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. 2018 Apr 13;92(9):e02065-17.
doi: 10.1128/JVI.02065-17. Print 2018 May 1.

HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection

Maria Isabel Costafreda  1 , Gerardo Kaplan  2
Affiliations

HAVCR1 (CD365) and Its Mouse Ortholog Are Functional Hepatitis A Virus (HAV) Cellular Receptors That Mediate HAV Infection

Maria Isabel Costafreda et al. J Virol. .

Abstract

The hepatitis A virus (HAV) cellular receptor 1 (HAVCR1), classified as CD365, was initially discovered as an HAV cellular receptor using an expression cloning strategy. Due to the lack of HAV receptor-negative replication-competent cells, it was not possible to fully prove that HAVCR1 was a functional HAV receptor. However, biochemistry, classical virology, and epidemiology studies further supported the functional role of HAVCR1 as an HAV receptor. Here, we show that an anti-HAVCR1 monoclonal antibody that protected African green monkey kidney (AGMK) cells against HAV infection only partially protected monkey Vero E6 cells and human hepatoma Huh7 cells, indicating that these two cell lines express alternative yet unidentified HAV receptors. Therefore, we focused our work on AGMK cells to further characterize the function of HAVCR1 as an HAV receptor. Advances in clustered regularly interspaced short palindromic repeat/Cas9 technology allowed us to knock out the monkey ortholog of HAVCR1 in AGMK cells. The resulting AGMK HAVCR1 knockout (KO) cells lost susceptibility to HAV infection, including HAV-free viral particles (vpHAV) and exosomes purified from HAV-infected cells (exo-HAV). Transfection of HAVCR1 cDNA into AGMK HAVCR1 KO cells restored susceptibility to vpHAV and exo-HAV infection. Furthermore, transfection of the mouse ortholog of HAVCR1, mHavcr1, also restored the susceptibility of AGMK HAVCR1 KO cells to HAV infection. Taken together, our data clearly show that HAVCR1 and mHavcr1 are functional HAV receptors that mediate HAV infection. This work paves the way for the identification of alternative HAV receptors to gain a complete understanding of their interplay with HAVCR1 in the cell entry and pathogenic processes of HAV.IMPORTANCE HAVCR1, an HAV receptor, is expressed in different cell types, including regulatory immune cells and antigen-presenting cells. How HAV evades the immune response during a long incubation period of up to 4 weeks and the mechanism by which the subsequent necroinflammatory process clears the infection remain a puzzle that most likely involves the HAV-HAVCR1 interaction. Based on negative data, a recent paper from the S. M. Lemon and W. Maury laboratories (A. Das, A. Hirai-Yuki, O. Gonzalez-Lopez, B. Rhein, S. Moller-Tank, R. Brouillette, L. Hensley, I. Misumi, W. Lovell, J. M. Cullen, J. K. Whitmire, W. Maury, and S. M. Lemon, mBio 8:e00969-17, 2017, https://doi.org/10.1128/mBio.00969-17) suggested that HAVCR1 is not a functional HAV receptor, nor it is it required for HAV infection. However, our data, based on regain of the HAV receptor function in HAVCR1 knockout cells transfected with HAVCR1 cDNA, disagree with their findings. Our positive data show conclusively that HAVCR1 is indeed a functional HAV receptor and lays the ground for the identification of alternative HAV receptors and how they interact with HAVCR1 in cell entry and the pathogenesis of HAV.

Keywords: African green monkey cells; CD365; CRISPR/Cas9; HAVCR1; KIM1; TIM1; Vero E6 cells; cellular receptor; gene editing; hepatitis A virus; human hepatoma Huh7 cells; mouse ortholog.

