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author:("balf, Peter")
1.  Combined Adenovirus Vector and Hepatitis C Virus Envelope Protein Prime-Boost Regimen Elicits T Cell and Neutralizing Antibody Immune Responses 
Journal of Virology  2014;88(10):5502-5510.
ABSTRACT
Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8+ and CD4+ T cell responses and functional Th1-type IgG responses. Immune sera neutralized luciferase reporter pseudoparticles expressing HCV envelope glycoproteins (HCVpp) and a diverse panel of recombinant cell culture-derived HCV (HCVcc) strains and limited cell-to-cell HCV transmission. This study demonstrated that combining adenovirus vector with protein antigen can induce strong antibody and T cell responses that surpass immune responses achieved by either vaccine alone.
IMPORTANCE HCV infection is a major health problem. Despite the availability of new directly acting antiviral agents for treating chronic infection, an affordable preventive vaccine provides the best long-term goal for controlling the global epidemic. This report describes a new anti-HCV vaccine targeting the envelope viral proteins based on adenovirus vector and protein in adjuvant. Rodents primed with the adenovirus vaccine and boosted with the adjuvanted protein developed cross-neutralizing antibodies and potent T cell responses that surpassed immune responses achieved with either vaccine component alone. If combined with the adenovirus vaccine targeting the HCV NS antigens now under clinical testing, this new vaccine might lead to a stronger and broader immune response and to a more effective vaccine to prevent HCV infection. Importantly, the described approach represents a valuable strategy for other infectious diseases in which both T and B cell responses are essential for protection.
doi:10.1128/JVI.03574-13
PMCID: PMC4019094  PMID: 24599994
2.  Hepatitis C Virus Infects the Endothelial Cells of the Blood-Brain Barrier 
Gastroenterology  2011;142(3):634-643.e6.
BACKGROUND & AIMS
Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS.
METHODS
We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture–derived HCV were used to study the ability of endothelial cells to support viral entry and replication.
RESULTS
Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hC-MEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis.
CONCLUSIONS
Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.
doi:10.1053/j.gastro.2011.11.028
PMCID: PMC3801216  PMID: 22138189
Virus Tropism; HCVpp; HCVcc; Neurologic Defect
3.  Hepatitis C Virus Induces CD81 and Claudin-1 Endocytosis 
Journal of Virology  2012;86(8):4305-4316.
Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.
doi:10.1128/JVI.06996-11
PMCID: PMC3318669  PMID: 22318146
4.  A dual role for hypoxia inducible factor-1α in the hepatitis C virus lifecycle and hepatoma migration 
Journal of Hepatology  2012;56(4):803-809.
Background & Aims
Hepatitis C virus (HCV) causes progressive liver disease and is a major risk factor for the development of hepatocellular carcinoma (HCC). However, the role of infection in HCC pathogenesis is poorly understood. We investigated the effect(s) of HCV infection and viral glycoprotein expression on hepatoma biology to gain insights into the development of HCV associated HCC.
Methods
We assessed the effect(s) of HCV and viral glycoprotein expression on hepatoma polarity, migration and invasion.
Results
HCV glycoproteins perturb tight and adherens junction protein expression, and increase hepatoma migration and expression of epithelial to mesenchymal transition markers Snail and Twist via stabilizing hypoxia inducible factor-1α (HIF-1α). HIF-1α regulates many genes involved in tumor growth and metastasis, including vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β). Neutralization of both growth factors shows different roles for VEGF and TGFβ in regulating hepatoma polarity and migration, respectively. Importantly, we confirmed these observations in virus infected hepatoma and primary human hepatocytes. Inhibition of HIF-1α reversed the effect(s) of infection and glycoprotein expression on hepatoma permeability and migration and significantly reduced HCV replication, demonstrating a dual role for HIF-1α in the cellular processes that are deregulated in many human cancers and in the viral life cycle.
