119GxE-Flag-Rb-Lx (LxGxERb- virus) as described in Materials and Methods and cells were fixed at 1 h p.i. sequentially probed with antibody against NF-κB-p65 and Alexa Fluor 594 labeled secondary antibody, counterstained with DAPI, and examined by confocal microscopy. Red, NF-κB-p65; Blue, DAPI. (B) Cells were counted (n = 500 cells/slide) and results are shown as percentage of cells expressing nuclear NF-κB-p65. Results are mean values from two independent experiments. p values for LxGxE virus/OFTu cells vs LxGxE virus/OFTuRb- cells, LxGxERb- virus/OFTu cells and LxGxERb- virus/OFTuRb- cells were not significant (P>0.05).(A) OFTu or OFTu(TIF)Click here for additional data file.S7 FigGxE-FlagLx (p119GxE-FlagLx) and harvested at 12h post transfection. Total cell lysate and proteins extracts immunoprecipitated with antibodies against anti-Flag (A) or anti-TRAF2 (B), were examined by Western blot (WB) using anti-TRAF2 or anti-Flag antibodies. Results are representative of two independent experiments.(A and B) 293T cells were transfected with control plasmid (pFlag) or pORFV119(TIF)Click here for additional data file.S8 FigFlag   or PBS  on the scarified skin of the right lower lip. (A). Clinical course of disease for OV-IA82-Δ119 (Δ119) and OV-IA82-RV119Flag (RV119). Results are shown for 5, 9, 16, and 21 days p.i. (B). Comparison of mean lesion length (in cm) at indicated time points.Sheep were topically inoculated with OV-IA82-Δ119 , OV-IA82-RV119(TIF)Click here for additional data file.


In situ delivery of RdB/IL12/shVEGF to tumor tissues resulted in massive infiltration of differentiated CD4+ T cells, CD8+ T cells, natural killer cells, and dendritic cells to tissues surrounding the necrotic region of tumor. Furthermore, RdB/IL12/shVEGF induced a potent tumor-specific T helper type 1 immune response, implying that attenuation of the immunosuppressive environment mediated by downregulation of VEGF can significantly enhance immune stimulatory functions in the tumor milieu. Collectively, these findings indicate the potential of inducing and restoring potent antitumor immunity using intratumorally administered oncolytic Ad co-expressing IL-12 and shVEGF.Tumor microenvironment is extremely immunosuppressive, preventing efficient induction of antitumor immunity. To overcome tumor-mediated immunosuppression and enhance the potency of immunogene therapy, oncolytic adenovirus (Ad) co-expressing interleukin (IL)-12 and vascular endothelial growth factor (VEGF)-specific short hairpin ribonucleic acid  was generated. Intratumoral injection of RdB/IL12/shVEGF induced a strong antitumor effect in an immune competent B16-F10 melanoma model. RdB/IL12/shVEGF restored immune surveillance function in tumor tissues and actively recruited immune cells by elevating the expression levels of IL-12 and interferon-γ. RdB/IL12/shVEGF efficiently suppressed expression of immunosuppressive VEGF, resulting in restoration of the antitumor immune response and prevention of thymic atrophy.  The field of cancer immunology and immunotherapy has been an important focus of basic and clinical research since early discoveries of tumor antigens and adaptive immunity –3. Underin vivo murine tumor models by inducing an immunomodulatory effect [Interleukin (IL)-12 is a heterodimeric protein produced by activated macrophages, monocytes, dendritic cells, and stimulated B lymphocytes. IL-12 induces T helper type 1 (Th1) immunity and cytolysis by cytotoxic T-lymphocytes (CTLs) . Furthery effect –15. Howey effect .Vascular endothelial growth factor (VEGF) is a pro-angiogenic protein that stimulates vasculogenesis, angiogenesis, and metastasis , 20. VEGOncolytic adenovirus (Ad)-mediated expression of cytokine genes is a promising approach to express cytokines at high levels in tumor tissue. A clear benefit of oncolytic Ad-mediated cytokine expression system is cancer-specific amplification of therapeutic genes, because replicating viruses infect and express these genes in neighboring tumor cells. Furthermore, cytokine-expressing oncolytic Ads can elicit sustained and preferential expression of cytokines in tumor tissue, resulting in a prolonged antitumor effect and diminished toxicity compared with systemically administered recombinant cytokines .