Quantitative proteomic comparison of myofibroblasts derived from bone marrow and cornea.

Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells. In the cornea, myofibroblasts are derived primarily from stromal keratocytes and from BM-derived fibrocytes after epithelial-stromal and endothelial-stromal injuries. Quantitative proteomic comparison of mature alpha-smooth muscle actin (α-SMA)+ myofibroblasts (verified by immunocytochemistry for vimentin, α-SMA, desmin, and vinculin) generated from rabbit corneal fibroblasts treated with transforming growth factor (TGF) beta-1 or generated directly from cultured BM treated with TGF beta-1 was pursued for insights into possible functional differences. Paired cornea-derived and BM-derived α-SMA+ myofibroblast primary cultures were generated from four New Zealand white rabbits and confirmed to be myofibroblasts by immunocytochemistry. Paired cornea- and BM-derived myofibroblast specimens from each rabbit were analyzed by LC MS/MS iTRAQ technology using an Orbitrap Fusion Lumos Tribrid mass spectrometer, the Mascot search engine, the weighted average quantification method and the UniProt rabbit and human databases. From 2329 proteins quantified with ≥ 2 unique peptides from ≥ 3 rabbits, a total of 673 differentially expressed (DE) proteins were identified. Bioinformatic analysis of DE proteins with Ingenuity Pathway Analysis implicate progenitor-dependent functional differences in myofibroblasts that could impact tissue development. Our results suggest BM-derived myofibroblasts may be more prone to the formation of excessive cellular and extracellular material that are characteristic of fibrosis.

The Efficacy of Topical HGF on Corneal Fibrosis and Epithelial Healing after Scar-Producing PRK Injury in Rabbits.

Purpose: To determine the effect of topical hepatocyte growth factor (HGF) on myofibroblast development and corneal opacity after fibrosis-producing photorefractive keratectomy (PRK). Methods: Twelve New Zealand rabbits had transepithelial PRK. Six rabbits received topical recombinant human HGF (rhHGF) (50 µL of 0.1 mg/mL) 3 times a day for 1 week beginning 6 hours prior surgery and until full closure of the epithelium, and 6 control rabbits received vehicle by the same schedule. Slit lamp photos were taken immediately and at 43 to 45 hours after surgery to determine the rate of epithelial healing. Slit lamp photographs and immunohistochemistry for α-smooth muscle actin were analyzed at 1 month in masked fashion. Results: The rhHGF group tended to have slower re-epithelization when compared with the controls, but no statistically significant difference was noted (P = 0.62). There was no significant difference in the density of myofibroblasts in the central stroma (P = 0.49) or corneal opacity (P = 0.84) between the HGF and control groups at 1 month after PRK. Conclusions: Topical rhHGF applied three times a day during the early postoperative period prior to epithelial closure did not significantly change the corneal epithelial healing rate, myofibroblast density, or opacity compared with vehicle after transepithelial -9.0 D PRK injury of the central cornea in rabbits. Translational Relevance: HGF has been reported to decrease myofibroblast generation and fibrosis in many organs, but topical HGF applied to the cornea until epithelial healing had no effect on scarring fibrosis in rabbit corneas.

miRNAs Involved in M1/M2 Hyperpolarization Are Clustered and Coordinately Expressed in Alcoholic Hepatitis.