Conclusions
These data provide new insights into the cancer-promoting effects of HCV infection on HCC migration and offer new approaches for treatment.
doi:10.1016/j.jhep.2011.11.018
PMCID: PMC3343261  PMID: 22178269
BC, bile canaliculi; CMFDA, 5-chloromethylfluorescein diacetate; HCC, hepatocellular carcinoma; EMT, epithelial to mesenchymal transition; HCVcc, hepatitis C virus cell culture; HIF-1α, hypoxia inducible factor 1 alpha; JFH-1, Japanese fulminant hepatitis-1; MRP-2, multidrug resistant protein-2; PHH, primary human hepatocytes; SR-BI, scavenger receptor class B member 1; TGFβ, transforming growth factor-beta; TNFα, tumor necrosis factor alpha; VEGF, vascular endothelial growth factor; VSV-G, vesicular stomatitis virus glycoprotein; Hepatitis C; Hypoxia; Invasion
5.  Hepatitis C virus infection of neuroepithelioma cell lines 
Gastroenterology  2010;139(4):1365-1374.
Background & Aims
Hepatitis C virus (HCV) establishes chronic infections in 3% of the world's population. Infection leads to progressive liver disease; hepatocytes are the major site of viral replication in vivo. However, chronic infection is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. We therefore screened a series of neural and brain-derived cell lines for their ability to support HCV entry and replication.
Methods
We used a panel of neural-derived cell lines, HCV pseudoparticles (HCVpp), and an infectious, HCV JFH-1 cell-culture system (HCVcc) to assess viral tropism.
Results
Two independently derived neuroepithelioma cell lines (SK-N-MC and SK-PN-DW) permitted HCVpp entry. In contrast, several neuroblastoma, glioma, and astrocytoma cell lines were refractory to HCVpp infection. HCVcc infected the neuroepithelioma cell lines and established a productive infection. Permissive neuroepithelioma cells expressed CD81, scavenger receptor BI (SR-BI), and the tight junction proteins Claudin-1 (CLDN1) and occludin, whereas non-permissive neural cell lines lacked CLDN1 and in some cases SR-BI. HCVpp infection of the neuroepithelioma cells was neutralized by antibodies to CD81, SR-BI, CLDN1 and HCV E2. Furthermore, anti-CD81, interferon and the anti-NS3 protease inhibitor VX-950 significantly reduced HCVcc infection of neuroepithelioma and hepatoma cells.
Conclusions
Neuroepithelioma-derived cell lines express functional receptors that support HCV entry at comparable levels to that of hepatoma cells. HCV infection in vitro is not restricted to hepatic-derived cells, so HCV might infect cells of the CNS in vivo.
doi:10.1053/j.gastro.2010.06.008
PMCID: PMC3298458  PMID: 20538002
OCLN; neurotropism; brain; therapy; replicon; Huh-7; VX-950
6.  Inferring viral quasispecies spectra from 454 pyrosequencing reads 
BMC Bioinformatics  2011;12(Suppl 6):S1.
Background
RNA viruses infecting a host usually exist as a set of closely related sequences, referred to as quasispecies. The genomic diversity of viral quasispecies is a subject of great interest, particularly for chronic infections, since it can lead to resistance to existing therapies. High-throughput sequencing is a promising approach to characterizing viral diversity, but unfortunately standard assembly software was originally designed for single genome assembly and cannot be used to simultaneously assemble and estimate the abundance of multiple closely related quasispecies sequences.