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Figures

FIG 1
FIG 1
HAVCR1 mediates HAV infection in cells expressing HAVCR1 and other alternative receptors. (A and B) Purification of exo-HAV and vpHAV from supernatants of AGMK cells infected with attenuated HAV-Bsd (A) or Huh7 cells infected with pathogenic HAV-Bsd (B) by isopycnic ultracentrifugation in iodixanol gradients. The HAV RNA in gradient fractions collected after isopycnic ultracentrifugation was quantified by RT-qPCR. Fractions 11 and 12, containing exo-HAV (density, 1.10 to 1.11 g cm−3), and fraction 19 or 20, containing vpHAV (density, 1.25 to 1.28 g cm−3), were collected and used for further experimentation. Arrows mark the exo-HAV and vpHAV peaks. GE, genome equivalents. (C to E) Expression of mkHAVCR1 at the cell surface of AGMK (C) and Vero E6 (D), and human HAVCR1 at the cell surface of Huh7 (E) cells stained with protective anti-HAVCR1 mAb 1D12 and analyzed by flow cytometry (black lines). Cells were also stained with an isotype mAb as a negative control (gray filled curves). (F to H) Protection of AGMK (F), Vero E6 (G), and Huh7 (H) cells against HAV infection by treatment with anti-HAVCR1 mAb 1D12. Cells pretreated with 50 μg/ml of mAb 1D12 (+) or medium (−) for 30 min at room temperature were infected with HAV-Bsd for 24 h, trypsinized, and selected with the antibiotic Bsd. At 12 days postinfection, cell colonies were fixed and stained with crystal violet (dark spots), and 6-well plates were imaged using a flatbed scanner. (I to K) The number of Bsd-resistant CFU was quantitated and graphed as the mean colony numbers from duplicate plates. Bars represent SDs for duplicate wells. Differences between control and mAb-treated cells were analyzed by Student's unpaired t test. *, P < 0.05; **, P < 0.01. Results are representative of those from two independent experiments.
FIG 2
FIG 2
Knockout of mkHAVCR1 in AGMK cells prevents HAV infection. (A) Two single guide RNAs (sgRNA) in exon 2 of the mkHAVCR1 gene, which codes for the IgV binding domain, were designed to knock out the gene in AGMK cells using the CRISPR/Cas9 gene editing technology. (B) Nucleotide sequence alignment of AGMK parental (wild type [WT]) and AGMK HAVCR1 knockout (AGMK HAVCR1 KO) cells shows a 100-nucleotide deletion in exon 2 resulting in a frameshift mutation. (C) Analysis of the expression of mkHAVCR1 in AGMK HAVCR1 KO cells. mkHAVCR1 expression at the cell surface of AGMK parental (black line) and AGMK HAVCR1 KO (red line) cells was determined by flow cytometry analysis using anti-HAVCR1 mAb 1D12. Parental AGMK cells were also stained with an isotype control (filled gray curve). (D) IF analysis of HAV-infected cells. AGMK parental and AGMK HAVCR1 KO cells were infected with HAV-Bsd for 4 days, and the cells were fixed and permeabilized, stained with anti-HAV neutralizing mAbs (green fluorescence), counterstained with DAPI nuclear dye (blue fluorescence), and observed under a fluorescence microscope. Fluorescence and phase-contrast micrographs from representative fields were taken at a magnification of ×400. The experiments whose results are shown in panels D and E were repeated at least 3 times with similar results.
FIG 3
FIG 3
HAVCR1 is required for infection of exo-HAV and vpHAV. Growth of exo-HAV and vpHAV in AGMK parental and AGMK HAVCR1 KO cells analyzed by RT-qPCR (A) and Bsd-resistant CFU assay (B). Infected cells were trypsinized at 12 to 72 h postinfection, 50% of the cells were used for RT-qPCR, and the remaining cells were seeded in 6-well plates for the Bsd-resistant CFU assay. RT-qPCR results are the mean ± SD for duplicate wells. Differences between means were analyzed by t test. **, P < 0.01. Dark dots in the Bsd-resistant CFU assay derive from HAV-infected cell colonies, and clear surfaces are free from cells. At 72 h postinfection, cell colonies formed an almost confluent monolayer. Data are representative of those from two experiments.
FIG 4
FIG 4
Transfection of HAVCR1 cDNA restores susceptibility to HAV infection. (A) Expression of HAVCR1 at the cell surface of vector-transfected (gray filled histogram) or HAVCR1-transfected (green line) AGMK HAVCR1 KO cells analyzed by flow cytometry. Transfectants were infected with an HAV preparation containing exo-HAV and vpHAV for 72 h and trypsinized. (B and C) Half of the cells were used for extraction of intracellular RNA for HAV-specific RT-qPCR (B), and the remaining cells were used for Bsd-resistant CFU analysis (C). RT-qPCR results are the mean ± SD for duplicate wells. Differences between means were analyzed by t test. *, P < 0.05. Dark dots in the Bsd-resistant CFU assay derive from HAV-infected cell colonies, and clear surfaces are free from cells. Data are representative of those from three experiments.
FIG 5
FIG 5
The mouse ortholog of HAVCR1, mHavcr1, is a functional HAV receptor. (A) Expression of mHavcr1 (left) or HAVCR1 (right) at the cell surface of AGMK HAVCR1 KO cells transfected with the cDNA of mHavcr1 (light blue line), HAVCR1 (green line), or the vector (filled gray histogram), as determined by flow cytometry. (B and C) Transfectants were infected with exo-HAV or vpHAV for 72 h, and virus growth was determined by RT-qPCR (B) and the Bsd-resistant CFU assay (C), as described in the legend to Fig. 4. (D) Isopycnic ultracentrifugation of purified vpHAV treated (blue line) or not treated (gray line) with 1% Sarkosyl in iodixanol gradients, as described in the legend to Fig. 1A. (E) HAV growth in cell transfectants infected with detergent-treated or untreated vpHAV from the assay whose results are shown in panel D, determined by RT-qPCR at 72 h postinfection. (F) IF analysis of cell transfectants infected with exo-HAV or vpHAV as described in the legend to Fig. 2E. RT-qPCR results are the mean ± SD for duplicate wells. Differences between means were analyzed by t test. *, P < 0.05; **, P < 0.01; ns, no significant difference. Data are representative of those from at least two different experiments.
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