Determinants in the Ig Variable Domain of Human HAVCR1 (TIM-1) Are Required To Enhance Hepatitis C Virus Entry

Abstract

Hepatitis C virus (HCV) is the leading cause of chronic hepatitis in humans. Several host molecules participate in HCV cell entry, but this process remains unclear. The complete unraveling of the HCV entry process is important to further understand viral pathogenesis and develop therapeutics. Human hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, functions as a phospholipid receptor involved in cell entry of several enveloped viruses. Here, we studied the role of HAVCR1 in HCV infection. HAVCR1 antibody inhibited entry in a dose-dependent manner. HAVCR1 soluble constructs neutralized HCV, which did not require the HAVCR1 mucinlike region and was abrogated by a mutation of N to A at position 94 (N94A) in the Ig variable (IgV) domain phospholipid-binding pocket, indicating a direct interaction of the HAVCR1 IgV domain with HCV virions. However, knockout of HAVCR1 in Huh7 cells reduced but did not prevent HCV growth. Interestingly, the mouse HAVCR1 ortholog, also a phospholipid receptor, did not enhance infection and a soluble form failed to neutralize HCV, although replacement of the mouse IgV domain with the human HAVCR1 IgV domain restored the enhancement of HCV infection. Mutations in the cytoplasmic tail revealed that direct HAVCR1 signaling is not required to enhance HCV infection. Our data show that the phospholipid-binding function and other determinant(s) in the IgV domain of human HAVCR1 enhance HCV infection. Although the exact mechanism is not known, it is possible that HAVCR1 facilitates entry by stabilizing or enhancing attachment, leading to direct interactions with specific receptors, such as CD81.IMPORTANCE Hepatitis C virus (HCV) enters cells through a multifaceted process. We identified the human hepatitis A virus cellular receptor 1 (HAVCR1), CD365, also known as TIM-1, as a facilitator of HCV entry. Antibody blocking and silencing or knockout of HAVCR1 in hepatoma cells reduced HCV entry. Our findings that the interaction of HAVCR1 with HCV early during infection enhances entry but is not required for infection support the hypothesis that HAVCR1 facilitates entry by stabilizing or enhancing virus binding to the cell surface membrane and allowing the correct virus-receptor positioning for interaction with the main HCV receptors. Furthermore, our data show that in addition to the phospholipid-binding function of HAVCR1, the enhancement of HCV infection involves other determinants in the IgV domain of HAVCR1. These findings expand the repertoire of molecules that HCV uses for cell entry, adding to the already complex mechanism of HCV infection and pathogenesis.

Keywords: hepatitis C virus; viral entry; viral receptors.

FIG 1

Inhibition of HCVcc entry into Huh7.5 cells by anti-HAVCR1 antibody. Huh7.5 cells were treated with decreasing concentrations of the following antibodies following 2-fold dilutions from 4 μg/ml to 0.0625 μg/ml: anti-HAVCR1 MAb (1D12) (A), anti-CD81 MAb (JS-81) (B), and isotype control (C). Cells were treated with antibodies 1 h prior to infection with J6/JFH1. After 72 h, HCVcc infection was evaluated by automatic counting of stained foci and the percentage of inhibition calculated by comparing focus counts in treated and mock-treated cells. (D) Huh7.5 cells were treated with increasing concentrations of anti-CD81 and anti-HAVCR1 MAbs individually or in combination. The percentage of inhibition and dose effect plot was created using CalcuSyn 2.0 software. Testing for all assays was performed in duplicate. Error bars represent standard errors of the means. Data are representative of three independent experiments.

FIG 2

Comparison of inhibitory activities and cell membrane localization of CD81 and HAVCR1. JFH1-Nanoluc was added to cells at 4°C for 1 h before moving the cells to 37°C to allow entry and infection to proceed. Antibody against CD81 (JS-81) (A), HAVCR1 (1D12) (B), or mouse isotype control (C) was added hourly to cells before and after HCVcc binding. The amount of antibody used was the concentration that was previously found to result in 50% inhibition of infection or the highest equivalent concentration in the case of the isotype control. The levels of luciferase expression at different time points were measured, and the percentages of inhibition were calculated by comparing the signals in mock-treated and infected cells at each time point. Error bars represent standard errors of the means. (D) Confocal analysis of Huh7.5 cells surface stained with 4′,6-diamidino-2-phenylindole (DAPI), anti-CD81 MAb (JS-81), and anti-HAVCR1 MAb (HAVCR1-1 MAb).

