In vitro characterization of six hepatitis B virus genotypes from clinical isolates using transfecting linear HBV genomes.

Hepatitis B virus (HBV) infection is a global public health problem with about 257 million chronically infected people and over 887000 deaths annually. In this study, 32 whole HBV genomes of various genotypes were amplified from clinical isolates to create transfection clones. The clones were sequenced, and their biological properties characterized by transfecting linear HBV clones into HepG2 cells. We analysed the SPI and SPII promotor regions, X-gene, BCP/PC sequences, core, preS/S and HBV polymerase sequences. HBV clones analysed in this study revealed differential replication kinetics of viral nucleic acids and expression of proteins. Sequence analysis of HBV clones revealed mutations in preS1, preS2 and S genes; deletion and insertion and point mutations in BCP/PC region; including novel and previously reported mutations. Among the patient samples tested, HBV genotype B clones were more likely to have higher frequencies of mutations, while sub-genotype A1 and A2 clones tended to have fewer mutations. No polymerase drug resistant mutations were seen. HBeAg mutations were primarily in the BCP/PC region in genotype B, but core truncations were found in genotype E. S gene mutations affecting HBsAg expression and detection were seen in all genotypes except A2. Using an HBV clone with repetitive terminal sequences and a SapI restriction site allowed us to analyse HBV analyte production in cell culture and characterize the genetics of viral phenotypes using complete HBV genomes isolated from serum/plasma samples of infected patients.

Distribution of hepatitis A antibodies in US blood donors.

BACKGROUND: Recently, there has been an increase in the number of hepatitis A outbreaks in the United States. Although the presence of hepatitis A virus (HAV) RNA in blood donors is known to be low, HAV antibody prevalence in this population is unknown. STUDY DESIGN AND METHODS: Samples from 5001 US blood donors collected primarily in the midwestern United States in 2015 were tested for the presence of HAV IgG antibodies using chemiluminescent microparticle immunoassays on the ARCHITECT platform (Abbott Laboratories). RESULTS: The overall prevalence of IgG anti-HAV was 60%. Only one specimen was IgM anti-HAV positive, for an incidence of 0.02%. IgG anti-HAV prevalence among donors aged 16 to 19 years was 67%, decreased to 54% among donors aged 40 to 49 years and increased to 70% among donors aged 80 to 93 years. No differences were seen by sex with overall IgG anti-HAV prevalence of 61% and 60% for males and females, respectively. Among the five states (Illinois, Indiana, Kansas, Kentucky, and Missouri) with the highest number of donors tested, IgG anti-HAV prevalence in Missouri (65%) was significantly higher (p <0.01) than that in Illinois (52%) or Kentucky (59%). No other significant differences between states were noted. CONCLUSION: This study demonstrates the overall high rates of IgG anti-HAV in US blood donors, with the low associated risk of HAV transfusion transmission likely the result of low incidence and effective vaccination.

Disparities in detection of antibodies against hepatitis E virus in US blood donor samples using commercial assays.

BACKGROUND: Reported hepatitis E virus (HEV) antibody assay performance characteristics are variable. Using a subset of surplus US blood donation samples, we compared assays for detecting anti-HEV immunoglobulin M (Ig)M and IgG or total anti-HEV antibodies. STUDY DESIGN AND METHODS: Samples from 5040 random blood donations, all HEV-RNA negative, collected primarily in the midwestern United States in 2015 were tested for anti-HEV IgM and IgG or total anti-HEV using assays manufactured by Diagnostic Systems, Wantai, and MP Biomedicals. RESULTS: Overall, the percentage of detection for anti-HEV IgG and total anti-HEV was 11.4%, and for anti-HEV IgM was 1.8%. Nine samples were reactive for anti-HEV IgM by all assays, giving a recent infection rate of 0.18%. Anti-HEV IgG/total anti-HEV detection rates increased with age. Interassay agreement was higher among the IgG anti-HEV/total anti-HEV assays (84%) than the IgM assays (22%). Regression analyses of signal-to-cutoff ratios from IgG/total antibody assay were heteroskedastic, indicating no constant variance among these assays, suggesting they may detect different epitopes or were affected by waning or less avid antibodies in the US donor population. CONCLUSIONS: Although similar percentages of IgG anti-HEV/total anti-HEV detection were observed across the three commercial assays, each assay detected a unique sample subpopulation and was heteroskedastic when compared pairwise. Discordance was higher among anti-HEV IgM assays, but a recent HEV infection rate of at least 0.18% was estimated based on assay concordance.

Suppression of interferon-α signaling by hepatitis E virus.

