Søren Nielsen

Association between Hepatitis C Virus and Very-Low-Density Lipoprotein (VLDL)/LDL Analyzed in Iodixanol Density Gradients

ABSTRACT Hepatitis C virus (HCV) RNA circulates in the blood of persistently infected patients in lipoviroparticles (LVPs), which are heterogeneous in density and associated with host lipoproteins and antibodies. The variability and lability of these virus-host complexes on fractionation has hindered our understanding of the structure of LVP and determination of the physicochemical properties of the HCV virion. In this study, HCV from an antibody-negative immunodeficient patient was analyzed using three fractionation techniques, NaBr gradients, isotonic iodixanol, and sucrose gradient centrifugation. Iodixanol gradients were shown to best preserve host lipoprotein-virus complexes, and all HCV RNA was found at densities below 1.13 g/ml, with the majority at low density, ≤1.08 g/ml. Immunoprecipitation with polyclonal antibodies against human ApoB and ApoE precipitated 91.8% and 95.0% of HCV with low density, respectively, suggesting that host lipoprotein is closely associated with HCV in a particle resembling VLDL. Immunoprecipitation with antibodies against glycoprotein E2 precipitated 25% of HCV with low density, providing evidence for the presence of E2 in LVPs. Treatment of serum with 0.5% deoxycholic acid in the absence of salt produced HCV with a density of 1.12 g/ml and a sedimentation coefficient of 215S. The diameters of these particles were calculated as 54 nm. Treatment of serum with 0.18% NP-40 produced HCV with a density of 1.18 g/ml, a sedimentation coefficient of 180S, and a diameter of 42 nm. Immunoprecipitation analysis showed that ApoB remained associated with HCV after treatment of serum with deoxycholic acid or NP-40, whereas ApoE was removed from HCV with these detergents.

Identification of a Residue in Hepatitis C Virus E2 Glycoprotein That Determines Scavenger Receptor BI and CD81 Receptor Dependency and Sensitivity to Neutralizing Antibodies

ABSTRACT Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.

Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity

ABSTRACT Viruses exploit signaling pathways to their advantage during multiple stages of their life cycle. We demonstrate a role for protein kinase A (PKA) in the hepatitis C virus (HCV) life cycle. The inhibition of PKA with H89, cyclic AMP (cAMP) antagonists, or the protein kinase inhibitor peptide reduced HCV entry into Huh-7.5 hepatoma cells. Bioluminescence resonance energy transfer methodology allowed us to investigate the PKA isoform specificity of the cAMP antagonists in Huh-7.5 cells, suggesting a role for PKA type II in HCV internalization. Since viral entry is dependent on the host cell expression of CD81, scavenger receptor BI, and claudin-1 (CLDN1), we studied the role of PKA in regulating viral receptor localization by confocal imaging and fluorescence resonance energy transfer (FRET) analysis. Inhibiting PKA activity in Huh-7.5 cells induced a reorganization of CLDN1 from the plasma membrane to an intracellular vesicular location(s) and disrupted FRET between CLDN1 and CD81, demonstrating the importance of CLDN1 expression at the plasma membrane for viral receptor activity. Inhibiting PKA activity in Huh-7.5 cells reduced the infectivity of extracellular virus without modulating the level of cell-free HCV RNA, suggesting that particle secretion was not affected but that specific infectivity was reduced. Viral particles released from H89-treated cells displayed the same range of buoyant densities as did those from control cells, suggesting that viral protein association with lipoproteins is not regulated by PKA. HCV infection of Huh-7.5 cells increased cAMP levels and phosphorylated PKA substrates, supporting a model where infection activates PKA in a cAMP-dependent manner to promote virus release and transmission.

Nuclear patterns of cyclin (PCNA) antigen distribution subdivide S-phase in cultured cells--some applications of PCNA antibodies.

Immunofluorescence studies using PCNA autoantibodies specific for the proliferation-sensitive protein cyclin have revealed dramatic changes in the nuclear distribution of this protein during the S-phase of normal and transformed cells. Patterns of cyclin antigen distribution subdivide S-phase and have provided new cell cycle landmarks. Some of these (nucleolar exclusion or staining), mimic topographical patterns of DNA synthesis thus arguing for a role of this protein in some specific aspect of DNA replication. Cells outside S-phase (G0 included) stain only weakly with PCNA antibodies, stressing the usefulness of this reagent for identifying proliferating cells (S-phase cells) of both normal and malignant origins.