The innate immune system, including monocytes/macrophages, is critical to the progression of alcoholic liver disease (ALD). In response to chronic ethanol, Kupffer cells, the resident macrophage of livers, and peripheral monocytes become sensitized to bacterial lipopolysaccharides (LPS), express more pro-inflammatory cytokines and exhibit macrophage M1/M2 hyperpolarization. Since miRNAs play an important role in the regulation of M1/M2 polarization, we hypothesized that miRNAs regulating macrophage polarization would be dysregulated after chronic ethanol consumption. miRNA sequencing data from Kupffer cells isolated from rats fed an ethanol diet vs. control diet and qPCR data from PBMCs isolated from alcoholic hepatitis (AH) patients and healthy controls were used to assess the role of miRNAs in macrophage hyperpolarization in ALD. Differential expression analyses revealed 40 misregulated miRNAs in Kupffer cells from the chronic ethanol-fed rats compared to pair-fed controls. Nine of these miRNAs are known to be associated with macrophage polarization and consist of a mixture of M1- and M2-associated miRNAs, indicative of hyperpolarization. Twenty-three of the 40 differentially expressed miRNAs were localized to miRNA clusters throughout the genome. Correlation analyses revealed that miRNAs in three of these clusters were co-regulated and located within antisense non-coding RNAs. Similar to Kupffer cells from ethanol-fed rats, M1 and M2 polarization markers, as well as sensitivity to LPS, were elevated in PBMCs from AH patients compared to healthy controls. These increases were associated with an up-regulation of polarization-associated miRNAs, including miR-125a-5p, a miRNA associated with hyperpolarization. miR-125a-5p is clustered in the genome with other miRNAs inside a host gene, Spaca6, which was also upregulated in PBMCs, as well as isolated monocytes, from AH patients. Finally, correlation analyses revealed co-regulation of human polarization-associated miRNA clusters. While expression of polarization-associated miRNAs in clusters was upregulated in AH compared to healthy controls, co-regulation of the miRNAs within a cluster was independent of disease state. Together, these results reveal that global changes in miRNA regulation are associated with polarization phenotypes in Kupffer cells from rat after chronic ethanol as well as in PBMCs from patients with AH. Importantly, polarization-associated miRNAs were localized to coordinately regulated clusters.

Specifically Sized Hyaluronan (35 kDa) Prevents Ethanol-Induced Disruption of Epithelial Tight Junctions Through a layilin-Dependent Mechanism in Caco-2 Cells.

BACKGROUND: Specific-sized species of the carbohydrate hyaluronan elicit a variety of cellular responses mediating tissue integrity and repair, as well as regulating inflammatory responses. Orally provided hyaluronan with an average molecular weight of 35 kDa (HA35) protects mice from short-term ethanol (EtOH)-induced liver injury. This protection was associated with maintenance of the colocalization of zonula occludens-1 (ZO-1) and occludin at tight junctions in the proximal colon. However, it is not known whether HA35 also protects other regions of the intestine or whether protection is due to a direct and/or indirect interaction of HA35 with the intestinal epithelium. METHODS: Female C57BL/6J mice were fed an EtOH containing diet or pair-fed control diet (4 days) and treated with or without HA35 via daily gavage during the last 3 days of EtOH feeding. Intestinal morphology and tight junction integrity were assessed. Differentiated Caco-2 cells were transfected or not with scrambled siRNA or siRNA targeting layilin, a hyaluronan receptor. Caco-2 cells were treated with or without HA35 prior to challenge with EtOH. Localization of tight junction proteins, fluorescein isothiocyanate (FITC)-dextran permeability, and transepithelial electrical resistance (TEER) were evaluated. RESULTS: While short-term EtOH did not result in any apparent changes in the gross morphology of the intestine, colocalization of ZO-1 and occludin at tight junctions was decreased in the proximal and distal colon. HA35 prevented these effects of EtOH. In differentiated Caco-2 cells, EtOH decreased the localization of ZO-1 and occludin at tight junctions and increased permeability of FITC-dextran. At higher concentrations, EtOH also decreased TEER. Pretreatment with HA35 prevented these changes. When the hyaluronan receptor layilin was knocked down in Caco-2 cells, HA35 no longer protected cells from EtOH-induced loss of tight junctions. CONCLUSIONS: Taken together, these data indicate that HA35 interacts with layilin on intestinal epithelial cells and maintains intestinal tight junction integrity during short-term EtOH exposure.

Descemet's Membrane Modulation of Posterior Corneal Fibrosis.

