TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions.
TIM1 (HAVCR1) Is Not Essential for Cellular Entry of Either Quasi-enveloped or Naked Hepatitis A Virions.
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DOI：
10.1128/mbio.00969-17
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发表时间：
2017-09-05
期刊：
影响因子：
6.4
通讯作者：
Lemon SM
中科院分区：
文献类型：
--
作者：
Das A;Hirai-Yuki A;González-López O;Rhein B;Moller-Tank S;Brouillette R;Hensley L;Misumi I;Lovell W;Cullen JM;Whitmire JK;Maury W;Lemon SM
受体分子在小核糖核酸病毒进入细胞中起关键作用，并且TIM 1（HAVCR 1）被广泛认为是甲型肝炎病毒（HAV）的受体，HAV是一种不常见的嗜肝性人类小核糖核酸病毒。然而，其作为肝病毒受体的鉴定早于肝病毒作为膜包被的、准包膜的HAV（eHAV）病毒体从感染细胞经历非裂解性释放的发现，所述病毒体通过与裸的、无包膜的病毒体不同的途径进入细胞。因此，我们重新审视了TIM 1在肝病毒进入中的作用，检查了具有成簇规则间隔短回文重复序列（CRISPR）/Cas9工程改造的TIM 1敲除的细胞中的粘附和感染/复制。在4°C下，细胞培养衍生的梯度纯化的eHAV结合Huh-7.5人肝癌细胞的效率低于裸HAV，但是消除TIM 1表达对任一形式的HAV的粘附没有引起差异，也没有对这些细胞中的感染和复制产生任何影响。相比之下，TIM 1缺陷型Vero细胞显示出半包膜eHAV（但非裸HAV）附着和复制的适度减少。因此，TIM 1促进准包膜eHAV进入Vero细胞，最有可能是通过结合eHAV膜上的磷脂酰丝氨酸（PtdSer）残基。Tim 1 −/− Ifnar 1 −/−和Tim 4 −/− Ifnar 1 −/−双基因敲除小鼠在用感染的肝匀浆静脉内攻击时对感染敏感，粪便HAV脱落和血清丙氨酸氨基转移酶（ALT）升高与Ifnar 1 −/−小鼠相似。然而，与用较低滴度的梯度纯化HAV或eHAV攻击的Ifnar 1 −/−小鼠相比，Tim 1 −/− Ifnar 1 −/−小鼠的肝内HAV RNA和ALT升高适度降低。我们的结论是，TIM 1是不是一个重要的肝病毒进入因子，虽然其PtdSer结合活性可能有助于准包膜病毒的传播和小鼠肝损伤。T细胞免疫球蛋白和含粘蛋白的结构域蛋白1（TIM 1）在20多年前被报道为甲型肝炎病毒（HAV）（肝病毒属中的一种小核糖核酸病毒）的必需细胞受体，导致其被人类基因组组织基因命名委员会命名为“甲型肝炎病毒细胞受体1”（HAVCR 1）。然而，最近的研究表明，HAV存在于自然界中的裸露，无包膜（HAV）病毒粒子和膜斗篷，准包膜感染性病毒（eHAV），促使我们重新审视TIM 1在病毒进入的作用。我们在这里表明，TIM 1（HAVCR 1）是不是一个重要的细胞受体HAV进入培养细胞或所需的病毒复制和发病机制在允许的小鼠品系，虽然它可能会促进感染的早期阶段，通过结合磷脂酰丝氨酸的eHAV表面。因此，这项工作纠正了已发表的记录，并为今后确定特定肝病毒进入因子的努力奠定了基础。
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. 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.