Mechanism of Eukaryotic RNA Polymerase III Transcription Termination

Stopping Transcription It is as important to terminate any biological process as it is to start it. Transcription, copying information encoded in genes into RNA, requires accurate and timely termination. Nielsen et al. (p. 1577) present a mechanism for transcription termination by RNA polymerase III, the enzyme that synthesizes the majority of RNA molecules in eukaryotes. In this scenario, the folding of the RNA as it is transcribed by polymerase into a highly structured transcript causes termination at the end of its synthesis. This mechanism may serve as a control of proper folding of structural or catalytic RNAs synthesized by RNA polymerase III. Comparison with other organisms suggests that this mechanism emerged before divergence of bacteria and eukaryotes. Formation of the secondary structure of the transcribed RNA facilitates termination during transcription. Gene expression in organisms involves many factors and is tightly controlled. Although much is known about the initial phase of transcription by RNA polymerase III (Pol III), the enzyme that synthesizes the majority of RNA molecules in eukaryotic cells, termination is poorly understood. Here, we show that the extensive structure of Pol III–synthesized transcripts dictates the release of elongation complexes at the end of genes. The poly-T termination signal, which does not cause termination in itself, causes catalytic inactivation and backtracking of Pol III, thus committing the enzyme to termination and transporting it to the nearest RNA secondary structure, which facilitates Pol III release. Similarity between termination mechanisms of Pol III and bacterial RNA polymerase suggests that hairpin-dependent termination may date back to the common ancestor of multisubunit RNA polymerases.

Intravascular transfer contributes to postprandial increase in numbers of very-low-density hepatitis C virus particles.

BACKGROUND & AIMSnThe physical association of hepatitis C virus (HCV) particles with lipoproteins in plasma results in distribution of HCV in a broad range of buoyant densities. This association is thought to increase virion infectivity by mediating cell entry via lipoprotein receptors. We sought to determine if factors that affect triglyceride-rich lipoprotein (TRL) metabolism alter the density and dynamics of HCV particles in the plasma of patients with chronic HCV infection.nnnMETHODSnFasting patients (n = 10) consumed a high-fat milkshake; plasma was collected and fractionated by density gradients. HCV- RNA was measured in the very-low-density fraction (VLDF, d < 1.025 g/mL) before and at 7 serial time points postprandially.nnnRESULTSnThe amount of HCV RNA in the VLDF (HCV(VLDF)) increased a mean of 26-fold, peaking 180 minutes after the meal (P < .01). Quantification of HCV RNA throughout the density gradient fractions revealed that HCV(VLDF) rapidly disappeared, rather than migrating into the adjacent density fraction. Immuno-affinity separation of the VLDF, using antibodies that recognize apolipoprotein B-100 and not apolipoprotein B-48, showed that HCV(VLDF) is composed of chylomicron- and VLDL-associated HCV particles; peaking 120 and 180 minutes after the meal, respectively. Plasma from fasting HCV-infected patients mixed with uninfected plasma increased the quantity of HCV(VLDF), compared with that mixed with phosphate-buffered saline, showing extracellular assembly of HCV(VLDF).nnnCONCLUSIONSnDietary triglyceride alters the density and dynamics of HCV in plasma. The rapid clearance rate of HCV(VLDF) indicates that association with TRL is important for HCV infectivity. HCV particles, such as exchangeable apolipoproteins, appear to reassociate with TRLs in the vascular compartment.

Characterization of hepatitis C RNA-containing particles from human liver by density and size.

Hepatitis C virus (HCV) particles found in vivo are heterogeneous in density and size, but their detailed characterization has been restricted by the low titre of HCV in human serum. Previously, our group has found that HCV circulates in blood in association with very-low-density lipoprotein (VLDL). Our aim in this study was to characterize HCV RNA-containing membranes and particles in human liver by both density and size and to identify the subcellular compartment(s) where the association with VLDL occurs. HCV was purified by density using iodixanol gradients and by size using gel filtration. Both positive-strand HCV RNA (present in virus particles) and negative-strand HCV RNA (an intermediate in virus replication) were found with densities below 1.08u2005gu2005ml−1. Viral structural and non-structural proteins, host proteins ApoB, ApoE and caveolin-2, as well as cholesterol, triglyceride and phospholipids were also detected in these low density fractions. After fractionation by size with Superose gel filtration, HCV RNA and viral proteins co-fractionated with endoplasmic reticulum proteins and VLDL. Fractionation on Toyopearl, which separates particles with diameters up to 200u2005nm, showed that 78u200a% of HCV RNA from liver was >100u2005nm in size, with a positive-/negative-strand ratio of 6u200a:u200a1. Also, 8u200a% of HCV RNA was found in particles with diameters between 40u2005nm and 70u2005nm and a positive-/negative-strand ratio of 45u200a:u200a1. This HCV was associated with ApoB, ApoE and viral glycoprotein E2, similar to viral particles circulating in serum. Our results indicate that the association between HCV and VLDL occurs in the liver.

