Cyclic compression increases F508 Del CFTR expression in ciliated human airway epithelium.

The mechanisms by which transepithelial pressure changes observed during exercise and airway clearance can benefit lung health are challenging to study. Here, we have studied 117 mature, fully ciliated airway epithelial cell filters grown at air-liquid interface grown from 10 cystic fibrosis (CF) and 19 control subjects. These were exposed to cyclic increases in apical air pressure of 15 cmH2O for varying times. We measured the effect on proteins relevant to lung health, with a focus on the CF transmembrane regulator (CFTR). Immunoflourescence and immunoblot data were concordant in demonstrating that air pressure increased F508Del CFTR expression and maturation. This effect was in part dependent on the presence of cilia, on Ca2+ influx, and on formation of nitrogen oxides. These data provide a mechanosensory mechanism by which changes in luminal air pressure, like those observed during exercise and airway clearance, can affect epithelial protein expression and benefit patients with diseases of the airways.

Augmentation of CFTR maturation by S-nitrosoglutathione reductase.

S-nitrosoglutathione (GSNO) reductase regulates novel endogenous S-nitrosothiol signaling pathways, and mice deficient in GSNO reductase are protected from airways hyperreactivity. S-nitrosothiols are present in the airway, and patients with cystic fibrosis (CF) tend to have low S-nitrosothiol levels that may be attributed to upregulation of GSNO reductase activity. The present study demonstrates that 1) GSNO reductase activity is increased in the cystic fibrosis bronchial epithelial (CFBE41o(-)) cells expressing mutant F508del-cystic fibrosis transmembrane regulator (CFTR) compared with the wild-type CFBE41o(-) cells, 2) GSNO reductase expression level is increased in the primary human bronchial epithelial cells expressing mutant F508del-CFTR compared with the wild-type cells, 3) GSNO reductase colocalizes with cochaperone Hsp70/Hsp90 organizing protein (Hop; Stip1) in human airway epithelial cells, 4) GSNO reductase knockdown with siRNA increases the expression and maturation of CFTR and decreases Stip1 expression in human airway epithelial cells, 5) increased levels of GSNO reductase cause a decrease in maturation of CFTR, and 6) a GSNO reductase inhibitor effectively reverses the effects of GSNO reductase on CFTR maturation. These studies provide a novel approach to define the subcellular location of the interactions between Stip1 and GSNO reductase and the role of S-nitrosothiols in these interactions.

Development of an AP-FRET based analysis for characterizing RNA-protein interactions in myotonic dystrophy (DM1).

Förster Resonance Energy Transfer (FRET) microscopy is a powerful tool used to identify molecular interactions in live or fixed cells using a non-radiative transfer of energy from a donor fluorophore in the excited state to an acceptor fluorophore in close proximity. FRET can be a very sensitive tool to study protein-protein and/or protein-nucleic acids interactions. RNA toxicity is implicated in a number of disorders; especially those associated with expanded repeat sequences, such as myotonic dystrophy. Myotonic dystrophy (DM1) is caused by a (CTG)n repeat expansion in the 3' UTR of the DMPK gene which results in nuclear retention of mutant DMPK transcripts in RNA foci. This results in toxic gain-of-function effects mediated through altered functions of RNA-binding proteins (e.g. MBNL1, hnRNPH, CUGBP1). In this study we demonstrate the potential of a new acceptor photobleaching assay to measure FRET (AP-FRET) between RNA and protein. We chose to focus on the interaction between MBNL1 and mutant DMPK mRNA in cells from DM1 patients due to the strong microscopic evidence of their co-localization. Using this technique we have direct evidence of intracellular interaction between MBNL1 and the DMPK RNA. Furthermore using the AP-FRET assay and MBNL1 mutants, we show that all four zinc-finger motifs in MBNL1 are crucial for MBNL1-RNA foci interactions. The data derived using this new assay provides compelling evidence for the interaction between RNA binding proteins and RNA foci, and mechanistic insights into MBNL1-RNA foci interaction demonstrating the power of AP-FRET in examining RNA-Protein interactions in DM1.

Inhibition of Hepatitis E virus replication using short hairpin RNA (shRNA).