Purpose: The purpose of this study was to evaluate the effect of removal of Descemet's basement membrane and endothelium compared with removal of the endothelium alone on posterior corneal fibrosis. Methods: Twelve New Zealand White rabbits were included in the study. Six eyes had removal of the Descemet's membrane-endothelial complex over the central 8 mm of the cornea. Six eyes had endothelial removal with an olive-tipped cannula over the central 8 mm of the cornea. All corneas developed stromal edema. Corneas in both groups were cryofixed in optimum cutting temperature (OCT) formula at 1 month after surgery. Immunohistochemistry (IHC) was performed for α-smooth muscle actin (SMA), keratocan, CD45, nidogen-1, vimentin, and Ki-67, and a TUNEL assay was performed to detect apoptosis. Results: Six of six corneas that had Descemet's membrane-endothelial removal developed posterior stromal fibrosis populated with SMA+ myofibroblasts, whereas zero of six corneas that had endothelial removal alone developed fibrosis or SMA+ myofibroblasts (P < 0.01). Myofibroblasts in the fibrotic zone of corneas that had Descemet's membrane-endothelial removal were undergoing both mitosis and apoptosis at 1 month after surgery. A zone between keratocan+ keratocytes and SMA+ myofibroblasts contained keratocan-SMA-vimentin+ cells that were likely CD45- corneal fibroblasts and CD45+ fibrocytes. Conclusions: Descemet's basement membrane has an important role in modulating posterior corneal fibrosis after injury that is analogous to the role of the epithelial basement membrane in modulating anterior corneal fibrosis after injury. Fibrotic areas had myofibroblasts undergoing mitosis and apoptosis, indicating that fibrosis is in dynamic flux.

IL-1 and TGF-ß Modulation of Epithelial Basement Membrane Components Perlecan and Nidogen Production by Corneal Stromal Cells.

Purpose: To determine whether (1) the in vitro expression of epithelial basement membrane components nidogen-1, nidogen-2, and perlecan by keratocytes, corneal fibroblasts, and myofibroblasts is modulated by cytokines/growth factors, and (2) perlecan protein is produced by stromal cells after photorefractive keratectomy. Methods: Marker-verified rabbit keratocytes, corneal fibroblasts, myofibroblasts were stimulated with TGF-ß1, IL-1α, IL-1ß, TGF-ß3, platelet-derived growth factor (PDGF)-AA, or PDGF-AB. Real-time quantitative RT-PCR was used to detect expression of nidogen-1, nidogen-2, and perlecan mRNAs. Western blotting evaluated changes in protein expression. Immunohistochemistry was performed on rabbit corneas for perlecan, alpha-smooth muscle actin, keratocan, vimentin, and CD45 at time points from 1 day to 1 month after photorefractive keratectomy (PRK). Results: IL-1α or -1ß significantly upregulated perlecan mRNA expression in keratocytes. TGF-ß1 or -ß3 markedly downregulated nidogen-1 or -2 mRNA expression in keratocytes. None of these cytokines had significant effects on nidogen-1, -2, or perlecan mRNA expression in corneal fibroblasts or myofibroblasts. IL-1α significantly upregulated, while TGF-ß1 significantly downregulated, perlecan protein expression in keratocytes. Perlecan protein expression was upregulated in anterior stromal cells at 1 and 2 days after -4.5 or -9 diopters (D) PRK, but the subepithelial localization of perlecan became disrupted at 7 days and later time points in -9-D PRK corneas when myofibroblasts populated the anterior stroma. Conclusions: IL-1 and TGF-ß1 have opposing effects on perlecan and nidogen expression by keratocytes in vitro. Proximate participation of keratocytes is likely needed to regenerate normal epithelial basement membrane after corneal injury.

Basement membranes in the cornea and other organs that commonly develop fibrosis.

Basement membranes are thin connective tissue structures composed of organ-specific assemblages of collagens, laminins, proteoglycan-like perlecan, nidogens, and other components. Traditionally, basement membranes are thought of as structures which primarily function to anchor epithelial, endothelial, or parenchymal cells to underlying connective tissues. While this role is important, other functions such as the modulation of growth factors and cytokines that regulate cell proliferation, migration, differentiation, and fibrosis are equally important. An example of this is the critical role of both the epithelial basement membrane and Descemet's basement membrane in the cornea in modulating myofibroblast development and fibrosis, as well as myofibroblast apoptosis and the resolution of fibrosis. This article compares the ultrastructure and functions of key basement membranes in several organs to illustrate the variability and importance of these structures in organs that commonly develop fibrosis.

