A <em>Gaussia</em> Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

<div><p>Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted <em>Gaussia</em> luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the <em>Gaussia</em> was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their <em>in vitro</em> infection and replication potential. Approximately 12% of the sera contained <em>in vitro</em> replication-competent viruses, as deduced by the <em>Gaussia</em> signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of <em>in vitro</em> replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.</p> </div

Productive replication of serum-derived HCV in Huh-7.5/EG(4A/4B)GLuc cells.

<p>(A&B) <i>Gaussia</i> activity and intracellular HCV RNA analysis in Huh-7.5/EG(4A/4B)GLuc cells inoculated with sera derived from patients 20 and 35 at the indicated time points. Results represent the mean values from duplicate wells, measured in duplicate (for both <i>Gaussia</i> and RNA), from a representative experiment of 3 (mean ± SD; n = 4) (C) Intracellular core expression in Huh-7.5/EG(4A/4B)GLuc cells inoculated with the indicated patient sera, JC1 virus or HCV negative Serum (HCV neg.) as negative control, as detected by core protein IF. Cells were stained with α-core specific antibodies (clone C7–50) and α-mouse Alexa-568 antibodies (red). Cell nuclei were counterstained with DAPI (blue), magnification 100x. (D) Core protein released in the supernatant from Huh-7.5/EG(4A/4B)GLuc cells infected by the indicated patient sera, JC1 virus or HCV neg. serum as negative control, 120 h post inoculation.</p

Overview of the screening strategy.

<p>(A) Eighty-six clinical sera were selected from patients as described in <i>Materials and Methods</i>. Patientś number rise relative to the viral load increase. Relative <i>Gaussia</i> expression scale: <i>Gaussia</i> activity by wells infected in parallel with the JC1 virus at an MOI of 0.5 TCID₅₀/cell was set at 100 while that by JFH-1/ΔGDD and/or HCV control negative serum (HCV neg.) was set at 0. (A) Results from 3 representative experiments. Means difference between the experiments were not significant (n.s.). Patients with <i>Gaussia</i> activity ≥40% are indicated. (B) Colours were drawn according to the mean of 3 independent experiments. In each experiment duplicate infections were performed and measured in duplicate (n = 4).</p

Schematic of dual-function reporter vectors used in the HCV NS3/4A protease activity assay.

<p>(A) The various recognition sites of NS3/4A protease, IPS_462–540, NLS-IPS_462–540, KDEL-DE_4x-4A/4B and DE_4x-4A/4B were inserted between the <i>EGFP</i> and the humanized <i>Gaussia luciferase</i> gene by an in-frame fusion. Arrows indicate the NS3/4A cleavage site. Expression of the reporter genes is under the human cytomegalovirus promoter (CMV) and the selection marker for the generation of the stable cell line is neomycin phosphotrasferase (Neo^R). SV40, simian virus 40. (B) Transiently trasfected Huh-7.5 cells were infected with JC1 virus at an MOI 0.5 TCID₅₀/cell or mock infected with JFH-1/ΔGDD supernatant. At 5 days post infection, the medium was harvested and <i>Gaussia</i> activity was measured. Results are expressed as the mean values from duplicate wells, measured in duplicates, from a representative experiment of 3 (mean ± SD; n = 4).*, <i>P</i><0.05.</p

List of patients with infectivity ≥ 40%.

a<p>Infectivity is expressed as a percentage (% ± SD) relevant to that obtained from cells infected in parallel with JC1 viruses.</p

Correlation between extracellular <i>Gaussia</i> activity and HCV RNA and proteins.

<p>Huh-7.5/EG(4A/4B)GLuc cells were infected with JC1 (A) or JFH-1 (B) viruses at an MOI of 0.5 TCID₅₀/cell. Extracellular <i>Gaussia</i> activity and intracellular RNA were analyzed at the given time points post infection. (C) Correlation between NS3 and core proteins levels with EG(4A/4B)GLuc cleavage efficiency. Huh-7.5/EG(4A/4B)GLuc cells were infected like in (A) and at the given time points expression levels of NS3, core and EG(4A/4B)GLuc were analyzed by Western blotting by anti-NS3, anti-core and anti-GFP antibodies, respectively.</p

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

<div><p>Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H³⁸⁶ and R⁴⁰⁸), and the two highly conserved basic residues H⁴⁸⁸ and R⁶⁴⁸ contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.</p> </div

Buoyant density infectivity and RNA of WT and mutant viruses.

<p>The indicated viruses were resolved using an iodixanol step gradient. For each fraction HCV infectivity in Huh-7.5 cells and RNA (RT-qPCR) were determined. Values are plotted against the density of the respective fraction. The infectivity is expressed as a percentage of the total infectivity obtained from all fractions. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of two independent experiments.</p

Density fraction neutralization of WT viruses by patient IgG, anti-SR-BI, anti-CD81, anti-LDL and anti-HDL.

<p>WT viruses were resolved using an iodixanol step gradient. (A) Infections of individual fractions in the presence of patient IgG, anti-SR-BI (C167) or anti-CD81 (JS-81). (B) Similar infections as described in (A) were performed with pre-incubated viruses with anti-ApoE, anti-HDL or anti-LDL. All points represent the mean of duplicate infections measured in duplicate (n = 4, ± SD). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052651#s3" target="_blank">Results</a> are drawn from a representative experiment of two independent experiments.</p

HCV E2 glycoprotein contains conserved basic residues in different regions.

<p>(A) Scheme of E2 putative GAG-binding sites and other regions important for entry and for proper protein folding. Amino acid numbers refer to positions in the polyprotein sequence of the H77 prototype isolate. N: glycosylation sites, HVR1, 2, 3: hyper-variable region 1, 2, 3, TM: transmembrane domain. (B) Frequency of basic residues at the positions analyzed in this study. The height of the box in each bar indicates the frequency of histidine (H, white box), lysine (K, light grey box) and arginine (R, dark grey box). The frequency of the basic residues at each position was calculated by dividing the number of basic residues by the total number of sequences (2073 sequences) and is expressed as a percentage.</p