UNLABELLED: The interferon (IFN) system is integral to the host response against viruses, and many viruses have developed strategies to overcome its antiviral effects. The effects of hepatitis E virus (HEV), the causative agent of hepatitis E, on IFN signaling have not been investigated primarily because of the nonavailability of an efficient in vitro culture system or small animal models of infection. We report here the generation of A549 cell lines persistently infected with genotype 3 HEV, designated as HEV-A549 cells and the effects HEV has on IFN-α-mediated Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling. Treatment of HEV-A549 cells with 250, 500, and 1000 U/mL of IFN-α for 72 hours showed a dose-dependent reduction in HEV RNA levels by 10%, 20%, and 50%, respectively. IFN-α-stimulated genes coding for the antiviral proteins dsRNA-activated protein kinase (PKR) and 2',5'-oligoadenylate synthetase (2',5'-OAS) were down-regulated in IFN-α-treated HEV-A549 cells. HEV infection also prevented IFN-α-induced phosphorylation of STAT1. Regulation of STAT1 by HEV was specific, as phosphorylation of STAT2, tyrosine kinase (Tyk) 2, and Jak1 by IFN-α was unaltered. Additionally, STAT1 levels were markedly increased in HEV-A549 cells compared with naive A549 cells. Furthermore, binding of HEV open reading frame (ORF)3 protein to STAT1 in HEV-A549 cells was observed. HEV ORF3 protein alone inhibited IFN-α-induced phosphorylation of STAT1 and down-regulated the IFN-α-stimulated genes encoding PKR, 2',5'-OAS, and myxovirus resistance A. CONCLUSION: HEV inhibits IFN-α signaling through the regulation of STAT1 phosphorylation in A549 cells. These findings have implications for the development of new strategies against hepatitis E.

A ceramide-binding C1 domain mediates kinase suppressor of ras membrane translocation.

Genetic and biochemical data support Kinase Suppressor of Ras 1 (KSR1) as a positive regulator of the Ras-Raf-MAPK pathway, functioning as a kinase and/or scaffold to regulate c-Raf-1 activation. Membrane translocation mediated by the KSR1 CA3 domain, which is homologous to the atypical PKC C1 lipid-binding domain, is a critical step of KSR1-mediated c-Raf-1 activation. In this study, we used an ELISA to characterize the KSR1 CA3 domain as a lipid-binding moiety. Purified GST-KSR1-CA3 protein effectively binds ceramide but not other lipids including 1,2-diacylglyceol, dihydroceramide, ganglioside GM1, sphingomyelin and phosphatidylcholine. Upon epidermal growth factor stimulation of COS-7 cells, KSR1 translocates into and is activated within glycosphingolipid-enriched plasma membrane platforms. Pharmacologic inhibition of ceramide generation attenuates KSR1 translocation and KSR1 kinase activation in COS-7 cells. Disruption of two cysteines, which are indispensable for maintaining ternary structure of all C1 domains and their lipid binding capability, mitigates ceramide-binding capacity of purified GST-KSR1-CA3 protein, and inhibits full length KSR1 membrane translocation and kinase activation. These studies provide evidence for a mechanism by which the second messenger ceramide can target proteins to subcellular compartments in the process of transmembrane signal transduction.

Kinase suppressor of Ras transphosphorylates c-Raf-1.

Whether kinase suppressor of Ras1 (KSR1) is an active kinase that phosphorylates c-Raf-1 or a scaffold that coordinates signaling along the Ras/ERK1 signaling module is actively debated. In this study, we generated a monoclonal antibody against a c-Raf-1 peptide containing phosphorylated Thr(269), the putative target for KSR1 kinase activity. We show that this antibody detects Thr(269)-phosphorylated c-Raf-1 in A431 cells upon epidermal growth factor (EGF) stimulation, preceding MEK1 activation. Furthermore, this antibody detects in vitro phosphorylation of FLAG-c-Raf-1 and kinase-dead FLAG-c-Raf-1(K375M) by immunopurified KSR1, but fails to detect phosphorylation of FLAG-c-Raf-1(K375M/T269V), engineered with a Thr(269) to valine substitution. To provide unequivocal evidence that KSR1 is a legitimate kinase, we purified KSR1 to homogeneity, confirmed by mass spectrometry, renatured it in-gel, and demonstrated that it phosphorylates BSA-conjugated c-Raf-1 peptide at Thr(269). These studies add to emerging data validating KSR1 as a kinase that phosphorylates c-Raf-1.

Permissive, in vitro replication of hepatitis B virus genotype E.