Results
In this paper, we introduce a new Viral Spectrum Assembler (ViSpA) method for quasispecies spectrum reconstruction and compare it with the state-of-the-art ShoRAH tool on both simulated and real 454 pyrosequencing shotgun reads from HCV and HIV quasispecies. Experimental results show that ViSpA outperforms ShoRAH on simulated error-free reads, correctly assembling 10 out of 10 quasispecies and 29 sequences out of 40 quasispecies. While ShoRAH has a significant advantage over ViSpA on reads simulated with sequencing errors due to its advanced error correction algorithm, ViSpA is better at assembling the simulated reads after they have been corrected by ShoRAH. ViSpA also outperforms ShoRAH on real 454 reads. Indeed, 7 most frequent sequences reconstructed by ViSpA from a real HCV dataset are viable (do not contain internal stop codons), and the most frequent sequence was within 1% of the actual open reading frame obtained by cloning and Sanger sequencing. In contrast, only one of the sequences reconstructed by ShoRAH is viable. On a real HIV dataset, ShoRAH correctly inferred only 2 quasispecies sequences with at most 4 mismatches whereas ViSpA correctly reconstructed 5 quasispecies with at most 2 mismatches, and 2 out of 5 sequences were inferred without any mismatches. ViSpA source code is available at http://alla.cs.gsu.edu/~software/VISPA/vispa.html.
Conclusions
ViSpA enables accurate viral quasispecies spectrum reconstruction from 454 pyrosequencing reads. We are currently exploring extensions applicable to the analysis of high-throughput sequencing data from bacterial metagenomic samples and ecological samples of eukaryote populations.
doi:10.1186/1471-2105-12-S6-S1
PMCID: PMC3194189  PMID: 21989211
7.  Neutralizing Antibody-Resistant Hepatitis C Virus Cell-to-Cell Transmission ▿ † ‡  
Journal of Virology  2010;85(1):596-605.
Hepatitis C virus (HCV) can initiate infection by cell-free particle and cell-cell contact-dependent transmission. In this study we use a novel infectious coculture system to examine these alternative modes of infection. Cell-to-cell transmission is relatively resistant to anti-HCV glycoprotein monoclonal antibodies and polyclonal immunoglobulin isolated from infected individuals, providing an effective strategy for escaping host humoral immune responses. Chimeric viruses expressing the structural proteins representing the seven major HCV genotypes demonstrate neutralizing antibody-resistant cell-to-cell transmission. HCV entry is a multistep process involving numerous receptors. In this study we demonstrate that, in contrast to earlier reports, CD81 and the tight-junction components claudin-1 and occludin are all essential for both cell-free and cell-to-cell viral transmission. However, scavenger receptor BI (SR-BI) has a more prominent role in cell-to-cell transmission of the virus, with SR-BI-specific antibodies and small-molecule inhibitors showing preferential inhibition of this infection route. These observations highlight the importance of targeting host cell receptors, in particular SR-BI, to control viral infection and spread in the liver.
doi:10.1128/JVI.01592-10
PMCID: PMC3014195  PMID: 20962076
8.  Hepatoma Cell Density Promotes Claudin-1 and Scavenger Receptor BI Expression and Hepatitis C Virus Internalization▿  
Journal of Virology  2009;83(23):12407-12414.
Hepatitis C virus (HCV) entry occurs via a pH- and clathrin-dependent endocytic pathway and requires a number of cellular factors, including CD81, the tight-junction proteins claudin 1 (CLDN1) and occludin, and scavenger receptor class B member I (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here, we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCV pseudoparticle (HCVpp) and HCV particle (HCVcc) infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in subconfluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.
doi:10.1128/JVI.01552-09
PMCID: PMC2786758  PMID: 19776133
9.  Claudin Association with CD81 Defines Hepatitis C Virus Entry 
The Journal of Biological Chemistry  2010;285(27):21092-21102.