+, CD8+ T cells, DCs, and NK cells), and efficient prevention of tumor-induced thymic atrophy. Together, these results indicate that oncolytic Ad-mediated coexpression of IL-12 and shVEGF has excellent therapeutic efficacy due to restoration of antitumor immune function in tumor tissues, making this oncolytic Ad a promising candidate for potential clinical cancer immunotherapy.In this present study, we generated oncolytic Ad co-expressing IL-12 and short-hairpin RNA-targeting VEGF (RdB/IL12/shVEGF). We hypothesized that this Ad would induce potent antitumor immunity and have good therapeutic efficacy by alleviating immunosuppression in the tumor microenvironment. Intratumoral administration of RdB/IL12/shVEGF elicited significant suppression of tumor growth in tumor-bearing immunocompetent mice. We demonstrate that the enhanced antitumor effect was associated with increased IFN-γ expression, induction of a tumor-specific immune response, recruitment of immune cells  by inserting the murine IL-12  and shVEGF genes into the E1 and E3 regions of oncolytic Ad (RdB) , respectP < 0.01), suggesting that additional expression of shVEGF in the E3 region of oncolytic Ad may positively stimulate the expression of IL-12 in a dose-dependent manner.To examine oncolytic Ad-mediated expression of IL-12 and downregulation of VEGF, B16-F10 melanoma cells were infected with RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF. As shown in Figure P < 0.01). In addition, IL-12 has been shown to elicit an antiangiogenic effect through IFN-γ-mediated production of monokine and IFN-inducible protein 10 [P < 0.01). Of note, RdB/IL12/shVEGF-treated cells exhibited the lowest VEGF expression level among all oncolytic Ad-treated groups .Furthermore, secretion level of VEGF was significantly attenuated in all oncolytic Ad-treated groups in comparison to the PBS-treated control group Figure . This inotein 10 . As expeTo confirm the oncolytic effect of oncolytic Ads, we evaluated their ability to induce cytopathic effects in a panel of murine cancer cell lines  and human cancer cell line (U87MG). As shown in 3 by 11 days following initial treatment , resulting in complete remission in five of eight mice treated with RdB/IL12 or RdB/IL12/shVEGF. In marked contrast, none of the mice in the other treatment groups  achieved complete remission.To compare the therapeutic efficacy of oncolytic Ads expressing shVEGF, IL-12, or shVEGF plus IL-12, B16-F10 melanoma tumors established in C57BL/6 mice were injected with RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF, along with PBS or RdB as controls. All mice in the PBS control group showed aggressive tumor growth, resulting in rapid formation of large tumors with a volume exceeding 3,000 mmt Figure . In cont9 VP of either virus, we further assessed antitumor efficacy at a 5-fold lower viral dose (6 × 108 VP). As shown in Figure P < 0.01) 21 days after the initial treatment. Taken together, these results suggest that RdB/IL12/shVEGF has prolonged and more potent therapeutic efficacy than oncolytic Ads expressing a single therapeutic gene (RdB/shVEGF or RdB/IL12).Because there was no difference in therapeutic efficacy between RdB/IL12 and RdB/IL12/shVEGF at 3 × 109 VP), RdB/shVEGF (3 × 109 VP), RdB/IL12 (6 × 108 VP), or RdB/IL12/shVEGF (6 × 108 VP), tumor tissues were harvested 12 days after initial viral injection. As seen in Figure P < 0.01 vs. RdB), whereas IL-12 expression was not detected in tumors treated with PBS, RdB, or RdB/shVEGF. Further, RdB/shVEGF-, RdB/IL12-, or RdB/IL12/shVEGF-treated tumors exhibited significantly lower levels of VEGF  than control RdB tumors . Importantly, RdB/IL12/shVEGF-treated tumors showed 3.5-fold higher IFN-γ expression than