Hepatitis C virus double‐stranded RNA is the predominant form in human liver and in interferon‐treated cells

Abstract

Hepatitis C virus (HCV) is unique among RNA viruses in its ability to establish chronic infection in the majority of exposed adults. HCV persists in the liver despite interferon (IFN)‐stimulated gene (ISG) induction; robust induction actually predicts treatment failure and viral persistence. It is unclear which forms of HCV RNA are associated with ISG induction and IFN resistance during natural infections. To thoroughly delineate HCV RNA populations, we developed conditions that fully separate the strands of long double‐stranded RNA (dsRNA) and allow the released RNAs to be quantified in reverse transcription/polymerase chain reaction assays. These methods revealed that dsRNA, a pathogen‐associated molecular pattern (PAMP), comprised 52% (standard deviation, 28%) of the HCV RNA in the livers of patients with chronic infection. HCV dsRNA was proportionally higher in patients with the unfavorable IL28B TT (rs12979860) genotype. Higher ratios of HCV double‐stranded to single‐stranded RNA (ssRNA) correlated positively with ISG induction. In Huh‐7.5 cells, IFN treatment increased the total amount of HCV dsRNA through a process that required de novo viral RNA synthesis and shifted the ratio of viral dsRNA/ssRNA in favor of dsRNA. This shift was blocked by ribavirin (RBV), an antiviral drug that reduces relapse in HCV patients. Northern blotting established that HCV dsRNA contained genome‐length minus strands. Conclusion: HCV dsRNA is the predominant form in the HCV‐infected liver and has features of both a PAMP and a genomic reservoir. Interferon treatment increased rather than decreased HCV dsRNA. This unexpected finding suggests that HCV produces dsRNA in response to IFN, potentially to antagonize antiviral defenses. (Hepatology 2017;66:357–370).

Reticulon-3 modulates the incorporation of replication competent hepatitis C virus molecules for release inside infectious exosomes

“…RTN3 has been shown to interact with HCV NS4B required for the formation of the membranous web that is obligatory for HCV replication [ 29 , 40 , 41 ]. However, the interaction between RTN3 and dsHCV RNA the predominant form in human liver and interferon‐treated cells [ 42 ] has not been demonstrated. Given our interest in deciphering the role of RTN3 in infectious exosomes loading, we assessed if RTN3 interacted with dsHCV RNA.…”

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes

“…Interestingly, we could detect significant amounts of negative-strand RNA in all sera, albeit at far lower and highly variable ratios compared with cell culture supernatants ( Figure 6E), indicating that serum predominantly contains infectious virus and that most dsRNA secreted in EVs does not reach the systemic circulation. Because a previous study observed increased levels of dsRNA in the liver of patients with chronic HCV depending on their IL28B gt, 24 it will be interesting to analyze in future studies whether this correlates with negative-strand levels in the serum.…”

“…We isolated EVs from Huh7-Lunet CD81high cells 18 infected with HCV (strain Jc1, Figure 1A), which likely also contained copurified infectious virus due to similar biophysical properties 10 and investigated the composition of HCV RNA in these vesicles relative to intracellular viral RNA by use of a strandspecific reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) assay (Supplementary Figure 1A and B). Because negative-strand RNA was used as a quantitative marker for dsRNA, we ensured that our assay reached a sensitivity of minus-strand detection comparable to a previously published protocol 24 (Supplementary Figure 1C). We found substantial amounts of negativestrands in the EV fraction of infected cells, with a far lower relative ratio of positive-to negative-strands than intracellularly (3.8 versus 29.8, respectively [ Figure 1B]).…”

Occult Hepatitis C Virus Infection: Are We Digging Too Deep?

“…11 A recent report suggests that HCV-positive and -negative RNA strands remain connected to form doublestranded RNA (dsRNA). 12 This dsRNA is copied multiple times by semiconservative replication to generate multiple progeny; particularly, positive-strand viral RNA genomes are present 5-to 10-fold molar excess over negative-strand RNA. 13 Excess positive-strand HCV RNA ensures efficient viral protein translation, viral genome encapsidation and virion production.…”

“…An alternative hypothesis would be that HCV dsRNAs remain protected inside the double membrane vesicles, even after a successful antiviral treatment, serving as genomic reservoirs not recognized by the endogenous viral-sensing pathways. 12 However, it has been shown recently that NS5A inhibitors (which are components of most of the direct-acting antiviral regimens) block early biogenesis of HCV-induced double membrane vesicles in vitro, 15 which would leave dsRNA finally exposed to host innate defense mechanisms. If this is the case in vivo, HCV RNA would persist in a yet unknown cell compartment.…”