Viruses initiate infection by attaching to molecules or receptors at the cell surface. Hepatitis C virus (HCV) enters cells via a multistep process involving tetraspanin CD81, scavenger receptor class B member I, and the tight junction proteins Claudin-1 and Occludin. CD81 and scavenger receptor class B member I interact with HCV-encoded glycoproteins, suggesting an initial role in mediating virus attachment. In contrast, there are minimal data supporting Claudin-1 association with HCV particles, raising questions as to its role in the virus internalization process. In the present study we demonstrate a relationship between receptor active Claudins and their association and organization with CD81 at the plasma membrane by fluorescence resonance energy transfer and stoichiometric imaging methodologies. Mutation of residues 32 and 48 in the Claudin-1 first extracellular loop ablates CD81 association and HCV receptor activity. Furthermore, mutation of the same residues in the receptor-inactive Claudin-7 molecule enabled CD81 complex formation and virus entry, demonstrating an essential role for Claudin-CD81 complexes in HCV infection. Importantly, Claudin-1 associated with CD81 at the basolateral membrane of polarized HepG2 cells, whereas tight junction-associated pools of Claudin-1 demonstrated a minimal association with CD81. In summary, we demonstrate an essential role for Claudin-CD81 complexes in HCV infection and their localization at the basolateral surface of polarized hepatoma cells, consistent with virus entry into the liver via the sinusoidal blood and association with basal expressed forms of the receptors.
doi:10.1074/jbc.M110.104836
PMCID: PMC2898367  PMID: 20375010
Fluorescence Resonance Energy Transfer (FRET); Receptor Structure-Function; Receptors; Tight Junction; Virus Entry
10.  Decay of K103N mutants in cellular DNA and plasma RNA after single-dose nevirapine to reduce mother-to-child HIV transmission 
AIDS (London, England)  2006;20(7):995-1002.
Objectives
Single-dose nevirapine (sd-NVP) for prevention of mother-to-child HIV-1 transmission is associated with selection of resistant viral variants, particularly the Lysine (K) to Asparagine (N) mutation at codon 103 (K103N) of reverse transcriptase. As this may influence subsequent treatment responses, a better understanding of the dynamics of decay and persistence of this variant is needed.
Design and methods
We measured the frequency of K103N mutants among a cohort of HIV-1-infected pregnant women recruited at an out-patient clinic in Johannesburg, South Africa. Samples taken 6 weeks, 3, 7 and 12 months after delivery from 67 HIV-1-infected women who received sd-NVP during labor to prevent transmission were analyzed. Quantification of K103N mutants in maternal plasma viral RNA and cellular DNA was done using an allele-specific real-time polymerase chain reaction assay capable of detecting codons AAC and AAT if their frequency was > 0.002 of the total viral population.
Results
Using the allele-specific assay, 87.1% (27/31) of RNA samples and 52.3% (23/44) of DNA samples collected 6 weeks after sd-NVP had detectable K103N variants. This declined to 65.4% (17/26), 38.9% (14/36), and 11.3% (6/53) in RNA at 3, 7 and 12 months respectively, and to 4.2% (2/48) in DNA at 12 months.
Conclusions
K103N resistant variants were present in almost all women at 6 weeks post-sd-NVP but declined rapidly over time. Resistant variants were detected less frequently in cellular DNA with persistence in this compartment by 12 months post-sd-NVP among only a minority.
doi:10.1097/01.aids.0000222071.60620.1d
PMCID: PMC2841686  PMID: 16603851
drug resistance; nevirapine; mother-to-child HIV transmission (MTCT)
11.  Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells▿  
Journal of Virology  2009;83(12):6211-6221.
The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.
doi:10.1128/JVI.00246-09
PMCID: PMC2687400  PMID: 19357163
12.  Mutations in Hepatitis C Virus E2 Located outside the CD81 Binding Sites Lead to Escape from Broadly Neutralizing Antibodies but Compromise Virus Infectivity▿  
Journal of Virology  2009;83(12):6149-6160.
Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.
doi:10.1128/JVI.00248-09
PMCID: PMC2687388  PMID: 19321602
13.  Identification of a Residue in Hepatitis C Virus E2 Glycoprotein That Determines Scavenger Receptor BI and CD81 Receptor Dependency and Sensitivity to Neutralizing Antibodies ▿  
Journal of Virology  2008;82(24):12020-12029.
