Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro.

丙型肝炎病毒（HCV）进入肝细胞是一个复杂的过程，涉及许多细胞因子，包括1类清道夫受体B型（SR-B1），四跨膜蛋白CD81和紧密连接（TJ）蛋白claudin-1（ CLDN1）和occludin（OCLN）。尽管表达了所有已知的HCV进入因子，但基于肝癌细胞系的体外模型不能完全重现肝细胞对原代HCV分离株的体内易感性，这意味着存在对于HCV进入和/或致死至关重要的其他宿主因子复制。同样，体内肝细胞癌（HCC）组织中的HCV复制受到严重损害，但目前尚不清楚造成这种限制的机制。在这里，我们确定肿瘤相关钙信号转导子2（TACSTD2）是原发性HCC组织中被下调最严重的基因之一，它是与CLDN1和OCLN相互作用并调节其细胞定位的宿主因子。TACSTD2基因沉默破坏了肝癌细胞和原代人肝细胞中沿细胞膜的CLDN1和OCLN的典型线性分布，概括了原发性HCC组织在体内观察到的模式。机理研究表明，TACSTD2参与了CLDN1和OCLN的磷酸化，这对于它们的适当细胞定位是必需的。TACSTD2的沉默通过在病毒进入水平发生的泛基因型效应，显着抑制HCV感染。我们的研究确定TACSTD2是两种主要的HCV输入因子CLDN1和OCLN的新型调节剂，在恶性肝细胞中它们被强烈下调。这些结果为HCV进入肝细胞的复杂过程提供了新的见识，并可能有助于开发更有效的HCV在体外传播的细胞系统。