Schwoerer Michael P, Carver Sebastian, Lin Aaron E, Liu Jianche, Cafiero Thomas R, Berggren Keith A, Dhawan Serene, Suzuki Saori, Heller Brigitte, Rodriguez Celeste, O'Connell Aoife K, Gertje Hans P, Crossland Nicholas A, Ploss Alexander
Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America.
Zhejiang University-University of Edinburgh Institute, Zhejiang University, Haining, China.
PLoS Pathog. 2025 Sep 8;21(9):e1013412. doi: 10.1371/journal.ppat.1013412. eCollection 2025 Sep.
Hepatitis C virus (HCV) exhibits a narrow species tropism, causing robust infections only in humans and experimentally inoculated chimpanzees. While many host factors and restriction factors are known, many more likely remain unknown, which has limited the development of mouse or other small animal models for HCV. One putative restriction factor, the black flying fox orthologue of receptor transporter protein 4 (RTP4), was previously shown to potently inhibit viral genome replication of several ER-replicating RNA viruses. Since the murine but not the human ortholog is a potent inhibitor of HCV, we aimed to analyze the potential role for RTP4 in restricting HCV replication in mice. We demonstrated that mouse RTP4 (mmRTP4) functions as a dominant inhibitor of HCV infection. Via interspecies domain-mapping, we identified the zinc-finger domain (ZFD) of murine RTP4 as essential for inhibiting HCV, consistent with prior work. Introducing mmRTP4 into HCV-infected Huh7 cells profoundly reduced HCV NS5A protein production and virion release, demonstrating that mmRTP4 can also disrupt already established HCV replication complexes. This inhibition of HCV was not driven by induction of interferon-stimulated genes based on bulk RNA-seq, suggesting that mmRTP4 might directly act on HCV replication. Indeed, by in situ proximity ligation, we found that mmRTP4 directly associates with the HCV NS5A protein significantly more than human RTP4 during infection. However, disrupting RTP4 expression in mice expressing humanized alleles of CD81 and occludin (OCLN) - the species specific cellular factors mediating HCV uptake - did not increase permissiveness irrespective of the immunocompetence of the mice. Collectively, our work provides detailed insights into the role of RTP4 in contributing to HCV's narrow host range and will inform downstream development of a more comprehensive small-animal model for this important pathogen.
丙型肝炎病毒（HCV）表现出狭窄的物种嗜性，仅在人类和经实验接种的黑猩猩中引发强烈感染。虽然已知许多宿主因子和限制因子，但很可能还有更多未知，这限制了HCV小鼠或其他小动物模型的开发。一种假定的限制因子，受体转运蛋白4（RTP4）的黑狐蝠直系同源物，先前已证明能有效抑制几种在内质网复制的RNA病毒的病毒基因组复制。由于小鼠而非人类直系同源物是HCV的有效抑制剂，我们旨在分析RTP4在限制小鼠中HCV复制的潜在作用。我们证明小鼠RTP4（mmRTP4）作为HCV感染的显性抑制剂发挥作用。通过种间结构域映射，我们确定小鼠RTP4的锌指结构域（ZFD）对抑制HCV至关重要，这与先前的研究一致。将mmRTP4引入HCV感染的Huh7细胞中，可显著降低HCV NS5A蛋白的产生和病毒粒子的释放，表明mmRTP4也能破坏已建立的HCV复制复合体。基于大量RNA测序，这种对HCV的抑制不是由干扰素刺激基因的诱导驱动的，这表明mmRTP4可能直接作用于HCV复制。事实上，通过原位邻近连接，我们发现感染期间mmRTP4与HCV NS5A蛋白的直接关联明显多于人类RTP4。然而，在表达介导HCV摄取的物种特异性细胞因子CD81和闭合蛋白（OCLN）人源化等位基因的小鼠中破坏RTP4表达，无论小鼠的免疫能力如何，都不会增加其易感性。总的来说，我们的工作为RTP4在导致HCV狭窄宿主范围中的作用提供了详细见解，并将为这一重要病原体更全面的小动物模型的下游开发提供信息。
