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. Our in
situ proximity ligation results demonstrate 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 the role of RTP4 in contributing to HCVs
narrow host range and will inform downstream development of a more
comprehensive small-animal model for this important disease.