Enhanced virus translation enables miR-122-independent Hepatitis C Virus propagation

Abstract

The 5’UTR of the Hepatitis C Virus genome forms RNA structures that regulate virus replication and translation. The region contains a viral internal ribosomal entry site and a 5’ terminal region. Binding of the liver specific miRNA, miR-122, to two conserved binding sites in the 5’ terminal region regulates viral replication, translation, and genome stability, and is essential for efficient virus replication, but its precise mechanism of its action is still under debate. A current hypothesis is that miR-122 binding stimulates viral translation by facilitating the viral 5’ UTR to form the translationally active HCV IRES RNA structure. While miR-122 is essential for detectable virus replication in cell culture, several viral variants with 5’ UTR mutations exhibit low level replication in the absence of miR-122. We show that HCV mutants capable of replicating independently of miR-122 also replicate independently of other microRNAs generated by the canonical miRNA synthesis pathway. Further, we also show that the mutant genomes display an enhanced translation phenotype that correlates with their ability to replicate independently of miR-122. Finally, we provide evidence that translation regulation is the major role for miR-122, and show that miR-122-independent HCV replication can be rescued to miR-122-dependent levels by the combined impacts of 5’ UTR mutations that stimulate translation, and by stabilizing the viral genome by knockdown of host exonucleases and phosphatases that degrade the genome. Thus, we provide a model suggesting that translation stimulation and genome stabilization are the primary roles for miR-122 in the virus life cycle.IMPORTANCEThe unusual role of miR-122 in promoting HCV propagation is incompletely understood but is essential for an HCV infection. To better understand its role, we have analyzed HCV mutants capable of replicating independently of miR-122. Our data show that the ability of viruses to replicate independently of miR-122 correlates with enhanced virus translation, but that genome stabilization is required to restore efficient HCV replication. This suggests that viruses must gain both abilities to escape the need for miR-122 and impacts the possibility that HCV can evolve to replicate outside of the liver.

MicroRNA-122 Regulation of HCV Infections: Insights from Studies of miR-122-Independent Replication

“…However, several studies on miR-122-independent replication also showed virus replication in Dicer and Drosha knockout cells, which are devoid of miRNAs that rely on the canonical miR-122 biogenesis pathways. HCV-S2-GGCGUG which has been reported previously for replicating in the absence of miR-122 was capable of replicating in Dicer KO Huh 7.5 cells, and several mutants were shown to replicate in Drosha knockout cells [19,65]. Additionally, another study showed that Ago2 interaction with HCV genome was not detected for miR-122independent replication of G28A, suggesting that miR-122 independent replication of HCV is not because of the binding of other microRNAs to the 5 UTR of the viral RNA and no other microRNA compensate for miR-122-independent replication of G28A HCV [71].…”

“…Further, knocking down of RNA degrading enzymes rescued replication of several mutants, some to miR-122-dependent levels, suggesting that the roles of miR-122 can be compensated by enhancing virus translation and stabilizing the viral genome. Experiments also demonstrated that enhanced translation rescued viral replication by approximately 100-fold, whereas enhanced stability rescued viral replication approximately 10-fold, and suggested that the major role for miR-122 is translation stimulation with stabilization being important but less potent [65].…”

“…Thus, it was proposed that 5 RNA structure may be dynamic and that mutations that allow for miR-122-independent replication shift the folding equilibrium toward that of the active IRES even in the absence of miR-122 [51]. In support of this, there was a correlation between the mutation induced enhancement of translation and the efficiency of miR-122-independent replication for the HCV mutants capable of miR-122-independent replication [53,65]. Further, knocking down of RNA degrading enzymes rescued replication of several mutants, some to miR-122-dependent levels, suggesting that the roles of miR-122 can be compensated by enhancing virus translation and stabilizing the viral genome.…”

“…DUSP11 has also been reported to directly act on the 5 end of HCV RNA rendering it susceptible to Xrn1-mediated degradation [63]. Further, depletion of all three enzymes, Xrn1, DOM3Z, and DUSP11, rescued HCV replication in the absence of miR-122 [62], however, knockdown of Xrn1, DOM3Z, and DUSP11 did not reinstate HCV replication to an miR-122 dependent level and suggests that miR-122 mediated protection of viral RNA is not the only mechanism by which miR-122 promotes viral propagation [62,65].…”

“…These mutants were generated by serially passaging HCV in miR-122 knockout Huh 7.5 cells expressing a mismatched miRNA [104] or through siRNA-knockdown mediated mutagenesis of the miR-122 binding region [51]. HCV mutations reported to provide an advantage to viral replication in the absence of miR-122 were identified in both miR-122 seed binding sites 1 and site 2 (C26G, U25C mutants, C37U, and A38U), miR-122 auxiliary binding site 1 [U4C], the nucleotide sequence between site 1 and site 2 (G28 mutants, G28U, G28C, G28DEL, G28A/A34G, C30U/A34G), and a combination of mutations present in Auxiliary site1, seed site 1, and seed site 2 (U4C/G28A/C37U) [51,65,71,104]. Another full-length HCV mutant reported to replicate independently from miR-122 was HCV-S2-GGCGUG, selected from a population of viral variants having all possible sequences at S2 [19], and in another report complete mutation of miR-122 binding site 2 to GUGAGG (HCV-S2C-GUGAGG) can also allow the virus to replicate independently of miR-122 [65].…”