Receptor transport protein 4 (RTP4)-mediated repression of hepatitis C virus replication in mouse cells

Hepatitis C virus (HCV) exhibits a narrow species tropism, causing robust infections only in humans and experimentally inoculated chimpanzees. The black flying fox orthologue of receptor transport protein 4 (RTP4) was previously shown to be a potent antiviral effector against several ER-replicating RNA viruses, inhibiting viral genome replication. Since the murine but not the human orthologue is a potent inhibitor of HCV we aimed to analyze the potential role for RTP4 in restricting HCV replication in mice. We demonstrate that mouse RTP4 (mmRTP4) functions as a dominant inhibitor of HCV infection. Via interspecies domain-mapping, we identify the zinc-finger domain (ZFD) of murine RTP4 as the essence of its specific HCV inhibition which is consistent with prior work showing that the 3CXXC zinc finger domain of black flying fox RTP4 is necessary and sufficient for antiviral activity. Expression of mouse RTP in HCV-infected Huh7 cells profoundly reduced HCV RNA, NS5A protein production and virion release demonstrating that mmRTP4 can also disrupt already established HCV replication complexes. Bulk RNAseq analysis of HCV-infected samples did not reveal any characteristic antiviral transcriptional signatures following mmRTP4 transduction, indicating that this antiviral activity cannot be readily attributed to induction of an antiviral response. In situ proximity ligation revealed that murine RTP4 associates with the HCV NS5A protein significantly more than human RTP4 during infection. Disrupting RTP4 expression in mice expressing humanized alleles of CD81 and occludin (OCLN) – the species specific cellular factors mediating HCV uptake – did not, however, increase permissiveness irrespective of the immunocompetence of the mice. Collectively, our work provides detailed insights into RTP4’s role in contributing to HCVs narrow host range and will inform downstream development of a more comprehensive small-animal model for this important disease.