Paramananda Saikia

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 IFNs antiviral action against HPV.

Epidermal Growth Factor Receptor Is Essential for Toll-Like Receptor 3 Signaling

TLR3-mediated antiviral responses require phosphorylation of TLR3 by a growth factor receptor and a nonreceptor tyrosine kinase. A Growth Factor Receptor Takes Its Toll Toll-like receptor 3 (TLR3) initiates innate immune responses to double-stranded RNA (dsRNA) released by viruses or apoptotic cells. Activation of TLR3 requires phosphorylation of two tyrosine residues in its cytoplasmic domain. Yamashita et al. identified the tyrosine kinases involved and dissected the temporal sequence of these phosphorylation events. In response to dsRNA, TLR3 was phosphorylated by the epidermal growth factor receptor ErbB1, a receptor tyrosine kinase that triggers cellular proliferation in response to ligand binding. This phosphorylation event was required to trigger phosphorylation of TLR3 at a different site by the tyrosine kinase Src. In cells lacking EGFR or treated with an inhibitor of EGFR, the TLR3-mediated antiviral response was impaired. These results indicate that TLR3-mediated antiviral responses require tyrosine kinases with established roles in regulating cell growth. 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-β) 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

ABSTRACT 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) E1s 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.

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.

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)

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 NH2 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.

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).

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.

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

ABSTRACT 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 Descemets 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. HIGHLIGHTSOverlying keratocytes undergo apoptosis following mechanical injury to corneal endothelial cells in rabbits.The epithelial basement membrane and Descemets basement membrane have high concentrations of nidogen‐1 in rabbits.There is higher nidogen‐1 in keratocytes and extracellular matrix in the posterior stroma compared to anterior stroma.

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.