Researchers map microRNA’s role in the human hepatitis c infection.


In the battle between a cell and a virus, either side may resort to subterfuge. Molecular messages, which control the cellular machinery both sides need, are vulnerable to interception or forgery. New research at Rockefeller University has revealed the unique twist on just such a strategy deployed by the liver-infecting Hepatitis C virus, one that may help explain the progression of liver disease and that the researchers suspect may be found more widely in the world of disease-causing viruses.  The opensource study is published in Cell.

The current study employed a powerful combination of techniques to map the interactions between the virus and a small piece of genetic material, known as miRNA-122, that is produced almost exclusively by liver cells, which normally use it to regulate expression of their own genes.

It is well known that once inside a liver cell, the hepatitis C virus must bind to miRNA-122 in order to establish a persistent infection. The researchers found an unanticipated consequence of this interaction, by binding to miRNA-122, the virus acts like a sponge, soaking up these gene-regulating molecules.  The experiments showed this has the effect of skewing gene activity in infected liver cells.

The team explain that the fight between an infecting virus and its host is often viewed as proteins fighting like soldiers. And soldiers on both sides must have orders, in this case the genetic information responsible for the production of proteins. This is where miRNA-122 comes in. It is a microRNA, a type of RNA encoded into the genome for the purpose of turning down the expression of genes. It does this job by guiding a complex of silencing proteins to an RNA transcript of a gene so as to prevent it from being turned into a protein. In this way, miRNA-122 appears to help control a number of normal functions, including cholesterol and iron metabolism, as well as circadian rhythms.

However,  miRNA-122 is also necessary for hepatitis C virus. Once in a liver cell, the viral RNA binds with miRNA-122, which stabilizes and protects the virus so it can replicate. Over the long term, the hepatitis C virus infections can lead to scarring of the liver and liver cancer.

In order to explore how the hepatitis C virus and miRNA-122 interact and the repercussions for the host liver cells, the researchers used a technique known as cross-linking and immunoprecipitation (CLIP), developed over the past 12 years by the lab, which was targeted to find Argonaute, one of the proteins involved in silencing. This way, the lab captured the miRNA-122 – Argonaute complexes and the sections of RNA transcript to which they bound. They then sequenced those RNA transcripts to see what genetic instructions they represented.

The team state that one microRNA can have hundreds of targets, but most often studies are driven by anecdotes focused on single interactions. By combining CLIP and RNA sequencing the researchers were able to take a global perspective and map out all of miRNA-122’s interactions across both the viral and infected host genomes.  The result is described as a rigorously constructed picture of what is actually going on in the cell.

Their maps showed a peak in miRNA-122 binding at one end of the viral genome, confirming previous work; miRNA-122 also interacted with the virus in a number of other places, for which the significance is not yet known. When the researchers looked at miRNA-122 interactions in an infected cell, they found lower levels as compared to uninfected cells. What’s more, a look at the expression of genes regulated by miRNA-122 confirmed this microRNA was less active because those genes had higher levels of activity.

The team theorise that chronic low levels of miRNA-122 many prompt changes that, over years, contribute to liver damage and cancer.  This could be a molecular link between the viral infection and the pathologies associated with hepatitis C adding that more work on miRNA-122 targets in hepatitis C-infected patients may clarify why some go on to develop liver cancer and others don’t.

The hepatitis C virus isn’t the only virus known to alter gene expression in host cells by sponging up their microRNAs. Human cytomegalovirus and herpesvirus saimiri, which infects New World monkeys, employ a similar strategy, producing RNAs specifically to bind up host microRNAs. But, unlike the others, the hepatitis C virus must bind to a microRNA to replicate, and when it does so, the new viral genomes sop up even more miRNA-122, creating a positive feedback.

The team suspect these three cases may just be the tip of the iceberg, that other viruses, perhaps those that replicate much more ferociously, may use similar microRNA-sponging strategies.  The researchers summise that the techniques that were used in the current study will make it possible to investigate the use of this strategy in an unbiased way.

Source:  The Rockefeller University

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5′UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.  Hepatitis C Virus RNA Functionally Sequesters miR-122.  Darnell et al 2015.

Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5′UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV. Hepatitis C Virus RNA Functionally Sequesters miR-122. Darnell et al 2015.

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