Posterior stromal cell apoptosis triggered by mechanical endothelial injury and basement membrane component nidogen-1 production in the cornea.

This study was performed to determine whether cells in the posterior stroma undergo apoptosis in response to endothelial cell injury and to determine whether basement membrane component nidogen-1 was present in the cornea. New Zealand White rabbits had an olive tip cannula inserted into the anterior chamber to mechanically injure corneal endothelial cells over an 8 mm diameter area of central cornea with minimal injury to Descemet's membrane. At 1 h (6 rabbits) and 4 h (6 rabbits) after injury, three corneas at each time point were cryopreserved in OCT for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemistry (IHC) for vimentin and nidogen-1, and three corneas at each time point were fixed for transmission electron microscopy (TEM). Uninjured corneas were controls. Stromal cells over approximately the posterior 25% of the stroma overlying to the site of corneal endothelial injury underwent apoptosis detected by the TUNEL assay. Many of these apoptotic cells were vimentin+, suggesting they were likely keratocytes or corneal fibroblasts. Stromal cells peripheral to the site of endothelial injury and more anterior stromal cells overlying the site of endothelial injury did not undergo apoptosis. Stromal cell death was confirmed to be apoptosis by TEM. No apoptosis of stromal cells was detected in control, uninjured corneas. Nidogen-1 was detected in the stroma of unwounded corneas, with higher nidogen-1 in the posterior stroma than the anterior stroma. After endothelial scrape injury, concentrations of nidogen-1 appeared to be in the extracellular matrix of the posterior stroma and, possibly, within apoptotic bodies of stromal cells. Thus, posterior stromal cells, likely including keratocytes, undergo apoptosis in response to corneal endothelial injury, analogous to anterior keratocytes undergoing apoptosis in response to epithelial injury.

Hyaluronic acid 35 normalizes TLR4 signaling in Kupffer cells from ethanol-fed rats via regulation of microRNA291b and its target Tollip.

TLR4 signaling in hepatic macrophages is increased after chronic ethanol feeding. Treatment of hepatic macrophages after chronic ethanol feeding with small-specific sized hyaluronic acid 35 (HA35) normalizes TLR4 signaling; however, the mechanisms for HA35 action are not completely understood. Here we used Next Generation Sequencing of microRNAs to identify negative regulators of TLR4 signaling reciprocally modulated by ethanol and HA35 in hepatic macrophages. Eleven microRNAs were up-regulated by ethanol; only 4 microRNAs, including miR291b, were decreased by HA35. Bioinformatics analysis identified Tollip, a negative regulator of TLR4, as a target of miR291b. Tollip expression was decreased in hepatic macrophages from ethanol-fed rats, but treatment with HA35 or transfection with a miR291b hairpin inhibitor restored Tollip expression and normalized TLR4-stimulated TNFα expression. In peripheral blood monocytes isolated from patients with alcoholic hepatitis, expression of TNFα mRNA was robustly increased in response to challenge with lipopolysaccharide. Importantly, pre-treatment with HA35 reduced TNFα expression by more than 50%. Taken together, we have identified miR291b as a critical miRNA up-regulated by ethanol. Normalization of the miR291b → Tollip pathway by HA35 ameliorated ethanol-induced sensitization of TLR4 signaling in macrophages/monocytes, suggesting that HA35 may be a novel therapeutic agent in the treatment of ALD.

MicroRNA 181b-3p and its target importin α5 regulate toll-like receptor 4 signaling in Kupffer cells and liver injury in mice in response to ethanol.

