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Q&A: Proteomic Screening Reveals Smallpox Virus Shuts Down Inflammation Pathway

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This article originally ran on May 27.

McFaddenpic.JPGName: Grant McFadden
Position: Professor, department of molecular genetics and microbiology, University of Florida, College of Medicine, 2006 to present
Background: Member, University of Florida Shands Cancer Center, 2006 to present; member, University of Florida McKnight Brain Institute, 2006 to present; director, Emerging Pathogens Initiative, University of Florida, College of Medicine, 2006 to 2007

Smallpox, historically among the most deadly diseases, was blamed for about 300 million deaths worldwide in the 20th century alone, according to some estimates.

In 1980 smallpox was declared eradicated by the World Health Organization and today only two sites worldwide are known to have live samples of the virus, the Centers for Disease Control and Prevention in Atlanta, Ga., and the State Research Center of Virology and Biotechnology in Koltsovo, Russia.

After the terrorist attacks of Sept. 11, 2001, however, fear of the virus reentered the public arena regarding its potential use as a bioterror weapon. The US government reacted by initiating programs targeted at trying to decipher the smallpox virus and possibly developing new vaccines and a treatment, which currently doesn't exist.

One company that received a grant to study the virus was Myriad Genetics in Salt Lake City, who has been collaborating with researchers at the University of Florida, the University of Alberta, WHO, and CDC on its work.

Last week the research team published a study in Proceedings of the National Academy of Sciences describing the identification of a virus-host protein interaction that disables a first line of defense in the human body, inflammation.

The study, according to the authors, is the "first systematic protein interaction screening" of the virus using yeast two-hybrid screening against "a variety of human cDNA libraries." Several protein-protein interactions were identified in their work, including one between variola G1R, an ankryin/F-box containing protein, and human nuclear factor kappa-B1/p105, representing "the first direct interaction between a pathogen-encoded protein and NF-kappa B1/p105," the authors said in the PNAS article.

This week ProteoMonitor spoke with Grant McFadden, the corresponding author of the study and a professor in the department of molecular genetics and microbiology at the University of Florida. Below is an edited version of the conversation.

Was the motivation behind this work the threat of the smallpox virus as a bioweapon?

About five, six years ago, after 9/11, the US government and NIH decided they wanted to investigate certain specific pathogens that were particularly worrisome for biodefense purposes.

They wanted to build up our understanding of some key pathogens, [including] smallpox and monkeypox viruses. The reason for that is smallpox is probably one of the most feared pathogens in history, yet we understand almost nothing about what it does or how it causes disease. And we have no drugs to treat it.

NIH made the decision to fund a number of rather large projects to different bidders … and the one I was involved in was a project that was given to … Myriad Genetics. … The reason they got a project to study a number of different pathogens, including smallpox, was they had a large army of robots that could do … yeast two-hybrid screening.

The project I was involved in and that this paper came from was a study that Myriad did looking at proteins that are made by smallpox virus and asking which of those proteins in a yeast cell can contact proteins from humans. The purpose of this was to try and understand better how smallpox virus is able to dismantle the human immune system.

As a result of this, and after many thousands of screenings with Myriad robots, a number of hits were detected, which is the subject of this paper. And one of those hits was for a viral molecule touching elements of the human inflammatory cascade called NF-kappa B, a signaling cascade.

And what we did in that paper was show that this particular interaction looks to be real and when this viral protein touches cells that have this pathway, it shuts that pathway down. The virus has developed its own anti-inflammatory drug against this particular pathway and our paper reported on the mechanism of that inhibition.

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Can you describe this mechanism?

You have to remember the inflammatory system is what we do to try and protect ourselves from danger, damage, and infection. It's sort of the first line of defense that humans have against infections.

So one of those pathways, they're called innate pathways, is the NF-kappa B signaling pathway. Basically, it's like an alarm bell in human cells … when it goes off, [it] tells the cells to start producing defensive cytokines and to go into defense mode.

This smallpox protein gets in and it dismantles that particular alarm bell in a novel way. And it does so by touching one of the elements of the cascade and prevents it from activating.

How unique is this? Have you or others seen similar types of mechanisms in other diseases?

People have seen other pathogens inhibit this pathway, but this is the first report of a pathogen going after this particular sensor, it's called the NF-kappa B1/p105.

We already have a vaccine for smallpox, right?

We have a vaccine and, in fact, the US stockpiles the vaccine. However, it's not a drug and it's actually a very dangerous vaccine. It has a very high complication rate. For a while the US was prophylactically vaccinating first responders. …They stopped doing it because of the secondary complication rates.