RdB/IL12-treated tumors (**P < 0.01). These results demonstrate that treatment of tumors with RdB/IL12/shVEGF tumors can induce high expression of IFN-γ and subsequently attenuate VEGF expression in tumor tissue, resulting in a shift in the T cell response toward the type 1 pattern for enhanced antitumor efficacy.IFN-γ is a cytokine that prevents tumor development and functions as an important mediator of tumor-specific immune responses . Furtherin vivo . TherefoAlthough controlled expression of antitumor cytokines at tumor tissue can ameliorate immunosuppression and induce antitumor immune response, high cytokine levels in serum can be detrimental and cause severe side effects. Thus, we next examined whether intratumoral administration of oncolytic Ads would lead to elevated cytokine level in serum. As shown in P < 0.01).The shift from Th1 to Th2 cytokine expression has been shown to promote progression of malignant tumors , 30. Fur+/CCR7+) in the spleen and lymph node in comparison to any other treatment groups (P < 0.001). In addition, immunosuppressive Th2 population (CD4+/CCR4+) was significantly lower in RdB/IL12/shVEGF-treated group in comparison to any other treatment groups (P < 0.001). Together, these results demonstrate that efficient secretion of IL-12 and downregulation of VEGF by RdB/IL12/shVEGF can induce polarization of naïve CD4+ T cells toward Th1 and conjunctively inhibit transition into immunosuppressive Th2 subset by increasing IFN-γ expression while suppressing IL-6 expression.To further assess the effect of oncolytic Ads on the polarization of naïve T cells into Th1 or Th2, we evaluated T cell population in the spleen and lymph nodes following treatment with oncolytic Ads. As shown in Figure P < 0.01), and a significantly greater number of IFN-γ-secreting immune cells was observed in the RdB/IL12/shVEGF-treated group than the RdB/IL12-treated group (**P < 0.01), suggesting that expression of IL-12 is more critical for induction of the Th1 immune responses than downregulation of VEGF alone. Importantly, RdB/IL12/shVEGF-treated group had significantly more IFN-γ-secreting immune cells than the groups treated with RdB/IL12 or RdB/shVEGF, indicating that the co-expression of IL-12 and shVEGF can induce a potent Th1-type immune response. Taken together, these results indicate that the observed potent antitumor efficacy of RdB/IL12/shVEGF in Figure To assess tumor-specific immune response, splenocytes from mice treated with RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF were further assessed for tumor cell-specific IFN-γ-secreting lymphocytes by IFN-γ ELISpot assay. As shown in Figure + T and CD8+ T cells are known to play a central role in regulating antigen-specific immune responses, including those mediated by tumor-associated antigens [+ T and CD8+ T cells were observed in the tumors treated with RdB/IL12/shVEGF in comparison to RdB-, RdB/shVEGF-, or RdB/IL12-treated tumor tissues  analysis of tumor lysates with NK cell marker further supported the notion that RdB/IL12/shVEGF treatment induced greatest infiltration of NK cells . The weight and size of thymuses were significantly higher in tumor-bearing mice treated with RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF than PBS-treated mice, suggesting that oncolytic Ad treatment could prevent thymic atrophy . These results were proportionally correlated with the VEGF suppression data shown in Figure Previous studies reported that thymic atrophy observed in tumor-bearing mice contributed to suppression of host immunity against the tumor , 33. Furin vivo . These fP < 0.01). Additionally, RdB/shVEGF-, RdB/IL12-, or RdB/IL12/shVEGF-treated mice had higher populations of thymocytes undergoing active proliferation than RdB-treated mice , which inversely correlates with the TUNEL data. Of note, RdB/IL12/shVEGF-treated thymuses had similar morphological attributes to the thymuses of healthy mice without tumors, suggesting that co-expression of IL-12 and shVEGF