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.
doi:10.1128/JVI.01569-08
PMCID: PMC2593310  PMID: 18829747
14.  Protein Kinase A-Dependent Step(s) in Hepatitis C Virus Entry and Infectivity▿  
Journal of Virology  2008;82(17):8797-8811.
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.
doi:10.1128/JVI.00592-08
PMCID: PMC2519651  PMID: 18579596
15.  CD81 and Claudin 1 Coreceptor Association: Role in Hepatitis C Virus Entry▿ †  
Journal of Virology  2008;82(10):5007-5020.
Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.
doi:10.1128/JVI.02286-07
PMCID: PMC2346731  PMID: 18337570
16.  Effect of Cell Polarization on Hepatitis C Virus Entry▿  
Journal of Virology  2007;82(1):461-470.
The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.
doi:10.1128/JVI.01894-07
PMCID: PMC2224355  PMID: 17959672
17.  Scavenger Receptor BI and BII Expression Levels Modulate Hepatitis C Virus Infectivity▿  
Journal of Virology  2007;81(7):3162-3169.
Hepatitis C virus (HCV) enters cells via a pH- and clathrin-dependent endocytic pathway. Scavenger receptor BI (SR-BI) and CD81 are important entry factors for HCV internalization into target cells. The SR-BI gene gives rise to at least two mRNA splice variants, SR-BI and SR-BII, which differ in their C termini. SR-BI internalization remains poorly understood, but SR-BII is reported to endocytose via a clathrin-dependent pathway, making it an attractive target for HCV internalization. We demonstrate that HCV soluble E2 can interact with human SR-BI and SR-BII. Increased expression of SR-BI and SR-BII in the Huh-7.5 hepatoma cell line enhanced HCV strain J6/JFH and JFH infectivity, suggesting that endogenous levels of these receptors limit infection. Elevated expression of SR-BI, but not SR-BII, increased the rate of J6/JFH infection, which may reflect altered intracellular trafficking of the splice variants. In human plasma, HCV particles have been reported to be complexed with lipoproteins, suggesting an indirect interaction of the virus with SR-BI and other lipoprotein receptors. Plasma from J6/JFH-infected uPA-SCID mice transplanted with human hepatocytes demonstrates an increased infectivity for SR-BI/II-overexpressing Huh-7.5 cells. Plasma-derived J6/JFH infectivity was inhibited by an anti-E2 monoclonal antibody, suggesting that plasma virus interaction with SR-BI was glycoprotein dependent. Finally, anti-SR-BI antibodies inhibited the infectivity of cell culture- and plasma-derived J6/JFH, suggesting a critical role for SR-BI/II in HCV infection.
doi:10.1128/JVI.02356-06
PMCID: PMC1866051  PMID: 17215280
18.  Diverse CD81 Proteins Support Hepatitis C Virus Infection▿  
Journal of Virology  2006;80(22):11331-11342.
Hepatitis C virus (HCV) entry is dependent on CD81. To investigate whether the CD81 sequence is a determinant of HCV host range, we expressed a panel of diverse CD81 proteins and tested their ability to interact with HCV. CD81 large extracellular loop (LEL) sequences were expressed as recombinant proteins; the human and, to a low level, the African green monkey sequences bound soluble HCV E2 (sE2) and inhibited infection by retrovirus pseudotype particles bearing HCV glycoproteins (HCVpp). In contrast, mouse or rat CD81 proteins failed to bind sE2 or to inhibit HCVpp infection. However, CD81 proteins from all species, when expressed in HepG2 cells, conferred susceptibility to infection by HCVpp and cell culture-grown HCV to various levels, with the rat sequence being the least efficient. Recombinant human CD81 LEL inhibited HCVpp infectivity only if present during the virus-cell incubation, consistent with a role for CD81 after virus attachment. Amino acid changes that abrogate sE2 binding (I182F, N184Y, and F186S, alone or in combination) were introduced into human CD81. All three amino acid changes in human CD81 resulted in a molecule that still supported HCVpp infection, albeit with reduced efficiency. In summary, there is a remarkable plasticity in the range of CD81 sequences that can support HCV entry, suggesting that CD81 polymorphism may contribute to, but alone does not define, the HCV susceptibility of a species. In addition, the capacity to support viral entry is only partially reflected by assays measuring sE2 interaction with recombinant or full-length CD81 proteins.
