PCSK9, apolipoprotein E and lipoviral particles in chronic hepatitis C genotype 3: evidence for genotype-specific regulation of lipoprotein metabolism.

BACKGROUND & AIMS: Hepatitis C virus (HCV) associates with lipoproteins to form "lipoviral particles" (LVPs) that can facilitate viral entry into hepatocytes. Initial attachment occurs via heparan sulphate proteoglycans and low-density lipoprotein receptor (LDLR); CD81 then mediates a post-attachment event. Proprotein convertase subtilisin kexin type 9 (PCSK9) enhances the degradation of the LDLR and modulates liver CD81 levels. We measured LVP and PCSK9 in patients chronically infected with HCV genotype (G)3. PCSK9 concentrations were also measured in HCV-G1 to indirectly examine the role of LDLR in LVP clearance. METHODS: HCV RNA, LVP (d<1.07g/ml) and non-LVP (d>1.07g/ml) fractions, were quantified in patients with HCV-G3 (n=39) by real time RT-PCR and LVP ratios (LVPr; LVP/(LVP+non-LVP)) were calculated. Insulin resistance (IR) was assessed using the homeostasis model assessment of IR (HOMA-IR). Plasma PCSK9 concentrations were measured by ELISA in HCV-G3 and HCV-G1 (n=51). RESULTS: In HCV-G3 LVP load correlated inversely with HDL-C (r=-0.421; p=0.008), and apoE (r=-0.428; p=0.013). The LVPr varied more than 35-fold (median 0.286; range 0.027 to 0.969); PCSK9 was the strongest negative predictor of LVPr (R(2)=16.2%; p=0.012). HOMA-IR was not associated with LVP load or LVPr. PCSK9 concentrations were significantly lower in HCV-G3 compared to HCV-G1 (p<0.001). PCSK9 did not correlate with LDL-C in HCV-G3 or G1. CONCLUSIONS: The inverse correlation of LVP with apoE in HCV-G3, compared to the reverse in HCV-G1 suggests HCV genotype-specific differences in apoE mediated viral entry. Lower PCSK9 and LDL concentrations imply upregulated LDLR activity in HCV-G3.

Generation and epitope mapping of a sub-group cross-reactive anti-respiratory syncytial virus G glycoprotein monoclonal antibody which is protective in vivo.

Passively administered antibodies to conserved epitopes on the attachment (G) glycoprotein of human respiratory syncytial virus (hRSV) have potential in the immunoprophylaxis of human infections. This study set out to generate monoclonal antibodies (MAbs) recognizing all prevalent lineages of HRSV and capable of immunoprophylaxis in mice. Two murine MAbs of broad specificity for prevalent virus strains were generated by immunization of mice with hRSV of sub-group A followed by selection of hybridomas on recombinant G glycoprotein from a sub-group B virus. The anti-G hybridomas generated secreted antibody of high affinity but negligible neutralizing capacity one of which was tested in mice and found to be protective against live virus challenge. Western blotting and partial epitope mapping on transiently expressed G-glycoprotein fragments indicate that these antibodies recognize a complex epitope on the protein backbone of the molecule involving residues both C'- and N-terminal to the central conserved motif.

Imaging analysis of human metapneumovirus-infected cells provides evidence for the involvement of F-actin and the raft-lipid microdomains in virus morphogenesis.

BACKGOUND: Due to difficulties of culturing Human metapneumovirus (HMPV) much of the current understanding of HMPV replication can be inferred from other closely related viruses. The slow rates of virus replication prevent many biochemical analyses of HMPV particles. In this study imaging was used to examine the process of HMPV morphogenesis in individually infected LLC-MK2 cells, and to better characterise the sites of HMPV assembly. This strategy has circumvented the problems associated with slow replication rates and allowed us to characterise both the HMPV particles and the sites of HMPV morphogenesis. METHODS: HMPV-infected LLC-MK2 cells were stained with antibodies that recognised the HMPV fusion protein (F protein), attachment protein (G protein) and matrix protein (M protein), and fluorescent probes that detect GM1 within lipid-raft membranes (CTX-B-AF488) and F-actin (Phalloidin-FITC). The stained cells were examined by confocal microscopy, which allowed imaging of F-actin, GM1 and virus particles in HMPV-infected cells. Cells co-expressing recombinant HMPV G and F proteins formed virus-like particles and were co-stained with antibodies that recognise the recombinant G and F proteins and phalloidin-FITC and CTX-B-AF594, and the distribution of the G and F proteins, GM1 and F-actin determined. RESULTS: HMPV-infected cells stained with anti-F, anti-G or anti-M revealed a filamentous staining pattern, indicating that the HMPV particles have a filamentous morphology. Staining of HMPV-infected cells with anti-G and either phalloidin-FITC or CTX-B-AF488 exhibited extensive co-localisation of these cellular probes within the HMPV filaments. This suggested that lipid-raft membrane domains and F-actin structures are present at the site of the virus morphogenesis, and are subsequently incorporated into the HMPV filaments. Furthermore, the filamentous virus-like particles that form in cells expressing the G protein formed in cellular structures containing GM1 and F-actin, suggesting the G protein contains intrinsic targeting signals to the sites of virus assembly. CONCLUSIONS: These data suggest that HMPV matures as filamentous particles and that virus morphogenesis occurs within lipid-raft microdomains containing localized concentrations of F-actin. The similarity between HMPV morphogenesis and the closely related human respiratory syncytial virus suggests that involvement of F-actin and lipid-raft microdomains in virus morphogenesis may be a common feature of the Pneumovirinae.

