Researchers from the University of North Carolina this week published new data on the role of microRNA-122 in hepatitis C infection, detailing the specific degradation pathways against which the miRNA protects the virus.
The findings, which appeared in the Proceedings of the National Academy of Sciences, suggest that miR-122 not only protects the HCV genome from enzymatic decay, but has a variety of additional functions that are vital for viral replication.
The importance of miR-122 in the HCV lifecycle was first established in 2005 by UNC's Stanley Lemon and collaborators at Stanford University, and earlier this year he and his team reported that the miRNA slows the decay of HCV RNA by recruiting the Argonaute 2 protein to its 5' UTR.
“These observations point to the importance of RNA decay pathways in HCV replication and suggest that miR-122 is likely to prevent degradation of viral RNA from its 5′ end,” Lemon and colleagues wrote in this week's PNAS.
They have also caught the attention of industry players aiming to use miRNAs as therapeutic targets. For instance, Santaris Pharma has advanced a locked nucleic acid-based inhibitor of miR-122 into phase II testing for HCV (GSN 4/26/2012), while Regulus Therapeutics has teamed up with GlaxoSmithKline to advance its own miR-122-targeting HCV drug, which is still in preclinical development (GSN 2/25/2010).
“However, the specific mechanisms involved in degradation of HCV RNA, or for that matter most positive-strand viral RNAs, have not been well studied and remain unclear. In addition, exactly how miR-122stabilizes the viral RNA remains to be elucidated,” they wrote.
To address these questions, the UNC scientists first looked at the decay of HCV RNA transfected into HeLa cells that do not express endogenous miR-122, specifically examining the role of the 5' exonuclease Xrn1. They found that transfected HCV RNA was degraded rapidly, as expected, but that Xrn1 knockdown significantly stabilized the RNA. Additional experimentation confirmed that the 3' exonuclease exosome complex also contributed to the degradation of transfected HCV RNA.
In order to examine HCV RNA decay under more natural conditions that reflect RNA decay pathways in replicating viral genomes, the team treated HCV-infected Huh-7.5 cells with PSI-6130, a potent and specific nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that blocks viral RNA synthesis.
Inhibiting Xrn1 slowed the rate of HCV RNA decay “impressively,” they noted in PNAS, whereas knocking down the exosome component PM/Scl-100 had no effect. “Thus, replicating HCV RNA is degraded by the 5′ exonuclease Xrn1, but not the exosome complex.”
The investigators added that HCV RNA was significantly stabilized by the addition of miR-122 in control cells where Xrn1 expression was unaffected, but that the miRNA offered no additional increase in RNA stability in cells where Xrn1 had been inhibited. As a result, they concluded that Xrn1 knockdown and miR-122 supplementation have “equal and redundant effects on the stability of RNA, from which we infer that miR-122 enhances HCV RNA stability by protecting it from Xrn1-mediated … degradation.”
Notably, Lemon and colleagues discovered that Xrn1 knockdown does not rescue replication of a viral mutant defective in miR-122 binding.
“That tells us that, in addition to stabilizing the RNA … miR-122 has a second, independent … direct effect on the ability of the virus to replicate,” Lemon told Gene Silencing News this week, adding that experiments detailed in PNAS suggest that is “not due to an increase of the translational activity of the viral RNA.”
Although Lemon said that he has no solid evidence on the nature of this additional role for miR-122, “my guess is that it is doing one of two things. It is either helping facilitate the assembly of the viral replicase … that we know involves several host protein factors … or it is directly involved in RNA synthesis. That could come about by modifying the template RNA on which viral RNA is transcribed.
“But we don't have molecular proof for either of those yet,” he cautioned. “We're looking now whether we can demonstrate directly an enhancement in RNA synthesis, and we have some preliminary results suggesting that does occur.”
Lemon and his colleagues are also using a proteomics approach to look at “exactly what's being recruited to the viral RNA along with miR-122,” he said.