Abstract
Hepatitis C virus (HCV) requires the liver specific micro-RNA (miRNA), miR-122, to replicate. This was considered unique among RNA viruses until recent discoveries of HCV-related hepaciviruses prompting the question of a more general miR-122 dependence. Among hepaciviruses, the closest known HCV relative is the equine non-primate hepacivirus (NPHV). Here, we used Argonaute cross-linking immunoprecipitation (AGO-CLIP) to confirm AGO binding to the single predicted miR-122 site in the NPHV 5’UTR in vivo. To study miR-122 requirements in the absence of NPHV-permissive cell culture systems, we generated infectious NPHV/HCV chimeric viruses with the 5’ end of NPHV replacing orthologous HCV sequences. These chimeras were viable even in cells lacking miR-122, although miR-122 presence enhanced virus production. No other miRNAs bound this region. By random mutagenesis, we isolated HCV variants partially dependent on miR-122 as well as robustly replicating NPHV/HCV variants completely independent of any miRNAs. These miRNA independent variants even replicate and produce infectious particles in non-hepatic cells after exogenous delivery of apolipoprotein E (ApoE). Our findings suggest that miR-122 independent HCV and NPHV variants have arisen and been sampled during evolution, yet miR-122 dependence has prevailed. We propose that hepaciviruses may use this mechanism to guarantee liver tropism and exploit the tolerogenic liver environment to avoid clearance and promote chronicity.
Highlights
Chronic Hepatitis C virus (HCV) infection is one of the most common liver diseases with ~71 million people persistently infected globally; a significant number of those will develop cirrhosis or liver cancer [1]
We show that minor changes in the 5’ untranslated region (5’UTR) of HCV and non-primate hepacivirus (NPHV)/HCV chimeras weaken or obviate the need for miR-122 for virus replication
Our data suggest that miR-122-independent hepaciviruses have likely been sampled during evolution, but hepaciviruses may have been selected to utilize miR-122 to restrict replication to the tolerogenic liver environment to help avoid immune clearance
Summary
Chronic HCV infection is one of the most common liver diseases with ~71 million people persistently infected globally; a significant number of those will develop cirrhosis or liver cancer [1]. The binding of liver specific miR-122 to HCV RNA is essential for viral replication [2]. This interaction is unusual in that two molecules of miR-122 bind to the 5’ untranslated region (5’UTR) of HCV using both seed and auxiliary pairing [3,4]. (ii) AGO/miR-122 binding can increase HCV internal ribosome entry site (IRES)-driven translation, promoting the HCV replication [11]. This process possibly works by switching the IRES from “closed” to “open” conformation [12,13]. This process possibly works by switching the IRES from “closed” to “open” conformation [12,13]. (iii) Competition between miR-122 and poly(rC)-binding protein (PCBP2) that binds and circularizes HCV RNA may act as a switch between translation and replication [14]. (iv) In addition, using AGO-CLIP and RNA-seq, we recently showed that HCV RNA can act as a miR-122 “sponge” in a positive feed-back loop to de-repress cellular mRNAs normally targeted by miR-122, thereby indirectly regulating hundreds of genes [15]
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