Differentiational regulation and phosphorylation of the fatty acid-binding protein from rat mammary epithelial cells

From the soluble protein fraction of lactating rat mammary epithelial cells, fatty acid-binding protein (FABP) was isolated by immunoaffinity chromatography. After digestion with trypsin, peptides were characterized with time-of-flight mass spectrometry and revealed identity with corresponding peptides derived from the heart-type FABP isolated from rat heart. In addition, by electrospray mass spectrometry the molecular mass has been determined to 14683.9 +/- 3 Da, further corroborating the identity. The content of FABP in mammary glands from virgin, pregnant and lactating rats was evaluated using two-dimensional gel electrophoresis and a FABP-specific immunosorbent assay. In the two-dimensional gels FABP was the apparently most abundant cytosolic protein in mammary epithelial cells from rats in late pregnancy as well as from lactating rats. The content of FABP was 59 +/- 19 microgram/mg (n = 11) of soluble proteins from the fully differentiated lactating mammary gland as determined by ELISA. This value represented an 80-fold increase compared with the FABP content of mammary gland from virgin rats, and is comparable with the level found in rat heart. Upon stimulation with insulin a small fraction of FABP was phosphorylated in lactating mammary epithelial cells. In conclusion, these findings indicate that the FABPs from rat mammary gland and heart are identical and further suggest that in mammary gland this FABP may play a role in signal transduction downstream from the insulin receptor.

Insulin resistance and low-density apolipoprotein B-associated lipoviral particles in hepatitis C virus genotype 1 infection

Background The density of hepatitis C virus (HCV) in plasma is heterogeneous but the factors which influence this are poorly understood. Evidence from animal models and cell culture suggest that low-density apolipoprotein B (apoB)-associated HCV lipoviral particles (LVP) are more infectious than high-density HCV. Objective To measure LVP in patients with chronic hepatitis C genotype 1 (CHC-G1) and examine metabolic determinants of LVP load. Patients 51 patients with CHC-G1 infection. Methods Fasting lipid profiles and homeostasis model assessment of insulin resistance (HOMA-IR) were determined in 51 patients with CHC-G1. LVP and non-LVP viral load were measured by real-time PCR of plasma at density <1.07u2005g/ml and >1.07u2005g/ml, respectively, following iodixanol density gradient ultracentrifugation. The LVP ratio was calculated using the formula: LVP/(LVP + non-LVP). Results The mean LVP ratio was 0.241 but varied 25-fold (from 0.029 to 0.74). Univariate analysis showed that the LVP ratio correlated with HOMA-IR (p=0.004) and the triglyceride/high-density lipoprotein cholesterol (TG/HDL-C) ratio (p=0.004), but not with apoB. In multivariate analysis, HOMA-IR was the main determinant of LVP load (log10IU/ml) (R2=16.6%; p=0.037) but the TG/HDL-C ratio was the strongest predictor of the LVP ratio (R2=24.4%; p=0.019). Higher LVP ratios were associated with non-response to antiviral therapy (p=0.037) and with greater liver stiffness (p=0.001). Conclusion IR and associated dyslipidaemia are the major determinants of low-density apoB-associated LVP in fasting plasma. This provides a possible mechanism to explain why IR is associated with more rapidly progressive liver disease and poorer treatment outcomes.

Characterization of a monoclonal antibody (BG3C8) that reacts with basal cells of stratified epithelia

Monoclonal antibodies were produced against a suspension of formaldehyde fixed human epidermal cells. The supernatant fluid of one clone (BG3C8) yielded a bright immunofluorescent staining of basal cells both in cryostat sections of human split skin and in preparations of purified basal cells. As determined by one‐ and two‐dimensional gel immunoblotting of epidermal basal cell proteins the antibody recognized a minor basic poiypeptide of 55000 apparent molecular weight that was not present in extracts of cultured cell lines of epithelial, fibroblast and lymphoid origin. The distribution of the 55000 molecular weight protein in normal human tissue was determined by immunohistological staining of cryostat tissue sections that included: central nervous, endocrine, female and male reproductive, alimentary, lymphatic‐haemopoietic, respiratory and urinary systems, skin and its appendages, mesenchymal tissue (bone, cartilage, muscle, connective tissue, blood vessels, nerves and synovia) as well as placenta and umbilical cord. The results showed a restricted distribution of this antigen which was found only in basal cells of most stratified or pseudostratified epithelia and in myoepithelial cells. This antibody may be useful in the study of normal and pathological differentiation in various epithelial disorders.