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.

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.Hepatitis C virus (HCV), the sole member of the Hepacivirus genus within the Flaviviridae, poses a global health burden, with an estimated 170 million infected individuals (according to the WHO). The majority of patients suffer a chronic infection that is associated with a progressive liver disease (1). HCV has a short positive-sense RNA genome encoding three structural (core protein, E1, and E2) glycoproteins (gps) and seven nonstructural proteins (p7 and NS2 to NS5) (40). The E1 and E2 gps interact with cell surface receptors to facilitate particle entry via low-pH and clathrin-dependent endocytosis (9,15,29,47,71). The recent discovery that the JFH-1 strain of HCV can replicate and assemble infectious particles in cultured cells (HCVcc) has allowed investigation into the viral life cycle for the first time since its identification almost 20 years ago (41,75,79).Early studies with truncated soluble HCV E2 (sE2) identified interactions with the tetraspanin CD81 and scavenger receptor class B type I (SR-BI) (56, 62). The recent availability of HCVcc and HCV pseudoparticles (HCVpp) provided the tools to validate receptor candidates. HCV entry is thought to require at least three cellular receptors: CD81, SR-BI, and the tight junction protein claudin-1 (reviewed in references 21 and 74). Other candidate components include glycosaminoglycans (5, 6, 51), low-density lipoprotein receptor (49,77), and the C...

NaHCO(3) transporters are involved in maintenance of intracellular pH and transepithelial HCO(3)(-) movement in many rodent tissues. To establish the human relevance of the many investigations on rodents, this study aimed to map these transporters and a related polypeptide, NaBC1 [solute carrier 4 (SLC4)A11], to several human tissues by using PCR on reverse transcribed human mRNA and immunoperoxidase histochemistry. The mRNA encoding the electroneutral Na(+):HCO(3)(-) cotransporter (NBCe1; SLC4A4), was expressed in renal cortex, renal medulla, stomach, duodenum, jejunum, ileum, colon, pancreas, choroid plexus, cerebellum, cerebrum, and hippocampus. NBCe2 (SLC4A5) and NBCn1 (SLC4A7) mRNAs were mainly found in kidney and brain tissues, as was mRNA encoding the Na(+)-dependent anion exchangers NCBE (SLC4A10) and NDCBE1 (SLC4A8). In addition to previous findings, NBCn1 protein was localized to human renal medullary thick ascending limbs and duodenal epithelial villus cells and NBCe2 protein to renal collecting ducts. Finally, the message encoding NaBC1 was found in kidney, stomach, duodenum, pancreas, and brain, and the corresponding protein in the anterior and posterior corneal epithelia, renal corpuscules, proximal tubules, collecting ducts, pancreatic ducts, and the choroid plexus epithelium. In conclusion, the selected human tissues display distinct expression patterns of HCO(3)(-) transporters, which closely resemble that of rodent tissues.

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, while not causing 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 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 multi-subunit RNA polymerases.Termination of transcription is an obligatory step following synthesis of the transcript, which leads to dissociation of RNA polymerase (RNAP) and the transcript from the template DNA. However, apparently different mechanisms are utilized by evolutionary conserved multi-subunit RNAPs from bacteria, archaea, and three eukaryotic RNAPs to terminate transcription (1-3). Pol III terminates after synthesis of a poly-U stretch (4, 5), and most studies have focused on the efficiency of recognition of the poly-T (on the nontemplate strand) termination signal (6). Both upstream and downstream sequences were shown to influence efficiency of recognition (7). However, the events leading to termination on the poly-T signal, i.e. dissociation of Pol III from the template, are not known.We investigated this problem by using assembled elongation complexes, a technique successfully used to investigate various RNAPs (8-11). These complexes, assembled with purified RNAP, synthetic complementary template and non-template DNA strands and RNA, allow skipping the step of initiation and, therefore, excluding any accessory factors from the reaction. Complexes were immobilized on streptavidin beads via biotin on the 5′ end of the non-template strand (scheme in Fig. 1A). The RNA in complexes was radioactively labeled by incorporation of radioactive NMP (12). First, we analyzed transcription through poly-T signals of various lengths by purified S. cerevisiae Pol III. As seen from Fig. 1A, at poly-T signals longer than 5 nucleotides, transcripts finishing at the end the poly-T signal were formed. On long poly-T signals (12T), transcription was stopping predominantly after 6 th -10 th T (T 12 template in Fig. 1B, lane 10). No stopping was observed on homopolymeric tracts other than poly-T (Fig. S1). We tested, if transcripts ending with a poly-U stretch were released from the template as a result of termination. This can be done by analysis of transcripts in the supernatant and immobilized fractions of the reaction ("super" and "beads" fractions, respectively, in scheme of Fig. 1B). As seen from Fig. 1B, while RNAs resulting from transcription to the end of tem...

Increasing tubular fluid flow rate has previously been shown to induce K+ secretion in mammalian cortical collecting duct. The mechanism responsible was examined in the present study using MDCK cells as a model. The change in membrane potential difference (EM) of MDCK cells was measured with a fluorescent voltage-sensitive dye, DiBAC4(3), when the cell's primary cilium was continuously bent with a micropipette or by the flow of perfusate. Bending the cilium produced a hyperpolarization of the membrane that lagged behind the increase in intracellular Ca2+ concentration by an average of 36 seconds. Gd3+, an inhibitor of the flow-induced Ca2+ increase, prevented the hyperpolarization. Blocking K+ channels with Ba2+ reduced the flow-induced hyperpolarization, implying that it resulted from activation of Ca2+-sensitive K+ channels. Further studies demonstrated that the hyperpolarization was diminished by the blocker of Ca2+-activated K+ channels, charybdotoxin, whereas iberiotoxin or apamin had no effect, results consistent with the activation of intermediate-conductance Ca2+-sensitive K+ channels. RT-PCR analysis and sequencing confirmed the presence of intermediate-conductance K+ channels in MDCK cells. We conclude that the increase in intracellular Ca2+ associated with bending of the primary cilium is the cause of the hyperpolarization and increased K+ conductance in MDCK cells.