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. 2017 Sep 5;8(5):e00969-17.
doi: 10.1128/mBio.00969-17.

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

Anshuman Das  1 , Asuka Hirai-Yuki  1 , Olga González-López  1 , Bethany Rhein  2 , Sven Moller-Tank  2 , Rachel Brouillette  2 , Lucinda Hensley  1 , Ichiro Misumi  1   3 , William Lovell  1 , John M Cullen  4 , Jason K Whitmire  1   5   3 , Wendy Maury  2 , Stanley M Lemon  6   3   7
Affiliations

TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions

Anshuman Das et al. mBio. .

Abstract

Receptor molecules play key roles in the cellular entry of picornaviruses, and TIM1 (HAVCR1) is widely accepted to be the receptor for hepatitis A virus (HAV), an unusual, hepatotropic human picornavirus. However, its identification as the hepatovirus receptor predated the discovery that hepatoviruses undergo nonlytic release from infected cells as membrane-cloaked, quasi-enveloped HAV (eHAV) virions that enter cells via a pathway distinct from naked, nonenveloped virions. We thus revisited the role of TIM1 in hepatovirus entry, examining both adherence and infection/replication in cells with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-engineered TIM1 knockout. Cell culture-derived, gradient-purified eHAV bound Huh-7.5 human hepatoma cells less efficiently than naked HAV at 4°C, but eliminating TIM1 expression caused no difference in adherence of either form of HAV, nor any impact on infection and replication in these cells. In contrast, TIM1-deficient Vero cells showed a modest reduction in quasi-enveloped eHAV (but not naked HAV) attachment and replication. Thus, TIM1 facilitates quasi-enveloped eHAV entry in Vero cells, most likely by binding phosphatidylserine (PtdSer) residues on the eHAV membrane. Both Tim1-/-Ifnar1-/- and Tim4-/-Ifnar1-/- double-knockout mice were susceptible to infection upon intravenous challenge with infected liver homogenate, with fecal HAV shedding and serum alanine aminotransferase (ALT) elevations similar to those in Ifnar1-/- mice. However, intrahepatic HAV RNA and ALT elevations were modestly reduced in Tim1-/-Ifnar1-/- mice compared to Ifnar1-/- mice challenged with a lower titer of gradient-purified HAV or eHAV. We conclude that TIM1 is not an essential hepatovirus entry factor, although its PtdSer-binding activity may contribute to the spread of quasi-enveloped virus and liver injury in mice.IMPORTANCE T cell immunoglobulin and mucin-containing domain protein 1 (TIM1) was reported more than 2 decades ago to be an essential cellular receptor for hepatitis A virus (HAV), a picornavirus in the Hepatovirus genus, resulting in its designation as "hepatitis A virus cellular receptor 1" (HAVCR1) by the Human Genome Organization Gene Nomenclature Committee. However, recent studies have shown that HAV exists in nature as both naked, nonenveloped (HAV) virions and membrane-cloaked, quasi-enveloped infectious virus (eHAV), prompting us to revisit the role of TIM1 in viral entry. We show here that TIM1 (HAVCR1) is not an essential cellular receptor for HAV entry into cultured cells or required for viral replication and pathogenesis in permissive strains of mice, although it may facilitate early stages of infection by binding phosphatidylserine on the eHAV surface. This work thus corrects the published record and sets the stage for future efforts to identify specific hepatovirus entry factors.

Keywords: hepatitis A virus; hepatovirus; phosphatidylserine; picornavirus; receptor; viral attachment.

