QB3 researchers probe details of how hepatitis C hijacks cells

By Robert Sanders, UC Berkeley Media Relations

The hepatitis C virus has found a clever way to hijack the body's cells and even make an end-run around defenses that cells throw up to stop its spread, according to new research by QB3 scientists.

While revealing the inventiveness of viruses, the finding also is leading the researchers to probe ever finer details of the process so they eventually can develop drugs to prevent the virus from taking over and establishing an infection.

"The goal of this work is to get enough detail on the mechanisms involved to design a drug to prevent the disease," said biochemist Jennifer Doudna, professor of chemistry and of molecular and cell biology at UC Berkeley and a Howard Hughes Medical Institute investigator.

"This is basic research, telling us how hepatitis C acts," added biophysicist Eva Nogales, associate professor of molecular and cell biology and also a Howard Hughes Medical Institute investigator. "The more we learn about how it does things, the easier it will be to block it without interfering otherwise with the normal mechanism of the cell."

The work by Doudna and Nogales, both QB3 affiliates and faculty scientists at Lawrence Berkeley National Laboratory (LBNL), appeared in the Dec. 2 issue of Science.

Scientists have known that the blood-borne virus hepatitis C and numerous other viruses - including the flu virus, polio, hepatitis A and the human immunodeficiency virus (HIV) - take over the machinery inside the cell that produces proteins and use it to make copies of themselves. Specifically, these viruses hijack the ribosome, a huge molecular complex that manufactures proteins based on a blueprint called messenger RNA (mRNA). The viruses not only force the ribosomes to manufacture viral proteins, they also suppress production of the cell's own proteins.

Moreover, hepatitis C does this in a way that circumvents the normal protein production process, so that even when the cell tries to shut down protein manufacturing by the ribosomes to fight the virus, the virus is able to hot-wire the ribosomes.

Doudna, Nogales and their colleagues found out how this happens, using a technique called cryo-electron microscopy to provide detailed pictures of the RNA and protein complexes involved.

The ribosome is composed of two pieces that must clamp together around a strand of mRNA in order to begin their work. In normal cells, a protein called eIF3 (eukaryotic translation initiation factor 3) latches onto one half of the ribosome, and together they scour the cell interior for an mRNA with a tag indicating it's legitimate. Only after this complex finds an mRNA with the proper tag, called a binding cap, does the other half of the ribosome come in to complete the ribosomal machine and start translating the mRNA into protein.

When hepatitis C invades a cell, however, a large piece of viral RNA called IRES (internal ribosome entry site) gloms onto eIF3 in imitation of the mRNA binding cap, essentially convincing the ribosome that it is one of the cell's own mRNAs. The viral mRNA, which lacks the binding cap of a cell's normal mRNA, is then able to feed through the machine to create its own proteins. The virus is thus able to get around the need for many other control proteins that are part of the well-regulated transcription process in the cell. For example, the binding cap protein that recognizes the binding cap tag on mRNA is no longer needed; the viral IRES substitutes for both the binding cap and the binding cap protein.

"It was known that the hepatitis C virus uses this IRES RNA in order to overcome the normal requirement for a very large number of cellular factors," Nogales said. "What was not known is how it did it and how it is able to overcome this requirement. What we have seen is that, basically, it does this by displacing the cap binding protein that normally selects the cellular mRNAs. That is the way it competes with the normal cellular mechanism and kidnaps the basic machinery for its own purpose."

This explains how, even when the cell tries to use these regulatory proteins to shut down the ribosome to stop infection, the hepatitis C virus is able to hijack the ribosomes anyway. Though other RNA viruses that target the ribosome may use this same technique, Doudna suspects that "nature has probably found a number of ways to skin the cat."

Nogales and Doudna revealed these details through cryo-electron microscopy, or cryo-EM, a technique Nogales has perfected that allows her to get images of large molecules in a liquid environment like that inside the cell. This contrasts with X-ray crystallography, where it is necessary to make a molecule condense into a hard crystal before it can be imaged.

"Cryo-EM is good because we don't have to crystallize the molecule, so we can deal with conformational flexibility, which is very important," Nogales said. "All these protein machines are moving, they have moving parts. Because we are not limited by having to crystallize our complexes, which really paralyzes them, we are able, with some ingenuity, to detect and describe the flex inherent to the complexes of the components. It is looking at complexes as they are in the cell."

The newly discovered details of how viral IRES latches onto the cell's normal eIF3 could lead to ways to foil this masquerade. Doudna, who has worked on RNA enzymes for most of her career and has collaborated with Nogales for nearly three years on this ribosome research, noted that small, single-nucleotide changes in the viral IRES RNA are known to inhibit binding to eIF3.

"Knowing how effective a small change in sequence can be means that it may be easy to find a small molecule that has a similar effect," Doudna said. "It's reasonable to think we might be able to come up with a small-molecule drug to inhibit IRES binding to the ribosome on eIF3."

At the moment, the team doesn't have sufficient detail about the viral-ribosome interactions, but it is working to improve the resolution of its cryo-EM models from 30 Angstroms to about 10 Angstroms. This would allow the researchers to see secondary protein structures which would give them a better understanding of the chemistry behind eIF3's interactions with other proteins and with RNA.

Co-authors with Doudna and Nogales are graduate student Bunpote Siridechadilok and post-doctoral researcher Christopher Fraser of UC Berkeley, and post-doc Richard Hall of LBNL. The work was supported by the Howard Hughes Medical Institute.