Characterization of miR-122-independent propagation of HCV

Abstract

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV 122KO , possessing G28A substitution in the 5'UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV 122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocytederived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. Author summaryA liver-specific microRNA, miR-122, is one of the key determinants of hepatitis C virus (HCV) hepatotropism and is required for efficient propagation of HCV. On the other hand, chronic infection with HCV is often associated with extrahepatic manifestations (EHMs), and a low level of HCV-RNA replication has been detected in some non-hepatic cells. Nonetheless, the detailed mechanisms underlying these phenomena remain unknown. Here, we show that miR-122 is dispensable for low-level replication or infectious particle formation, and a mutant virus adapted to miR-122-knockout cells exhibited efficient but miR-122-independent propagation. The adaptive virus of HCV genotype 2a possessed a G28A substitution in the 5'UTR and facilitated efficient replication complex formation under an miR-122-deficient condition, while it propagated at a level comparable to the wild type HCV in the presence of miR-122. Moreover, various adaptive mutations including C30U were introduced into genotype...

Various miRNAs are involved in efficient HCV replication

“…miR-6880-5p was identified as a genotype 1b specific non-miR-122 type miRNA (S6A Fig). To examine the enhancement of replication of genotype 1b HCV by miRNAs, 751-122KO cells transfected with mimics of miRNAs (S6A and S6B Figs) were infected with chimeric HCV of genotype 1b Con1 strain and genotype 2a JFH1 strain (Con1C3/JFH) [23]. Among the miRNAs we examined, transfection of mimics of miR-504-3p and miR-574-5p exhibited slight but significant enhancement of replication of the chimeric HCV compared to control mimics, in contrast to large enhancement by that of miR-122 (S6C Fig).…”

“…For a rapid identification of 5’UTR sequence of HCV, RNAs extracted from 100 µl of virus-containing supernatants or PBMCs were amplified by using a PrimeScript ® RT reagent Kit (Perfect Real Time) (Takara Bio) and 5’RACE was performed by using a 5’RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Life Technologies) as described by Ono et al [23].…”

“…Interestingly, the G28A mutation does not occur under miR-122-abundant conditions. Moreover, in clinical samples obtained from patients infected with HCV genotype 2, the rate of G28A mutation was higher in miR-122-deficient peripheral blood mononuclear cells (PBMCs) than in sera that mainly included hepatocyte-derived HCV [23]. Therefore, in this study, we examined the possibility of HCV-RNA replication in miR-122-deficient cells, such as non-hepatic cells, and of participation of miRNAs other than miR-122 to support replication of HCV-RNA.…”

Impact of a Patient-Derived Hepatitis C Viral RNA Genome with a Mutated MicroRNA Binding Site

“…Curiously, the C3U mutation has not been observed during selection experiments in cultured cells [17, 24, 25] and no naturally occurring C3U HCV genotypes have been deposited into Gene bank. Ono et al observed a viral G28A mutation that arose in the serum and peripheral blood monocytes of type 2-infected patients [25], however, the C3U variant has only been observed in patient serum after several weeks in anti-miR treatment, and only transiently. Whether the extrahepatic C3U genomes reflect growth of the C3U variant in the liver of re-bounding patients is not known, because liver biopsies are not available.…”

High-Throughput Sequencing of Putative Novel microRNAs in Rhesus Monkey Peripheral Blood Mononuclear Cells following EV71 and CA16 Infection

“…Moreover, a growing body of evidence has demonstrated that miRNAs can play a key role in the regulation of viral replication. For example, the liver-specific miR-122 interacts with the 5′-UTR of the genome of the hepatitis C virus (HCV) and increases viral replication [12]. The H1N1 influenza A virus-induced miR-323, miR-491, and miR-654 inhibit viral replication by directly binding to the PB1 gene [13].…”