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.

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Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections in infants and young children. In addition, RSV causes significant morbidity and mortality in hospitalized elderly and immunocompromised patients. Currently, only palivizumab, a monoclonal antibody against the RSV fusion (F) protein, and inhaled ribavirin are approved for the prophylactic and therapeutic treatment of RSV, respectively. Therefore, there is a clinical need for safe and effective therapeutic agents for RSV infections. H uman respiratory syncytial virus (RSV) is the predominant cause of bronchiolitis and pneumonia in infants and young children (1). Those most at risk for severe RSV disease are infants born prematurely (Ͻ34 weeks gestation) or less than 6 weeks of age and children with underlying medical conditions, such as bronchopulmonary dysplasia, congenital heart disease, or immunodeficiency (1-3). Severe RSV infection in children less than 1 year old is associated with recurrent wheezing and asthma later in life (4). RSV is also an important cause of lower respiratory tract infections in immunocompromised individuals and the elderly, often resulting in significant morbidity and mortality (5-7).Currently, there is no effective vaccine available for the prevention of RSV infection. Current approved therapeutic options for RSV include palivizumab (Synagis), a neutralizing monoclonal antibody against the RSV F protein, and inhaled ribavirin (Virazole), a broad-spectrum nucleoside analog targeting RNA transcription/replication. Palivizumab is approved for the prophylactic treatment of pediatric patients at high risk of developing severe RSV infection, whereas ribavirin is the only antiviral approved for RSV treatment (8). However, contradictory observations regarding the efficacy, concerns about tolerability, and challenging routes of administration have significantly limited the use of inhaled ribavirin (9). Therefore, there is a clinical need for a safe and effective therapeutic for pediatric and adult RSV infections. Recently, a number of small-molecule RSV inhibitors have been identified. These inhibitors partition into three categories based upon their different mechanisms of action: (i) nucleocapsid protein inhibitors (RSV604) (10), (ii) RNA-dependent RNA polymerase inhibitors (YM-53403, BI-D, and ALS-8176) (11-13), and (iii) fusion inhibitors (VP-14637, TMC-353121, BMS-433771, and GS-5806) (14-17). Among these, RSV fusion inhibitors are the most potent class in vitro and exhibit efficacy in animal models of RSV infection (14-17). Currently, only ALS-8176 and GS-5806 are being clinically developed for the treatment of RSV infection.RSV is an enveloped virus with a negative-sense, single-stranded RNA genome. RSV infection is initiated by attachment of the viral glycoprotein (G) to the cell surface. Following attachment, the RSV fusion protein (F) mediates fusion of the viral and cellular membranes, allowing the viral replication complex to enter the cell. Spread of RSV infection occurs either through cel...

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.Hepatitis C virus (HCV) is an enveloped positive-stranded RNA virus and the sole member of the genus Hepacivirus within the Flaviviridae. Approximately 170 million individuals are infected with HCV worldwide, and the majority are at risk for developing serious progressive liver disease. The HCV RNA genome of approximately 9.6 kb encodes a polyprotein of around 3,000 amino acids, which is cleaved by viral and cellular proteases to generate the structural and nonstructural (NS) proteins. The amino terminus of the polyprotein sequence contains the structural proteins including the core, the envelope glycoproteins (GPs) E1 and E2, and p7. The NS proteins including NS2 through NS5 are located at the carboxy terminus of the polyprotein. Much of our current understanding of HCV replication has been gained through the use of genomic replicons (reviewed in reference 7). The recent development of an infectious system allowing the generation of HCV particles in cell culture (HCVcc) has enabled the complete viral life cycle to be explored (63,99,110).

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 1.08 g ml−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 200 nm, showed that 78 % of HCV RNA from liver was >100 nm in size, with a positive-/negative-strand ratio of 6 : 1. Also, 8 % of HCV RNA was found in particles with diameters between 40 nm and 70 nm and a positive-/negative-strand ratio of 45 : 1. 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.

Atherosclerosis has been described as a liver disease of the heart [1]. The liver is the central regulatory organ of lipid pathways but since dyslipidaemias are major contributors to cardiovascular disease and type 2 diabetes rather than liver disease, research in this area has not been a major focus for hepatologists. Virus-host interaction is a continuous co-evolutionary process [2] involving the host immune system and viral escape mechanisms [3]. One of the strategies HCV has adopted to escape immune clearance and establish persistent infection is to make use of hepatic lipid pathways. This review aims to: • update the hepatologist on lipid metabolism • review the evidence that HCV exploits hepatic lipid pathways to its advantage • discuss approaches to targeting host lipid pathways as adjunctive therapy.

In the absence of satisfactory cell culture systems for hepatitis C virus (HCV), virtually all that is known about the proteins of the virus has been learned by the study of recombinant proteins. Characterization of virus proteins from patients with HCV has been retarded by the low virus titre in blood and limited availability of infected tissue. Here, the authors have identified a primary infection in a liver transplanted into an immunodeficient patient with chronic HCV. The patient required re-transplant and the infected liver, removed 6 weeks after the initial transplant, had a very high titre of HCV, 5610 9 International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1?04 g ml "1 with 73 % of virus below 1?08 g ml "1 . Full-length HCV RNA was detected by Northern blotting and the ratio between positive-and negative-strand HCV RNA was determined as 60. HCV was partially purified by precipitation with heparin/Mn 2+ and a single species of each of the three structural proteins, core, E1 and E2, was detected by Western blotting. The molecular mass of core was 20 kDa, which corresponds to the mature form from recombinant sources. The molecular mass of glycoprotein E1 was 31 kDa before and 21 kDa after deglycosylation with PNGase F or endoglycosidase H. Glycoprotein E2 was 62 kDa before and 36 kDa after deglycosylation, but E2-P7 was not detected. This was in contrast to recombinant sources of E2 which contain E2-P7. INTRODUCTIONInfection with hepatitis C virus (HCV) occurs worldwide and afflicts approximately 170 million people (Anonymous, 1997). Chronic infection, which occurs in 80 % of cases, can result in chronic active hepatitis, cirrhosis and hepatocellular carcinoma and, less commonly, extrahepatic autoimmune or immune complex diseases (reviewed by Major et al., 2001). Gene fragments from the virus were initially identified by screening a lambda phage expression library of cDNA from the nucleic acid extracted from infected chimpanzee serum against serum from a non-A non-B hepatitis patient (Choo et al., 1989). Subsequently, the whole virus genome of approximately 9500 nt was rescued, sequenced and shown to exhibit a similar genome organization to the flaviviruses and pestiviruses (Choo et al., 1991). This insight, the in vitro transcription of the genome and extensive studies of viral protein expression in eukaryotic expression systems indicate that the genome consists of a single open reading frame encoding a 3000 aa polyprotein flanked by highly conserved 59 and 39 untranslated regions. The putative structural proteins of the virus, core protein and two membrane glycoproteins, E1 and E2, lie at the amino-terminal end of the polyprotein and are released co-translationally by host cell signal peptidase. The nonstructural proteins of the virus, NS2 to NS5B, are located at the C-terminal end of the polyprotein and are released by virus proteases (reviewed by Major et al., 2001;Bartenschlager & Lohmann, 2000). In between the structural and non-structural...

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