doi:10.1128/JVI.00104-06
PMCID: PMC1642177  PMID: 16943299
19.  Blinded, Multicenter Comparison of Methods To Detect a Drug-Resistant Mutant of Human Immunodeficiency Virus Type 1 at Low Frequency 
Journal of Clinical Microbiology  2006;44(7):2612-2614.
We determined the abilities of 10 technologies to detect and quantify a common drug-resistant mutant of human immunodeficiency virus type 1 (lysine to asparagine at codon 103 of the reverse transcriptase) using a blinded test panel containing mutant-wild-type mixtures ranging from 0.01% to 100% mutant. Two technologies, allele-specific reverse transcriptase PCR and a Ty1HRT yeast system, could quantify the mutant down to 0.1 to 0.4%. These technologies should help define the impact of low-frequency drug-resistant mutants on response to antiretroviral therapy.
doi:10.1128/JCM.00449-06
PMCID: PMC1489464  PMID: 16825395
20.  Molecular Characterization of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus in Paid Blood Donors and Injection Drug Users in China 
Journal of Virology  2004;78(24):13591-13599.
China is facing a rapid upsurge in cases of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection due to large numbers of paid blood donors (PBD), injection drug users (IDU), and sexual partners of infected individuals. In this report, a total of 236 HIV-1-positive blood samples were collected from PBD, IDU, and their sexual partners in the most severely affected provinces, such as Henan, Yunnan, Guangxi, and Xinjiang. PCR was used to amplify the p17 region of gag and the C2-V3 region of env of HIV-1 and the 5′ noncoding region and a region of E1/E2 of HCV. Genetic characterization of viral sequences indicated that there are two major epidemics of HIV-1 and multiple HCV epidemics in China. The PBD and transfusion recipients in Henan harbored HIV-1 subtype B′, which is similar to the virus found in Thailand, and HCV genotypes 1b and 2a, whereas the IDU in Yunnan, Guangxi, and Xinjiang carried HIV-1 circulating recombinant forms 07 and 08, which resemble those in India, and HCV genotypes 1b, 3a, and 3b. Our findings show that the epidemics of HIV-1 and HCV infection in China are the consequences of multiple introductions. The distinct distribution patterns of both the HIV-1 and HCV genotypes in the different high-risk groups are tightly linked to the mode of transmission rather than geographic proximity. These findings provide information relevant to antiviral therapy and vaccine development in China and should assist public health workers in implementing measures to reduce the further dissemination of these viruses in the world's most populous nation.
doi:10.1128/JVI.78.24.13591-13599.2004
PMCID: PMC533913  PMID: 15564470
21.  Origin of Human Immunodeficiency Virus Type 1 Quasispecies Emerging after Antiretroviral Treatment Interruption in Patients with Therapeutic Failure 
Journal of Virology  2002;76(14):7000-7009.
The emergence of antiretroviral (ARV) drug-resistant human immunodeficiency virus type 1 (HIV-1) quasispecies is a major cause of treatment failure. These variants are usually replaced by drug-sensitive ones when the selective pressure of the drugs is removed, as the former have reduced fitness in a drug-free environment. This was the rationale for the design of structured ARV treatment interruption (STI) studies for the management of HIV-1 patients with treatment failure. We have studied the origin of drug-sensitive HIV-1 quasispecies emerging after STI in patients with treatment failure due to ARV drug resistance. Plasma and peripheral blood mononuclear cell samples were obtained the day of treatment interruption (day 0) and 30 and 60 days afterwards. HIV-1 pol and env were partially amplified, cloned, and sequenced. At day 60 drug-resistant variants were replaced by completely or partially sensitive quasispecies. Phylogenetic analyses of pol revealed that drug-sensitive variants emerging after STI were not related to their immediate temporal ancestors but formed a separate cluster, demonstrating that STI leads to the recrudescence and reemergence of a sequestrated viral population rather than leading to the back mutation of drug-resistant forms. No evidence for concomitant changes in viral tropism was seen, as deduced from env sequences. This study demonstrates the important role that the reemergence of quasispecies plays in HIV-1 population dynamics and points out the difficulties that may be found when recycling ARV therapies with patients with treatment failure.
