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A model of the flu virus showing how the capsid (yellow-green layer) protects the virus RNA. (Image: http://visualscience.ru)





> PERSPECTIVE IN SCIENCE

October 30, 2014

New opportunities for anti-viral intervention


Patrick Matthias from the FMI, together with scientists from the ETH Zurich, has discovered that the influenza virus takes advantage of the cellular process that deals with misfolded proteins, to crack open its capsid. They could show that the viral capsid, by carrying unanchored ubiquitin, mimics misfolded protein aggregates. This form of ubiquitin is recognized with high affinity by a cytoplasmic enzyme, HDAC6, which is therefore recruited to the envelope encasing the viral genome. In addition, HDAC6 binds to cytoplasmic motor proteins, dynein and myosin. These four proteins create a molecular module with HDAC6 at its center. The motor proteins promote movement along microtubules and actin filaments, thereby generating mechanical stress that pulls the viral capsid apart. These unexpected findings have just been published in Science.

Virus infections always follow a similar course: For its own multiplication, the pathogen needs to infiltrate the host cells to use their replication and protein production machinery. The virus overcomes the first barrier, the cell membrane, by docking to the surface, which prompts the cell to engulf the virus in a vesicle and transport it towards the cell nucleus. During this journey, the inside of the vesicle becomes increasingly acidic, which ultimately causes the fusion of the virus’ outer shell with the membrane of the vesicle. In a second step, RNA viruses like the influenza virus have to release their genome, which is packed into a second envelop. This so-called capsid keeps the RNA intact when moving from cell to cell, and protects the viral genome against premature degradation.

Until now, very little has been known about how the capsid of influenza virus is cracked open. A team of scientists from the ETH Zurich, the Friedrich Miescher Institute for Biomedical Research in Basel and the Biological Research Center in Szeged (Hungary) has now discovered how this key aspect of a flu infection works: the capsid of the influenza A virus imitates a bundle of misfolded protein waste, called an aggresome. A cytoplasmic enzyme called histone deacetylase 6 (HDAC6) is central to aggresome formation and subsequent disassembly. By containing unanchored ubiquitin, the influenza virus loosely mimics the aggresome and takes advantage of the HDAC6-dependent machinery to crack open its capsid and boost its infectivity. This discovery has been published recently in the journal Science.

The team of scientists under the leadership of ETH professor Ari Helenius could show that the virus capsid itself carries cellular waste ‘labels’ on its surface, as well as inside. These waste labels, called unanchored ubiquitins, call into action HDAC6, which binds to ubiquitin with high affinity. At the same time, HDAC6 also interacts with scaffolding motor proteins. These motor proteins promote movement in divergent directions along microtubules and actin filaments, generating mechanical stress that pulls the viral capsid apart and releases the virus’ genetic material.

This finding came as a great surprise to the researchers. “This is another important cellular process that the influenza virus hitchhikes for its purposes,” said Patrick Matthias. “The HDAC-6 dependent aggresome assembly and disassembly machinery is essential for taking care of misfolded proteins in a cell. That the influenza virus uses this system to destabilize and open its capsid is exciting news.”

A mouse model in which the protein HDAC6 was absent provided further evidence for the importance of HDAC6 in these processes. In these mice the flu infection was significantly weaker. How these findings might impact therapy remains to be seen, as an absence of HDAC6 in mice moderates the infection rather than prevents it. Although HDAC inhibitors are considered for therapeutic intervention in a wide range of conditions, from cancer to neurodegeneration and autoimmunity, the known HDAC6 inhibitors target its two active sites, which are not involved in the viral uncoating process. This is because catalytic deacetylase activity and ubiquitin binding by HDAC6 are two independent processes. “We would need a molecule that prevents HDAC6 from binding to ubiquitin, not one that inhibits the enzymatic activity,” said Matthias. The 3D structure of HDAC6 in complex with ubiquitin indicates that this is possible, and follow-up experiments are already planned. “Antiviral therapies targeting cellular proteins offer multiple advantages, as they are less prone to mutations in the virus, and might target a broad range of viruses. We are examining how broadly the HDAC6/ubiquitin node is used by viruses beyond influenza”.

Finally, the researchers were also surprised by the time it takes to open the viral capsid : around 20-30 minutes. The total infection period – from docking onto the cell’s surface to the RNA entering the cell nucleus – is two hours. “The process is gradual and more complex than we thought,” said Yohei Yamauchi, a former postdoc with Helenius. He initially detected HDAC6 in a screen for human proteins involved in influenza infections. In follow-up work, Indranil Banerjee, lead author of the study and currently postdoc at the FMI, with critical help from Yasuyuki Miyake from the Matthias lab, determined the nuts and bolts of how the flu virus tricks HDAC6 to open its capsid.

Text based on news release of the ETH Zurich

Original publication
Banerjee I, Miyake Y, Nobs SP, Schneider C, Horvath P, Kopf M, Matthias P, Helenius A, Yamauchi Y. Influenza A virus uses the aggresome processing machinery for host cell entry. Science, published online 24th October 2014 DOI: 10.1126/science.1257037

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