FIG 3

Dose-dependent inhibition of HCVcc entry with soluble HAVCR1 receptors, phosphatidyl serine, or annexin V. HCVcc was incubated 1 h prior to infection of Huh7.5 cells with increasing amounts of soluble HAVCR1 receptor containing the IgV- and mucinlike domains (HAVCR1-Fc) (A), soluble monkey HAVCR1 (mkHAVCR1-Fc) or mouse Havcr1 ortholog (mHavcr1-Fc) (B), HAVCR1-Fc or soluble HAVCR1 containing the IgV-like domain [HAVCR1(IgV)-Fc] (C), HAVCR1-Fc or the same receptor with an N94A mutation in the phosphatidylserine binding domain (HAVCR1-N94A-Fc) (D), phosphatidylserine (PS) or phosphatidylcholine (PC) liposomes (E), or annexin V (F). Testing for all assays was performed in duplicate. Error bars represent standard errors of the means. Data are representative of three independent experiments. All experiments were performed with HCVcc J6/JFH1 except for that shown in panel A, which used HCVcc chimeric viruses 1a/2a, 1b/2a, 2a/2a (J6/JFH1), and 3a/2a.

FIG 4

Reduced HAVCR1 expression on Huh7.5 cells results in reduced levels of HCVcc and HCVpp entry. Huh7.5 HAVCR1-1 knockdown (KD) cells were generated by transfection of parent Huh7.5 cells with a plasmid expressing HAVCR1 shRNA. (A) Percentages of parent and KD cells positive for HAVCR1, CD81, and SRB1 surface expression. Bars represent the mean values from 5 independent experiments for HAVCR1 and CD81 and 4 independent experiments for SRB1. (B) Mean fluorescence signals of HAVCR1, CD81, and SRB1 surface expression on parent and KD cells. Bars represent the mean values from 5 independent experiments for HAVCR1 and CD81 and 4 independent experiments for SRB1. (C) Western blot detection of claudin and occludin after cell surface biotinylation of Huh7.5 and HAVCR1 KD cells. Whole-cell lysate (WCL) is cell lysate prior to pulldown treatment. Nonbiotinylated control represents cells treated exactly as for the biotinylated cells but without the addition of EZ-Link sulfo-NHS-LCLC-biotin for cell surface biotinylation. Anti-claudin antibody was used for detection and shows the specificity of the NeutrAvidin beads for precipitation. (D) Luciferase activities at 24, 48, and 72 h postinfection in lysates of parent and KD cells infected with JFH1-Nanoluc. Bars represent the mean relative light units (RLU) calculated from 4 independent experiments using 9 replicates per experiment. (E) Titers of virus recovered from the supernatants of parent and KD cells at 72 h postinfection with JFH1-Nanoluc. Bars represent the mean titers from 4 independent experiments. (F) Luciferase activities in parent and KD cells 48 h after infection with HCVpp. (G) Luciferase activities in parent and KD cells 48 h after infection with VSV-G. Bars represent the mean values from 4 independent experiments. Error bars in all graphs represent standard errors of the means. Statistical analyses were performed using the Mann-Whitney test.

FIG 5

Complete silencing of HAVCR1 expression in Huh7.5 cells significantly reduces HCVcc infection. Surface expression levels of receptors on Huh7.5 cells, Huh7-A-I cells, and different clones of Huh7-A-I HAVCR1 knockout (KO) cells were assessed by FACS using MAbs specific to HAVCR1 (REA383) (A), CD81 (JS-81) (B), and SRB1 (m1B9) (C). Data for positive cells are shown in dark gray, and data for mouse PE-isotype control are shown in light gray. Data are representative of three independent experiments. (D) Western blot detection of claudin and occludin after cell surface biotinylation of Huh7-A-I and clone 12 KO cells. Whole-cell lysate (WCL) is cell lysate prior to pulldown treatment. Nonbiotinylated control represents cells treated exactly as for the biotinylated cells but without the addition of EZ-Link sulfo-NHS-LCLC-biotin for cell surface biotinylation. Anticlaudin antibody was used for detection and shows the specificity of the NeutrAvidin beads for precipitation. Antiactin antibody was used for detection of actin, a cytoplasmic protein that is not expressed on the cell surface, demonstrating biotinylation of surface proteins only. (E) Luciferase activities in lysates from Huh7-A-I HAVCR1 KO clones and parent cell line at 72 h postinfection with JFH1-Nanoluc. Data were obtained from 3 independent experiments, and each experiment was carried out using 12 replicates for each cell clone. Each symbol represents an individual well from the 3 independent experiments. Horizontal bars represent the mean luciferase activities. P values were calculated using the mean values from each of the 3 independent experiments. (F) Time course of luciferase expression relative to the expression at 3 h posttransfection in Huh7-A-I HAVCR1 KO clones 2 and 12 and parent cells following transfection with JFH1-Nanoluc RNA transcribed in vitro. Data are the mean values from 6 replicates at each time point. Error bars represent standard errors of the means. Data are representative of two independent experiments. RLU, relative light units.