doi:10.1128/JVI.76.14.7000-7009.2002
PMCID: PMC136319  PMID: 12072500
22.  Antigenic Variation within the CD4 Binding Site of Human Immunodeficiency Virus Type 1 gp120: Effects on Chemokine Receptor Utilization 
Journal of Virology  2001;75(12):5593-5603.
To assess the antigenicity of envelope glycoproteins derived from primary human immunodeficiency virus type 1 populations, their interactions with the receptor CD4, and their coreceptor usage, we have cloned and expressed multiple gp120 proteins from a number of primary virus isolates. Characterization of these proteins showed a high degree of antigenic polymorphism both within the CD4 binding site and in defined neutralization epitopes, which may partially account for the general resistance of primary isolates to neutralizing agents. Furthermore, chimeric viruses expressing gp120 proteins with reduced CD4 binding abilities are viable, suggesting that primary viruses may require a less avid interaction with the receptor CD4 to initiate infection than do their laboratory-adapted counterparts. The coreceptor usage of chimeric viruses was related to the ability of the virus to bind CD4, with reduced CD4 binding correlating with preferential usage of CXCR4. Changes in coreceptor usage mapped to sequence changes in the C2 and V4 regions, with no changes seen in the V3 region.
doi:10.1128/JVI.75.12.5593-5603.2001
PMCID: PMC114272  PMID: 11356967
23.  In silico directed mutagenesis identifies the CD81/claudin-1 hepatitis C virus receptor interface 
Cellular Microbiology  2012;14(12):1892-1903.
Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62–66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCVglycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.
doi:10.1111/cmi.12008
PMCID: PMC3549482  PMID: 22897233
24.  Paracrine Signals From Liver Sinusoidal Endothelium Regulate Hepatitis C Virus Replication 
Hepatology (Baltimore, Md.)  2013;59(2):375-384.
Hepatitis C virus (HCV) is a major cause of global morbidity, causing chronic liver injury that can progress to cirrhosis and hepatocellular carcinoma. The liver is a large and complex organ containing multiple cell types, including hepatocytes, sinusoidal endothelial cells (LSEC), Kupffer cells, and biliary epithelial cells. Hepatocytes are the major reservoir supporting HCV replication; however, the role of nonparenchymal cells in the viral lifecycle remains largely unexplored. LSEC secrete factors that promote HCV infection and transcript analysis identified bone morphogenetic protein 4 (BMP4) as a candidate endothelial-expressed proviral molecule. Recombinant BMP4 increased HCV replication and neutralization of BMP4 abrogated the proviral activity of LSEC-conditioned media. Importantly, BMP4 expression was negatively regulated by vascular endothelial growth factor A (VEGF-A) by way of a VEGF receptor-2 (VEGFR-2) primed activation of p38 MAPK. Consistent with our in vitro observations, we demonstrate that in normal liver VEGFR-2 is activated and BMP4 expression is suppressed. In contrast, in chronic liver disease including HCV infection where there is marked endothelial cell proliferation, we observed reduced endothelial cell VEGFR-2 activation and a concomitant increase in BMP4 expression. Conclusion: These studies identify a role for LSEC and BMP4 in HCV infection and highlight BMP4 as a new therapeutic target for treating individuals with liver disease.
doi:10.1002/hep.26571
PMCID: PMC3992845  PMID: 23775568

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