There is a surprising new role for a microRNA in the accumulation of hepatitis C virus in the liver, a study in
While previous work has shown that microRNAs can cleave mRNA or repress its translation by binding with mRNA's 3' noncoding region, this is the first time such a molecule has been found to interact with the 5' region in an animal cell, or to positively regulate gene expression. "In the last couple of years, many people have found new and varied forms in which miRNAs can interact with the replication of animal viruses. This is a new and quite different example," said John Taylor, of the Fox Chase Cancer Center, who did not participate in the study.
The human genome contains about 800 genes that code for microRNAs. The authors chose to look at miR-122, which accounts for 70% of microRNAs found in the liver, to see if the liver-based RNA virus hepatitis C might be using the host microRNA "for its own good," said Sarnow.
To determine if miR-122 is needed to regulate hepatitis C gene expression, the researchers sequestered miR-122 by transfecting complementary oligonucleotides in Huh7 liver cell lines expressing a hepatitis C RNA replicon. They found that the amount of hepatitis C RNA was reduced by about 80% when miR-122 was inactivated. "So we knew the miRNA is important for hepatitis C abundance, [but we] didn't know whether it was a direct interaction or whether the miRNA interacts with a cellular target," said Sarnow.
They next introduced mutations into the two potential miR-122 binding sites in the noncoding region of the viral RNA genome. While mutations at the 3' noncoding region binding site had no effect on hepatitis C RNA accumulation, they found that RNA with mutations in the 5' noncoding region binding site failed to accumulate. But when they ectopically expressed miR-122 with the corresponding 5' mutations in these cells, levels of hepatitis C RNA were restored, suggesting that the putative base-to-base binding interaction between the miRNA and its mRNA target had been rescued and arguing against the possibility that the 5' mutation lowered RNA levels by causing misfolding.
From this result, which Taylor called "the most convincing evidence" in the study, Sarnow and his team concluded that there is a direct genetic interaction between miR-122 and hepatitis C that is essential for the virus to replicate.
Because other miRNAs are known to reduce the translational efficiency of their mRNA targets, the researchers then evaluated amounts of core protein translated from viral RNA with and without miRNA binding sites; the results suggest that miRNA regulates viral RNA at the replication level.
The mechanism for this new miRNA role is still very much unclear, though Sarnow speculates that miR-122 may recruit the viral RNA into a replication complex or help in RNA folding. He plans to use colocalization experiments to investigate the nature of the interaction.
According to the paper, hepatitis C mRNA can also replicate in non-liver cells, such as those in the kidney and cervix. "It's a puzzle that hepatitis C needs miR-122 when grown in Huh7 cells but doesn't need it when it's grown in those other cells," said Taylor. Sarnow pointed out that "that kind of makes sense" because initial infection with hepatitis C can occur through peripheral cells before the virus reaches the liver.
Scientists "could potentially design ways to manipulate this miRNA to prevent virus replication in [the] host," said Jian-Kang Zhu, at the University of California, Riverside, who did not participate in this study. Sarnow said it will also be important to determine the normal function of the highly abundant miR-122 in the liver, particularly if the microRNA is targeted for antiviral therapies.