FIG 6

Expression of HAVCR1 in KO clone 12 enhances HCV infection. HAVCR1 KO clone 12 cells were transfected with plasmids expressing HAVCR1, mutated forms of HAVCR1 (E88A, N94A, Y330A, Y336A, and del324–331 mutations), or vector. (A) Expression of HAVCR1 on the surface of clone 12 cells transfected with vector or plasmid expressing HAVCR1 48 h posttransfection was assessed using anti-HAVCR1-1 MAb. (B) Apoptotic binding activities of clone 12 cells transfected with vector or plasmid expressing HAVCR1. (C) Luciferase expression in cell lysates of clone 12 cells transfected with HAVCR1 expression plasmid and infected with JFH1-Nanoluc. Data at 24 h postinfection are shown. Four independent experiments were performed; each time point included 3 replicates. (D) Expression of E88A or N94A mutant on the surface of transfected clone 12 cells 48 h posttransfection assessed using anti-HAVCR1-1 MAb. (E) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing E88A and N94A mutant forms of HAVCR1. (F) Luciferase expression in cell lysates of clone 12 cells transfected with plasmids expressing E88A or N94A mutants and infected with JFH1-Nanoluc. Data are from 4 independent experiments, each including 3 replicates, at 24 h postinfection. (G) Expression of Y330A, Y336A, or del324–331 mutant on the surface of transfected clone 12 cells 48 h posttransfection was assessed using anti-HAVCR1-1 MAb. (H) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing Y330A, Y336A, and del324–331 mutant forms of HAVCR1. (I) Luciferase expression in cell lysates of clone 12 cells transfected with Y330A, Y336A, or del324–331 mutant plasmids and infected with JFH1-Nanoluc. Data are from 4 independent experiments, each including 3 replicates, at 24 h postinfection. Horizontal bars represent the mean values. Asterisks represent significance values obtained using the Mann-Whitney test to compare the mean values for vector-transfected cells with the mean values for each of the other transfectants. NS, not significant; **, P < 0.001; ***, P < 0.0001. For FACS histograms, HAVCR1 expression on transfected cells is shown as filled dark-gray areas, and data for untransfected cells stained with anti-HAVCR1-1 MAb are shown as filled light-gray areas. RLU, relative light units; MFI, mean fluorescence intensity.

FIG 7

Schematic representation of the HAVCR1, mHavcr1, and chimeric proteins used in transient-transfection experiments. (A) Receptor domains and mutations in the human and mouse proteins. (B) Sequence alignment of HAVCR1, mouse Havcr1 (mHavcr1), and chimeric protein. Yellow highlighting denotes residues conserved between the molecules. The different receptor regions are defined by colored underlining as follows: red, IgV domain; blue, mucin domain; green, transmembrane domain; black, cytoplasmic domain. Mutated or deleted residues in HAVCR1 are shown in black boxes.

FIG 8

The IgV domain of human HAVCR1 is responsible for facilitating HCV entry. (A) Expression of HAVCR1 or mHavcr1 on the surface of transfected clone 12 cells 48 h posttransfection. Receptor expression is shown as filled dark-gray areas, and data for stained, untransfected cells are shown as filled light-gray areas. (B) Apoptotic binding of clone 12 cells transfected with vector or plasmids expressing HAVCR1, mHavcr1, or chimeric human/mouse receptor. MFI, mean fluorescence intensity. (C) Luciferase expression in cell lysates of clone 12 cells transfected with expression plasmids and infected with JFH1-Nanoluc. RLU, relative light units. Data shown are from 5 experiments with transient transfection of the control vector and HAVCR1 and 3 independent experiments for mHavcr1, including 3 replicates each, at 24 h postinfection. Asterisks represent significance values obtained using the Mann-Whitney test to compare the data for vector-transfected cells with the data for each of the other transfectants or the parent cell line. NS, not significant; **, P < 0.001; ***, P < 0.0001.