can alleviate thymic atrophy. Overall, these results demonstrate that RdB/IL12/shVEGF can effectively prevent thymic atrophy in tumor-bearing mice via the inhibition of apoptosis and induction of proliferation in the thymus.To further assess morphologic and biologic changes in the thymus, we performed histological and immunohistochemical staining of thymic tissue collected from tumor-bearing mice treated with PBS (control) or various oncolytic Ads. We observed clearly segregated areas between the cortex and medulla zones in thymus tissues from tumor-bearing mice treated with RdB/shVEGF-, RdB/IL12-, or RdB/IL12/shVEGF, whereas the thymic structure was abnormally disrupted in mice treated with PBS or RdB on day 12 following initial treatment Figure . These rWeakened immune function in cancer patients is well-characterized . One of IL-12 facilitates Th1 differentiation and augments the cytolytic effect of NK cells and CTL, resulting in enhanced antitumor immunity , 38. ImpRecombinant cytokines have been reported to induce potent immunomodulation in tumor-bearing mice, resulting in potent antitumor efficacy . HoweverIn this present report, we generated an oncolytic Ad co-expressing IL-12 and shVEGF (RdB/IL12/shVEGF) to ameliorate the immunosuppressive tumor microenvironment and cytokine-mediated induction of antitumor immunity. Cancer cells infected with RdB/IL12/shVEGF showed significantly higher IL-12 expression than those infected with RdB/IL12 Figure , implyinAn aberrant Th1/Th2 cytokine balance, in which Th2 cytokines are overexpressed, has been shown to promote progression of cancers in patients, and a strong correlation between Th1/Th2 cytokine balance and the efficacy of cancer treatments has been reported , 30. IFN+T cell, CD8+ T cell, and NK cells  staining, cell culture, and polymerase chain reaction.Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum , L-glutamine (2 mmol/L), penicillin (100 IU/mL), and streptomycin (50 mg/mL) was used as the culture medium. HEK293 , U87MG (human glioma cell line), BNL (murine liver cancer cell line), B16-F10 (murine melanoma cell line), LLC (murine lung cancer cell line), and CMT-93 (murine polyploid carcinoma cell line) were obtained from the American Type Culture Collection . All cell lines tested negative for Six- to eight-week-old male C57BL/6 mice were purchased from Orient Bio Inc.  and maintained in a laminar air-flow cabinet under a specific pathogen-free environment. All facilities were approved by the AAALAC . All animal experiments were conducted according to the institutional guidelines established by the Hanyang University Institutional Animal Care and Use Committee.Escherichia coli BJ5183 for homologous recombination, generating the pRdB/shVEGF Ad vector . Double-stranded RNA oligonucleotides corresponding to nucleotides 92-112 of murine VEGF mRNA (GenBank accession number gi: 70608153) were synthesized using a Silencer™ siRNA construction kit (Ambion) . To gener Figure . To consr Figure . As cont5 cells per well, and then infected with RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF at 50 MOI. At 48 hr after infection, the supernatants were collected. Levels of IL-12 and shVEGF were determined by ELISA according to the manufacturer's instructions .B16-F10 melanoma cells were plated onto six-well plates at 3 × 105) were injected subcutaneously into the right abdomen of 6- to 7-week-old male C57BL/6 mice. When the tumor volume reached around 100 mm3, mice were sorted into groups with similar mean tumor volumes, and different doses of oncolytic Ads suspended in a total volume of 50 μl PBS  were injected intratumorally three times every other day. Tumor growth was monitored every day by measuring two perpendicular tumor diameters using electronic calipers . Tumor volume was calculated using the following formula: volume = 0.523L (W)2, where L is length and W is width.B16-F10 cells  in ice-cold radioimmunoprecipitation assay buffer  with a proteinase inhibitor cocktail . Levels of