Hepatitis E virus (HEV) is a non-enveloped, single-stranded, positive sense RNA virus, which is a major cause of water-borne hepatitis. RNA interference (RNAi) is a sequence-specific cellular antiviral defence mechanism, induced by double-stranded RNA, which we used to investigate knockdown of several genes and the 3' cis-acting element (CAE) of HEV. In the present report, shRNAs were developed against the putative helicase and replicase domains and the 3'CAE region of HEV. Production of siRNA was confirmed by northern hybridization. The possible innate response induction due to shRNA expressions was verified by transcript analysis for interferon-beta and 2',5'-oligoadenylate synthetase genes and was found to be absent. Initially, the selected shRNAs were tested for their efficiency against the respective genes/3'CAE using inhibition of fused viral subgenomic target domain-renilla luciferase reporter constructs. The effective shRNAs were studied for their inhibitory effects on HEV replication in HepG2 cells using HEV replicon and reporter replicon. RNAi mediated silencing was demonstrated by reduction of luciferase activity in subgenomic target-reporter constructs and reporter replicon. The real time PCR was used to demonstrate inhibition of native replicon replication in transfected cells. Designed shRNAs were found to be effective in inhibiting virus replication to a variable extent (45-93%).

Subcellular localization of hepatitis E virus (HEV) replicase.

Hepatitis E virus (HEV) is a hepatotropic virus with a single sense-strand RNA genome of approximately 7.2 kb in length. Details of the intracellular site of HEV replication can pave further understanding of HEV biology. In-frame fusion construct of functionally active replicase-enhanced green fluorescent protein (EGFP) gene was made in eukaryotic expression vector. The functionality of replicase-EGFP fusion protein was established by its ability to synthesize negative-strand viral RNA in vivo, by strand-specific anchored RT-PCR and molecular beacon binding. Subcellular co-localization was carried out using organelle specific fluorophores and by immuno-electron microscopy. Fluorescence Resonance Energy Transfer (FRET) demonstrated the interaction of this protein with the 3' end of HEV genome. The results show localization of replicase on the endoplasmic reticulum membranes. The protein regions responsible for membrane localization was predicted and identified by use of deletion mutants. Endoplasmic reticulum was identified as the site of replicase localization and possible site of replication.

Role of surface promoter mutations in hepatitis B surface antigen production and secretion in occult hepatitis B virus infection.

The production, secretion, and localization of surface proteins of hepatitis B virus (HBV) and the ratio of large to small surface protein S was studied in HepG2 cells transfected with the wild-type and mutant pre-S1 and pre-S2/S promoters of HBV molecular clones 313.1 (GenBank accession no. AY161147) and 761.1 (GenBank accession no. AY161159) from two patients with occult HBV infection. Fusion constructs were made by in frame fusion of the wild-type surface gene to the mutant pre-S1 and pre-S2/S promoters and wild-type promoter so that the structural part of the small surface protein remains identical. HepG2 cells transfected transiently were used for analysis. HBV surface proteins production and secretion was determined by enzyme linked immuno assay (ELISA) and localization by immunofluorescence. Immunoprecipitation of the large, middle, and small surface protein was carried out in transient transfected and metabolically labeled cells to determine the ratio of the large to small surface protein. The results indicate that HepG2 cells transfected with mutant HBV promoters had reduced HBV surface proteins secretion compared to wild-type HBV. HepG2 cells transfected with mutant HBV pre-S1 and pre-S2/S promoters showed cytoplasmic aggregation of HBV surface proteins compared to wild-type HBV promoters, which showed diffuse cytoplasmic localization. In all cases, the HBV surface proteins localized to the endoplasmic reticulum. The ratio between the large and small surface protein was 1.89 and 0.56 with mutant HBV 313.1 and 761.1 pre-S1 and pre-S2/S promoters, respectively, compared to 0.17 in wild-type. Thus, the aggregation of surface proteins, altered ratio and secretion of surface proteins were possibly the causes of occult hepatitis B infection.

Hepatitis E virus.

Hepatitis E virus (HEV) is the aetiological agent of non-HAV enterically transmitted hepatitis. It is the major cause of sporadic as well as epidemic hepatitis, which is no longer confined to Asia and developing countries but has also become a concern of the developed nations. In the Indian subcontinent, it accounts for 30-60% of sporadic hepatitis. It is generally accepted that hepatitis E is mostly self-limited and never progresses to chronicity. It has a higher mortality in pregnant women where the disease condition is accentuated with the development of fulminant liver disease. Currently, no antiviral drug or vaccine is licensed for HEV, although a vaccine candidate is in clinical trials. HEV genome is 7.2kb in size with three open reading frames (ORFs) and 5' and 3' cis acting elements, which have important roles to play in HEV replication and transcription. ORF1 codes for methyl transferase, protease, helicase and replicase; ORF2 codes for the capsid protein and ORF3 for a protein of undefined function. HEV has recently been classified in the genus Hepevirus of the family Hepeviridae. There are four major recognised genotypes with a single known serotype. The absence of a reliable in vitro propagation system is an obstacle to deciphering HEV biology. The genome of HEV has been cloned, sequenced and the infectious nature of these replicons has been established. However, questions related to replication, transcription, virus-host interactions and pathogenesis remain to be answered. This comprehensive review summarises the progress made so far in HEV research, and addresses some of the unanswered questions.

Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.

Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.