A cloned stable cell line, HepG2-HBVE6, was established following transfection of HepG2 cells with a retroviral plasmid into which a 1.1-fold genomic construct of hepatitis B virus (HBV) belonging to genotype E (HBV/E) was inserted. The cell line retains the entire HBV/E insert, and produces episomal HBV DNA. It expresses HBV pregenomic, preS1 and preS2/S transcripts, and sheds hepatitis B surface and e antigens as well as structures resembling HBV-subviral and Dane particles. The HepG2-HBVE6 cell line, in permitting recapitulation of the HBV life cycle, may be used for studying viral characteristics, therapeutic and preventative outcomes and for preparing reagents specific to HBV genotype E.

Reevaluation of a North India isolate of hepatitis E virus based on the full-length genomic sequence obtained following long RT-PCR.

The genomic cloning and sequence of hepatitis E virus (HEV) from an epidemic in North India is reported. We describe here a simple method wherein the viral RNA was reverse transcribed and then amplified in a single step using an extra long polymerase chain reaction procedure. The full genome nucleotide sequence of this HEV isolate (called Yam-67) was made up of 7191 nucleotides, excepting the poly(A) tail and had three open reading frames: ORF1 coding for 1693 amino acids (aa), ORF2 coding for 659 aa and ORF3 coding for 122 aa. This North Indian isolate of HEV showed close sequence homology to other HEV isolates from India and Asia, but was distant from the Chinese genotype 4, Japanese, Mexican and US isolates. There is no indication from sequence analysis that this may be an atypical strain of HEV, as reported earlier.

A C-Terminal Hydrophobic Region is Required for Homo-Oligomerization of the Hepatitis E Virus Capsid (ORF2) Protein.

Hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The open reading frame 2 (ORF2) of HEV encodes the viral capsid protein, which can self-oligomerize into virus-like particles. To understand the domains within this protein important for capsid biogenesis, we have carried out in vitro analyses of association and folding patterns of wild type and mutant ORF2 proteins. When expressed in vitro or in transfected cells, the ORF2 protein assembled as dimers, trimers and higher order forms.While N-terminal deletions upto 111 amino acids had no effect, the deletion of amino acids 585-610 led to reduced homo-oligomerization. This deletion also resulted in aberrant folding of the protein, as determined by its sensitivity to trypsin. This study suggests that a C-terminal hydrophobic region encompassing amino acids 585-610 of the ORF2 protein might be critical for capsid biogenesis.

Downregulation of KSR1 in pancreatic cancer xenografts by antisense oligonucleotide correlates with tumor drug uptake.

While antisense oligonucleotide (AS-ODN) technology holds promise for the treatment of cancer, to date there have been no clinical successes. Unfortunately, current assays are not sufficiently sensitive to measure tissue ODN levels. Hence it has not been possible to ascertain whether treatment failures result from failure of drug delivery. To investigate the relationship between drug uptake and therapeutic effect, we developed an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay (NCHL-ELISA) to quantify Kinase Suppressor of Ras1 (KSR1) AS-ODN drug uptake in plasma and tumor tissues. In mice harboring PANC-1 pancreatic cancer xenografts and continuously infused with AS-ODN, our ELISA detects plasma and tumor KSR1 AS-ODN levels over an extended range, from 0.05 nM to 20 nM. Using this sensitive assay, we demonstrate that KSR1 repression in pancreatic cancer xenografts correlates highly with AS-ODN uptake into tumor tissues. In contrast, plasma drug levels do not correlate with tumor drug content or target downregulation. These studies indicate the efficacy of our ELISA, and suggest that tumor biopsy material will need to be procured to estimate the potential of this antisense technology.

The phosphorylated form of the ORF3 protein of hepatitis E virus interacts with its non-glycosylated form of the major capsid protein, ORF2.

Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser(80) of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

Acidic pH enhances structure and structural stability of the capsid protein of hepatitis E virus.

Hepatitis E virus (HEV) is enterically transmitted and endemic to tropical areas of the world. The major capsid protein of HEV is pORF2 ( approximately 74 kDa), encoded by open reading frame 2 (ORF2). When expressed in insect cells, it is processed into a approximately 55 kDa form (n-pORF2). We also generated a mutant, m-pORF2, lacking a C-terminal hydrophobic region shown earlier to be required for its homo-oligomerization. Circular dichroism was used to measure the secondary structure and stability of these proteins as a function of pH and temperature. With decreasing pH both proteins acquired increasing alpha-helicity and thermal stability in terms of midpoint of denaturation and the Gibbs energy change.