IL-12, VEGF, and IFN-γ were measured by conventional ELISA kits . In addition, Th1/Th2 type cytokine expression profiles from supernatant co-cultured with splenocytes and irradiated B16-F10  were estimated using a Th1/Th2 CBA kit .At 7 days following treatment with PBS, RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF, spleens were obtained aseptically from tumor-bearing mice, and unicellular splenocytes were prepared as described previously . Prepare+ T cell population, splenocytes or lymphocytes were gated by plotting forward vs. side scatter followed by gating on CD4+ cells. Gated cells were then analyzed for CCR4+ or CCR7+ cells. Samples were analyzed using BD Biosciences BD-LSR II Analytic Flow Cytometer and FACSDiva software (BD Biosciences Pharmingen).Spleen and draining lymph nodes (DLNs) were harvested at 12 days following first viral inoculation of the B16-F10 tumor-bearing mice. Before staining, cells were treated with saturating anti-CD16/CD32  in staining buffer (2% FBS and 0.02% sodium azide in PBS). Cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (eBioscience), percp-CY5.5-conjugated anti-CD4 (BD Biosciences Pharmingen), PE-CY7-conjugated anti-CCR4 (Biolegend), and/or PE-conjugated anti-CCR7 (Biolegend). To determine the percentage of CCR4- or CCR7-expressing CD4® image analysis software . Results are expressed as the mean optical density of five different digital images.Tumor or thymus tissues collected from tumor-bearing mice treated with PBS, RdB, RdB/shVEGF, RdB/IL12, or RdB/IL12/shVEGF were fixed in 10% neutral buffered formalin , embedded in paraffin, and cut into 4-μm-thick sections. Representative sections were stained with H & E, and then examined by light microscopy. To detect lymphocytes and DCs, tumor tissues were frozen in Optimal Cutting Temperature compound  and cut into 10-μm-thick sections. The cryosections were incubated with rat anti-mouse CD4 monoclonal Ab (BD Biosciences Pharmingen), rat anti-mouse CD8 monoclonal Ab (BD Biosciences Pharmingen), or mouse anti-mouse PCNA monoclonal Ab  as the primary Ab, and then treated with horseradish peroxidase-conjugated goat anti-rat IgG (BD Biosciences Pharmingen) or horseradish peroxidase-conjugated goat anti-mouse IgG  as the secondary Ab. Diaminobenzidine/hydrogen peroxidase (DAKO) was used as the chromogen substrate. All slides were counterstained with Meyer's hematoxylin (Sigma). The expression levels of CD4, CD8, and PCNA were semi-quantitatively analyzed using MetaMorphFor immunofluorescence staining of CD11c, CD86, and NK-1.1 cells, cryosections were treated with hamster anti-mouse CD11c monoclonal Ab (BD Biosciences Pharmingen), rat anti-mouse CD86 monoclonal Ab (BD Biosciences Pharmingen), or mouse anti-mouse NK-1.1 monoclonal Ab  and incubated overnight at 4°C, and then treated with Alexa Fluor® 568 (Red)-labeled goat anti-hamster IgG , Alexa Fluor® 488 (Green)-labeled goat anti-rat IgG (Invitrogen), or Alexa Fluor® (Green)-labeled goat anti-mouse IgG (Invitrogen) at room temperature for 1 hr, respectively. For counterstaining, the samples were incubated with 4,6-diamidino-2-phenylindole (Sigma). Slides were mounted with Vectashield mounting medium  and cells were viewed under a confocal laser-scanning microscope .® image analysis software . Results are expressed as the mean optical density of five different digital images.The apoptotic thymocyte population was assessed by TUNEL assay as described previously . ApoptotOn day 12 after initial viral treatment, all thymuses were removed and immediately placed in Roswell Park Memorial Institute 1640 medium (GIBCO), and the weight of each thymus was determined using an electronic scale . The size of the thymus was semi-quantitatively analyzed using ImageJ software .P values less than 0.05 were considered statistically significant .Data are expressed as means ± standard deviation (SD). Statistical comparisons were made using Stat View software  and the Mann-Whitney test (non-parametric rank sum test).