Apolipoprotein-E and hepatitis C lipoviral particles in genotype 1 infection: evidence for an association with interferon sensitivity.

BACKGROUND & AIMS: Hepatitis C virus (HCV) interacts with apolipoproteins B (apoB) and E (apoE) to form infectious lipoviral particles (LVP). Response to peginterferon is influenced by interferon-stimulated genes (ISGs) and IL28B genotype. LDL cholesterol (LDL-C) also predicts interferon response, therefore we hypothesised that LVP may also be associated with interferon sensitivity. METHODS: LVP (HCV RNA density ≤1.07 g/ml) and 'non-LVP' (d >1.07 g/ml) were measured in 72 fasted HCV-G1 patients by iodixanol density gradient ultracentrifugation and the LVP ratio (LVP/LVP+non-LVP) was calculated. Fasting lipid profiles and apolipoproteins B and E were measured. Interferon-gamma-inducible protein 10 kDa (IP10), a marker of ISGs, was measured by ELISA. RESULTS: Complete early virological response (EVR) was associated with lower apoE (23.9±7.7 vs. 36.1±15.3 mg/L, p=0.013), higher LDL-C (p=0.039) and lower LVP ratios (p=0.022) compared to null responders. In multivariate linear regression analysis, apoE was independently associated with LVP (R(2) 19.5%, p=0.003) and LVP ratio (p=0.042), and negatively with LDL-C (p<0.001). IP10 was significantly associated with ApoB (p=0.001) and liver stiffness (p=0.032). IL28B rs12979860 CC was associated with complete EVR (p=0.044), low apoE (CC 28±11 vs. CT/TT 35±13 mg/L, p=0.048) and higher non-LVP (p=0.008). Logistic regression analysis indicated that patients with high LVP ratios were less likely to have EVR (odds ratio 0.01, p=0.018). CONCLUSIONS: In HCV-G1, interferon sensitivity is characterised by low LVP ratios and low apoE levels in addition to higher LDL-C and IL28B rs12979860 CC. Null-response is associated with increased LVP ratio. The association of apoE and LVP with peginterferon treatment response suggests that lipid modulation is a potential target to modify interferon sensitivity.

The characterization of monoclonal antibodies to human metapneumovirus and the detection of multiple forms of the virus nucleoprotein and phosphoprotein.

Little is known of the proteome of human metapneumovirus (HMPV). In this study a panel of monoclonal antibodies to the virus have been characterized and used to identify viral proteins present in infected cell lysates. Of thirteen anti-HMPV monoclonal antibodies four reacted with recombinant fusion glycoprotein and one with recombinant G glycoprotein by immunofuorescence but not in western blots suggesting that they recognize conformation dependent epitopes. The specificity of the remaining antibodies were determined by MALDI/TOF analysis of the proteins they immunoprecipitated from HMPV infected cell lysates and by western blotting. Five MAbs bound to the nucleoprotein and three to the phosphoprotein. In western blots of lysates of cells infected with low passage HMPV, the anti-nucleoprotein MAbs stained a single polypeptide corresponding in size to the full length nucleocapsid protein. On repeated passage of the virus in cell culture, however, a second, smaller band appeared which may result from internal initiation of translation within the nucleocapsid gene as described for avian metapneumovirus. Antibodies to the phosphoprotein, besides the full length form, also recognized multiple polypeptides in infected cell lysates, with patterns differing for the two subtypes A and B. The possibility that these too may derive by internal initiation of translation is discussed.

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 <1.07 g/ml and >1.07 g/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.

HCV and the hepatic lipid pathway as a potential treatment target.

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.

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

BACKGROUND & AIMS: The 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. METHODS: Fasting 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. RESULTS: The 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). CONCLUSIONS: Dietary 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.

Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity.

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.

Molecular epidemiology and evolution of human respiratory syncytial virus and human metapneumovirus.

Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are ubiquitous respiratory pathogens of the Pneumovirinae subfamily of the Paramyxoviridae. Two major surface antigens are expressed by both viruses; the highly conserved fusion (F) protein, and the extremely diverse attachment (G) glycoprotein. Both viruses comprise two genetic groups, A and B. Circulation frequencies of the two genetic groups fluctuate for both viruses, giving rise to frequently observed switching of the predominantly circulating group. Nucleotide sequence data for the F and G gene regions of HRSV and HMPV variants from the UK, The Netherlands, Bangkok and data available from Genbank were used to identify clades of both viruses. Several contemporary circulating clades of HRSV and HMPV were identified by phylogenetic reconstructions. The molecular epidemiology and evolutionary dynamics of clades were modelled in parallel. Times of origin were determined and positively selected sites were identified. Sustained circulation of contemporary clades of both viruses for decades and their global dissemination demonstrated that switching of the predominant genetic group did not arise through the emergence of novel lineages each respiratory season, but through the fluctuating circulation frequencies of pre-existing lineages which undergo proliferative and eclipse phases. An abundance of sites were identified as positively selected within the G protein but not the F protein of both viruses. For HRSV, these were discordant with previously identified residues under selection, suggesting the virus can evade immune responses by generating diversity at multiple sites within linear epitopes. For both viruses, different sites were identified as positively selected between genetic groups.

Molecular epidemiology of human metapneumovirus in Ireland.

Human metapneumovirus (hMPV) is a cause of respiratory illness ranging from wheezing to bronchiolitis and pneumonia in children. A quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was developed for the detection of all four main genetic lineages of hMPV and employed to validate an indirect immunofluorescence (IF) assay to detect hMPV positive specimens. The IF assay detected 24 positives from a screen of 625 randomly selected pediatric respiratory specimens collected (3.8% prevalence). From this cohort of 625 specimens, 229 were also tested by real-time RT-PCR assay. This included the 24 IF positive specimens and 205 randomly selected specimens from both study periods. In addition to confirming all the IF positives, the real-time assay detected an additional six hMPV positive specimens giving rise to a combined prevalence of 4.8%. Phylogenetic analysis showed that hMPV subtypes A2b and B2 to be the most prevalent genotypes circulating in our population and surprisingly no hMPV subgroups A1 or B1 were detected during this study period. Based on this phylogenetic analysis, we propose the existence of sub-clusters of hMPV genotype B2 present in our population which we term subtypes B2a and B2b. The mean log 10 copies/ml of quantitative RT-PCR determinations from these 30 hMPV positive respiratory specimens was 6.35 (range = 4.44-8.15). Statistical analysis of quantitative RT-PCR determinations of viral load from these 30 respiratory specimens suggests that hMPV genotype B specimens have a higher viral load than hMPV genotype A isolates (P < 0.03).

Binding of liver derived, low density hepatitis C virus to human hepatoma cells.

HCV recovered from low density fractions of infected blood is associated with lipid and host apo-lipoproteins in lipo-viro-particles (LVP). It has been proposed that these particles are capable of binding and entering hepatocytes by viral glycoprotein independent mechanisms utilizing uptake pathways of normal host lipoproteins after binding to cell surface glycosaminoglycans (GAG), the low density lipoprotein receptor (LDL-r) or scavenger receptor B1 (SR-B1). In this study binding to human hepatoma cells of HCV low density RNA containing particles, semi-purified from macerates of infected human liver, is compared with that of normal host low density lipoprotein (LDL). Binding of both LDL and HCV low density RNA containing particles paralleled LDL-r but not SR-B1 expression on the recipient cells. Binding of both particle types was sensitive to suramin at 0 degrees C but less so at 37 degrees C suggesting that they both bind initially to GAG but, at 37 degrees C, are internalized or transferred to a suramin resistant receptor. Suramin resistant uptake of both particles was blocked in the presence of excess LDL or oxidized LDL. However, whilst LDL uptake was blocked by anti-apoB-100, HCV low density RNA uptake was enhanced by anti-apoB100 and further enhanced by a cocktail of anti-apo-B100 and anti-apoE. Pre-incubation of HCV low density RNA containing particles with antibodies to the E2 glycoprotein had little or no effect on uptake. These data indicate that whilst liver derived HCV RNA containing particles are taken up by HepG2 cells by a virus glycoprotein independent mechanism, the mechanism differs from that of LDL uptake.

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 1.08 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.

Association between hepatitis C virus and very-low-density lipoprotein (VLDL)/LDL analyzed in iodixanol density gradients.

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, < or =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.

Characterization of the genome and structural proteins of hepatitis C virus resolved from infected human liver.

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, 5 x 10(9) International Units (IU) per gram of liver. The density distribution of HCV in iodixanol gradients showed a peak at 1.04 g x ml(-1) with 73 % of virus below 1.08 g x 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.