Increased inflammatory signaling by Kupffer cells contributes to alcoholic liver disease (ALD). Here we investigated the impact of small, specific-sized hyaluronic acid of 35 kD (HA35) on ethanol-induced sensitization of Kupffer cells, as well as ethanol-induced liver injury in mice. Unbiased analysis of microRNA (miRNA) expression in Kupffer cells identified miRNAs regulated by both ethanol and HA35. Toll-like receptor 4 (TLR4)-mediated signaling was assessed in primary cultures of Kupffer cells from ethanol- and pair-fed rats after treatment with HA35. Female C57BL6/J mice were fed ethanol or pair-fed control diets and treated or not with HA35. TLR4 signaling was increased in Kupffer cells by ethanol; this sensitization was normalized by ex vivo treatment with HA35. Next generation sequencing of Kupffer cell miRNA identified miRNA 181b-3p (miR181b-3p) as sensitive to both ethanol and HA35. Importin α5, a protein involved in p65 translocation to the nucleus, was identified as a target of miR181b-3p; importin α5 protein was increased in Kupffer cells from ethanol-fed rats, but decreased by HA35 treatment. Overexpression of miR181b-3p decreased importin α5 expression and normalized lipopolysaccharide-stimulated tumor necrosis factor α expression in Kupffer cells from ethanol-fed rats. In a mouse model of ALD, ethanol feeding decreased miR181b-3p in liver and increased expression of importin α5 in nonparenchymal cells. Treatment with HA35 normalized these changes and also protected mice from ethanol-induced liver and intestinal injury. CONCLUSION: miR181b-3p is dynamically regulated in Kupffer cells and mouse liver in response to ethanol and treatment with HA35. miR181b-3p modulates expression of importin α5 and sensitivity of TLR4-mediated signaling. This study identifies a miR181b-3p-importin α5 axis in regulating inflammatory signaling pathways in hepatic macrophages. (Hepatology 2017;66:602-615).

Myeloid Mixed Lineage Kinase 3 Contributes to Chronic Ethanol-Induced Inflammation and Hepatocyte Injury in Mice.

Proinflammatory activity of hepatic macrophages plays a key role during progression of alcoholic liver disease (ALD). Since mixed lineage kinase 3 (MLK3)-dependent phosphorylation of JNK is involved in the activation of macrophages, we tested the hypothesis that myeloid MLK3 contributes to chronic ethanol-induced inflammatory responses in liver, leading to hepatocyte injury and cell death. Primary cultures of Kupffer cells, as well in vivo chronic ethanol feeding, were used to interrogate the role of MLK3 in the progression of liver injury. Phosphorylation of MLK3 was increased in primary cultures of Kupffer cells isolated from ethanol-fed rats compared to cells from pair-fed rats. Kupffer cells from ethanol-fed rats were more sensitive to LPS-stimulated cytokine production; this sensitization was normalized by pharmacological inhibition of MLK3. Chronic ethanol feeding to mice increased MLK3 phosphorylation robustly in F4/80(+) Kupffer cells, as well as in isolated nonparenchymal cells. MLK3(-/-) mice were protected from chronic ethanol-induced phosphorylation of MLK3 and JNK, as well as multiple indicators of liver injury, including increased ALT/AST, inflammatory cytokines, and induction of RIP3. However, ethanol-induced steatosis and hepatocyte apoptosis were not affected by MLK3. Finally, chimeric mice lacking MLK3 only in myeloid cells were also protected from chronic ethanol-induced phosphorylation of JNK, expression of inflammatory cytokines, and increased ALT/AST. MLK3 expression in myeloid cells contributes to phosphorylation of JNK, increased cytokine production, and hepatocyte injury in response to chronic ethanol. Our data suggest that myeloid MLK3 could be targeted for developing potential therapeutic strategies to suppress liver injury in ALD patients.

Receptor interacting protein 3 protects mice from high-fat diet-induced liver injury.