What sort of complications are we talking about?

Historically, there's a series of complication rates that come from the vaccine, and some of them have been well-known for a long period of time. For example, people who are immuno-suppressed or who have eczema can get very serious complications that can lead to death.

Is the ultimate goal of this current work into smallpox to develop new, safer therapies and vaccines?

Some people are looking at new categories of vaccines and new categories of drug therapeutics. This particular study was more basic: it was to try and understand how this virus causes disease and why it is so potent in human beings,

So your work is still a few steps away from any new drug development.

Based on this, yes, but it does open doors a little bit more for us to understand why it was such an extreme pathogen of man.

Your paper says some inflammation is part of the body's natural defense system but some inflammation can actually be harmful. Is it clear that this mechanism described in your paper blocks inflammation that is good or inflammation that is not so good?

It's not inflammation that's harmful — it's what pathogens do to the pathway that can be harmful. When these pathways work properly, their job is to protect us from infection. But successful pathogens have learned how to get in there and preemptively disrupt those pathways.

Part of our job is to figure out what are those strategies that are being used, because our idea is that if we understand what the virus is dismantling, we may be able to learn how, in turn, to bolster those pathways.

Do your findings have more of an implication for one strain of smallpox versus another, variola major versus variola minor?

It doesn't really shed much light on variola major vs. minor. Both of those viruses have this inhibitory mechanism.

Do your findings have implications for other diseases?

In some ways, they might. When a pathogen learns to shut off an inflammatory pathway, sometimes we can find other ways of mimicking that for other kinds of purposes.

For example, there are a number of diseases that are associated with hyperactivated inflammation. There are actually quite a few diseases like that, everything from rheumatoid arthritis to atherosclerosis. Now we're learning some tricks from a pathogen about how to shut those pathways off, so we might be able to utilize the lessons from that shut-off to devise clinical strategies to shut them off when diseases are caused by those pathways going wild.

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You used the yeast two-hybrid assay, which is a fairly common method for protein-protein interaction work. Were there any important or notable steps in your work that other people doing protein-protein interaction studies can learn from?

One of the key things we learned is that if you develop a hit, let's say, by yeast two-hybrid, what it shows is that before you can know what it means, you have to validate that hit with a completely different strategy or different mechanism.

But once you validate it, you can begin to study it as a biologic process.

Can you describe any kind of a validation or verification work that you did or are doing?

In this paper, we actually expressed the protein and showed that it would block this pathway. We went beyond just protein-protein interaction, which is really showing that two things stick together, and showed that it has biologic consequence in a real human cell.

You have to do this in order to really know if a hit is real or a false negative.

There have been other protein-protein interaction methods that have been formulated. Have you found any that are particularly interesting to you that may shed light that the Y2H method didn't?

I'm always interested in new technologies of protein-protein interactions and my lab has been exploring a number of them. For example, [we're exploring] protein microarrays; we're exploring using in vivo reconstitutions of fluorescent proteins; we're exploring some technologies like alpha screening, which is a technology for protein-protein interaction using beads.

And I guess you would say that my lab is very interested in being at the leading edge of protein-protein interactions of viruses with human cells.

Are you developing new technologies yourself?

No, we're not really developing new technologies. We're happy to be beta-testers of new technologies.

What are some new ones that interest you?

I would say right now, the protein microarrays and the alpha screenings have turned out to be very useful in looking at hits.

Are the protein microarrays Invitrogen's ProtoArray or some other product?

We're collaborating with Harvard's proteomics facility. They have a technique called NAPPA [or Nucleic Acid-Programmable Protein Array]. We're collaborating with Josh [LaBaer, who had headed the Harvard facility and is now director of Arizona State University's Biodesign Institute's Virginia G. Piper Center for Personalized Diagnostics]. We're actually adapting his method.

Have you been able to detect other kinds of interactions aside from the one described in the PNAS paper that you will be following up on?

Yes … for example, no one can work with smallpox virus. It's a very difficult virus to work with; it's very dangerous, and there are only two places in the world that are allowed to work with live virus.

We're very interested in this family and so we've been studying its related family members in two other viruses that we can work with in the laboratory. One of them is a virus called cowpox.

Cowpox has a very closely related version of this protein out of smallpox. We've deleted that gene out of cowpox and we're looking at how cowpox that's lost this particular gene … becomes crippled.

Can you share any findings with me?

We've just written that paper and sent it out, and so I shouldn't comment until the reviewers have signed off on it.

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