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Figures

FIG 1
FIG 1
Impact of TIM1 knockout on eHAV and HAV infection of human Huh-7.5 cells. (A) Human HAVCR1 gene structure (NCBI Homo sapiens annotation release 108, accession no. XM_017009340.1; map not drawn to scale). The red arrows show the locations of CRISPR-induced disruption of the TIM1 sequence in exons 2 (KO#1) and 3 (KO#2 and KO#3). (B) Immunoblots of TIM1 and actin (loading control) in lysates of control Huh-7.5 cells and CRISPR/Cas9-generated Huh-7.5 TIM1-KO cells. Anti-TIM1 reactive bands appear at ∼38.7 kDa (predicted TIM1 molecular mass) and ∼55 kDa. (C) Surface expression of TIM1 on control Huh-7.5 and TIM1-KO cells quantified by flow cytometry. “Isotype” refers to the immunoglobulin control. (D) Distribution of HAV RNA in an isopycnic iodixanol density gradient loaded with supernatant fluids of HM175/18f-infected Huh-7.5 cells. The abundance of eHAV, the predominant form of virus present in supernatant fluids, peaked in fraction 8 (1.056 g/cm3), whereas naked HAV formed a small peak in fraction 18 (1.260 g/cm3). (E) Adherence of HAV and eHAV to control Huh-7.5 and Huh-7.5 TIM1-KO cells at 4°C determined by RT-qPCR specific for viral RNA. Differences in residual virus bound to parental versus TIM1-KO cells did not achieve statistical significance. Error bars = SEM; n = 6 (2 independent experiments, each with 3 technical replicates). (F) Accumulation of intracellular HAV RNA in Huh-7.5 and related TIM1-KO cells following infection at 37°C with eHAV or HAV inocula containing similar amounts of HAV RNA. Viral RNA in HAV-infected cells significantly exceeded that in eHAV-infected cells at 24 and 48 h, but differences between control and any TIM1-KO cell line infected with the same inoculum did not achieve statistical significance. Error bars = SEM; n = 4 (2 independent experiments each with 2 technical replicates). Values in panels E and F that were significantly different for eHAV versus HAV by two-way analysis of variance (ANOVA) are indicated by bars and asterisks as follows: *, P < 0.05; ***, P < 0.001.
FIG 2
FIG 2
Impact of TIM1 knockout on HAV infection of Vero cells. (A) African green monkey HAVCR1 gene structure (NCBI Chlorocebus sabaeus annotation release 100, accession no. XM_008015132.1; map not drawn to scale). The red arrow shows the location of CRISPR-induced disruption of the TIM1 sequence in exon 3 as determined by DNA sequencing. (B) Expression of TIM1 on the surfaces of control and TIM1-KO Vero cells quantified by flow cytometry. “Isotype” refers to the immunoglobulin control. (C) Adherence of HAV and eHAV to Vero control and two distinct TIM1-KO cell lines at 4°C, determined by RT-qPCR specific for viral RNA. Error bars = SEM; n = 5 (2 independent experiments, each with 2-3 technical replicates). *, P < 0.05; ***, P < 0.001. (D) Accumulation of intracellular HAV RNA in control and TIM1-KO Vero cells following infection at 37°C with eHAV or HAV inocula containing similar amounts of HAV RNA. Viral RNA in quasi-enveloped eHAV-infected control cells exceeded that in TIM1-KO cells at 24 and 48 h (TIM1-KO#1 [P < 0.05] and TIM1-KO#2 [P < 0.01] by two-way ANOVA with Tukey’s multiple-comparison test). Error bars = SEM; n = 4 (2 independent experiments each with 2 technical replicates). There was no significant difference (ns) between HAV RNA levels in control and KO cell lines infected with naked HAV.
FIG 3
FIG 3
Hepatitis A infection in Tim1−/− Ifnar1−/− (n = 4), Tim4−/− Ifnar1−/− (n = 2), and Ifnar1−/− (n = 4) mice inoculated intravenously (i.v.) with ∼108 genome equivalents (GE) of fifth mouse-passage HM175 virus (mp5 unfractionated liver homogenate). (A) Fecal shedding of HAV by Tim1−/− Ifnar1−/−and Tim4−/− Ifnar1−/−double-knockout mice was similar to single Ifnar1−/− knockouts, but significantly elevated in Tim1−/− Ifnar1−/− versus Ifnar1−/− mice on day 7 postinfection (P < 0.01). (B) Intrahepatic HAV RNA abundance at day 14 (d14) postinfection. (C) Serum ALT on days 7 and 14 postinfection. Bar = mean. (D) H&E-stained liver sections from mock-infected Tim1−/− Ifnar1−/− mice, mp5 HM175 virus-infected Tim1−/− Ifnar1−/− mice, and Ifnar1−/− mice. Virus-infected Tim1−/− Ifnar1−/− and Ifnar1−/− livers show diffuse small inflammatory cell infiltrates with apoptotic hepatocytes (white arrows) scattered throughout the parenchyma. Only minimal numbers of periportal lymphocytes are evident in mock-infected liver tissue. Bar, 100 µm.
FIG 4
FIG 4
Infection in Ifnar1−/− and Tim1−/− Ifnar1−/− mice (four mice in each group) initiated by gradient-isolated quasi-enveloped and naked, nonenveloped fifth mouse-passage virus. (A) HAV RNA in fractions of an isopycnic iodixanol gradient loaded with a lysate of infected Mavs−/− mouse liver (2). Fractions 10 (eHAV) and 18 (HAV) were used to inoculate mice with comparable amounts of virus based on RNA quantitation (∼108 genome equivalents). (B) Fecal HAV shedding in mice infected by i.v. inoculation of HAV or eHAV (fractionated as described for panel A). Fecal shedding was significantly greater in HAV-infected than eHAV-infected animals at 7 and 10 days postinfection, regardless of the presence or absence of TIM1 (P < 0.001 by two-way ANOVA). Ifnar1−/− and Tim1−/− Ifnar1−/− mice differed in fecal shedding only at day 10 in HAV-infected, not eHAV-infected animals (P < 0.05). There were four mice in each group. (C) Intrahepatic HAV RNA at day 14 postinfection. Less viral RNA was present in Tim1−/− Ifnar1−/− mice than in Ifnar1−/− mice infected with either inoculum (P < 0.05 by two-way ANOVA). (D) Serum ALT on days 7 and 14 postinfection. ALT elevations were consistently lower in Tim1−/− Ifnar1−/− mice compared to Ifnar1−/− mice, but this difference achieved statistical significance only at day 14. Error bars = SEM. *, P < 0.05; ***, P < 0.001.
FIG 5
FIG 5
Interferon-stimulated gene expression and histopathology in the livers of eHAV- or HAV-infected Tim1−/− Ifnar1−/− mice versus Ifnar1−/− mice, 14 days postinfection. (A) Intrahepatic MCP-1, CCL5 (Rantes), and IFIT-2 mRNA expression. Data shown represent the mean ± SEM fold increase compared to uninfected control mice. There were four mice in each group. *, P < 0.05 by two-way ANOVA; **, P < 0.01 by two-way ANOVA. (B, C) H&E-stained liver sections from eHAV-infected mice: (B) Tim1−/− Ifnar1−/− double-knockout (ALT = 249 IU/ml) showing a single focus of inflammation, characterized by an apoptotic hepatocyte and a surrounding aggregate of infiltrating lymphocytes; (C) Ifnar1−/− single-knockout (ALT = 309 IU/ml). (D, E) Similar sections from naked HAV virion-infected (D) Tim1−/− Ifnar1−/− (ALT = 290 IU/ml) and (E) Ifnar1−/− (ALT = 270 IU/ml) mice, each showing a diffuse inflammatory infiltrate of lymphocytes and scattered apoptotic hepatocytes. Bar, 100 µm.
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