Multiple pathways of programmed cell death are important in liver homeostasis. Hepatocyte death is associated with progression of nonalcoholic fatty liver disease, and inhibition of apoptosis partially protects against liver injury in response to a high-fat diet (HFD). However, the contribution of necroptosis, a caspase-independent pathway of cell death, to HFD-induced liver injury is not known. Wild-type C57BL/6 and receptor interacting protein (RIP) 3-/- mice were randomized to chow or HFD. HFD-fed C57BL/6 mice increased expression of RIP3, the master regulator of necroptosis, as well as phosphorylated mixed lineage kinase domain-like, an effector of necroptotic cell death, in liver. HFD did not increase phosphorylated mixed lineage kinase domain-like in RIP3-/- mice. HFD increased fasting insulin and glucose, as well as glucose intolerance, in C57BL/6 mice. RIP3-/- mice were glucose-intolerant even on the chow diet; HFD further increased fasting glucose and insulin but not glucose intolerance. HFD also increased hepatic steatosis, plasma alanine aminotransferase activity, inflammation, oxidative stress, and hepatocellular apoptosis in wild-type mice; these responses were exacerbated in RIP3-/- mice. Importantly, increased inflammation and injury were associated with early indicators of fibrosis in RIP3-/- compared to C57BL/6 mice. Culture of AML12 hepatocytes with palmitic acid increased cytotoxicity through apoptosis and necrosis. Inhibition of RIP1 with necrostatin-1 or small interfering RNA knockdown of RIP3 reduced palmitic acid-induced cytotoxicity. CONCLUSION: Absence of RIP3, a key mediator of necroptosis, exacerbated HFD-induced liver injury, associated with increased inflammation and hepatocyte apoptosis, as well as early fibrotic responses; these findings indicate that shifts in the mode of hepatocellular death can influence disease progression and have therapeutic implications because manipulation of hepatocyte cell death pathways is being considered as a target for treatment of nonalcoholic fatty liver disease. (Hepatology 2016;64:1518-1533).

Linking Pathogenic Mechanisms of Alcoholic Liver Disease With Clinical Phenotypes.

Alcoholic liver disease (ALD) develops in approximately 20% of alcoholic patients, with a higher prevalence in females. ALD progression is marked by fatty liver and hepatocyte necrosis, as well as apoptosis, inflammation, regenerating nodules, fibrosis, and cirrhosis.(1) ALD develops via a complex process involving parenchymal and nonparenchymal cells, as well as recruitment of other cell types to the liver in response to damage and inflammation. Hepatocytes are damaged by ethanol, via generation of reactive oxygen species and induction of endoplasmic reticulum stress and mitochondrial dysfunction. Hepatocyte cell death via apoptosis and necrosis are markers of ethanol-induced liver injury. We review the mechanisms by which alcohol injures hepatocytes and the response of hepatic sinusoidal cells to alcohol-induced injury. We also discuss how recent insights into the pathogenesis of ALD will affect the treatment and management of patients.

Antiviral innate immunity disturbs podocyte cell function.

Immunoglobulin A nephropathy (IgAN) is the most common form of glomerulonephritis throughout the world. A majority (approx. 60%) of patients with IgAN experience disease exacerbations associated with an acute respiratory or gastrointestinal illness that appears to represent a viral infection. However, the exact mechanism of the disease exacerbation by viral infection is not understood, especially at the cellular and molecular levels. Here we report that glomerular podocytes express the major sensors for double-stranded RNA (dsRNA), a common byproduct of viral replication. In addition to these receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene 1 (RIG-I)-like helicases (RLHs), podocytes express the collateral proteins required to support intracellular signaling. The pathways that mediate responses to dsRNA are fully functional in podocytes. The transcription factor interferon regulatory factor 3 (IRF3) and nuclear factor kappa B (NF-ĸB) are phosphorylated and translocate to the nucleus, and dsRNA increases synthesis of proteins driven by IRF3 (P54, P56 and P60) or NF-ĸB (interleukin 8 and A20). Furthermore, dsRNA suppresses podocyte cell migration, alters the expression of a panel of podocyte essential proteins (nephrin, podocin and CD2-associated protein or CD2AP) and changes transepithelial albumin flux. These effects are dsRNA sensor-specific: TLR3-/- podocytes do not respond to extracellular dsRNA, while intracellular dsRNA has no effect on podocytes bearing a dominant negative form of the major active RLH. These results demonstrate that innate responses to viruses can disturb podocyte cell function in vitro.

Epidermal growth factor receptor is essential for Toll-like receptor 3 signaling.

Toll-like receptors (TLRs) recognize specific microbial products and elicit innate immune signals to activate specific transcription factors that induce protective proteins, such as interferon. TLR3 is localized to endosomes and recognizes double-stranded RNA (dsRNA), which is generated by virally infected or apoptotic cells. TLR3 has been genetically linked to several human diseases, including some without viral etiology. Unlike other TLRs, TLR3 requires phosphorylation of two specific tyrosine residues in its cytoplasmic domain to recruit the adaptor protein TRIF (Toll-interleukin-1 receptor domain-containing adaptor protein inducing interferon-ß) and initiate the antiviral response. We showed that two protein tyrosine kinases, the epidermal growth factor receptor (EGFR) ErbB1 and Src, bound sequentially to dsRNA-activated TLR3 and phosphorylated the two tyrosine residues. In cells lacking EGFR or treated with an inhibitor of EGFR, viral replication was enhanced and induction of antiviral genes was impaired. Thus, these results reveal a connection between antiviral innate immunity and cell growth regulators.

The inhibitory action of P56 on select functions of E1 mediates interferon's effect on human papillomavirus DNA replication.

The interferon (IFN)-induced protein P56 inhibits human papillomavirus (HPV) DNA replication by binding to HPV E1, which has several distinct functions in initiating viral DNA replication. Here, we determined that P56 inhibited HPV type 18 (HPV18) E1's DNA helicase activity, E2 binding, and HPV Ori sequence-specific DNA binding but not nonspecific DNA binding. We observed that deletion of a single amino acid, F399, produced an E1 mutant that could not bind P56. This E1 mutant retained its ability to support Ori DNA replication, but this activity was not inhibited by IFN, demonstrating that P56 is the principal executor of the anti-HPV action of IFN.

Interferon-inducible protein, P56, inhibits HPV DNA replication by binding to the viral protein E1.

Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the expression of which is strongly induced by IFN, double-stranded RNA and viruses, mediates the anti-HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN-treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein-protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin-binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N-terminal region of P56 and the C-terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1-mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.

Identification of functional domains of phosphoproteins of two morbilliviruses using chimeric proteins.

The paramyxovirus P protein is an essential component of the transcriptase and replicase complex along with L protein. In this article, we have examined the functional roles of different domains of P proteins of two closely related morbilliviruses, Rinderpest virus (RPV) and Peste des petits ruminants virus (PPRV). The PPRV P protein physically interacts with RPV L as well as RPV N protein when expressed in transfected cells, as shown by co-immunoprecipitation. The heterologous L-P complex is biologically active when tested in a RPV minigenome replication/transcription system, only when used with PPRV N protein but not with RPV N protein. Employing chimeric PPRV/RPV cDNAs having different coding regions of P protein in the minigenome replication/transcription system, we identified a region between 290 and 346 aa in RPV P protein necessary for transcription of the minigenome.

Phosphorylation status of the phosphoprotein P of rinderpest virus modulates transcription and replication of the genome.

The phosphoprotein P of paramyxoviruses is known to play more than one role in genome transcription and replication. Phosphorylation of P at the NH(2) terminus by cellular casein kinase II has been shown to be necessary for transcription of the genome in some of the viruses, while it is dispensable for replication. The phosphorylation null mutant of rinderpest virus P protein, in which three serine residues have been mutated, has been shown earlier to be non-functional in an in vivo minigenome replication/transcription system. In this work, we have shown that the phosphorylation of P protein is essential for transcription, whereas the null mutant is active in replication of the genome in vivo. The null mutant P acts as a transdominant repressor of transcriptional activity of wild-type P and as an activator of replication carried out by wild-type P protein. These results suggest the phosphorylation status of P may act as a replication switch during virus replication. We also show that the phosphorylation null mutant P is capable of interacting with L and N proteins and is able to form a tripartite complex of L-(N-P) when expressed in insect cells, similar to wild-type P protein.