New tool to figure out what the flu virus does (and what it needs to do it)

In my regular science trawling I noticed a fascinating paper in Nature (epub ahead of print) that I haven't seen anything about in the news. It seems to me it's worth a discussion, if for no other reason than it uses a relatively new approach, small interfering RNA (siRNA), to dissect the functions in the host cell the virus needs for the only thing it wants to do, make a copy of itself. It also lets me try out on you a new analogy I cooked up for a short talk on flu for high school students and their parents and teachers. It turns out that parts of it will be useful to explain the new siRNA paper.

For the talk I used the example of their school, but consider instead your office or workplace. If you landed here from a distant galaxy and knew nothing about life forms on earth, an objective description of what goes on in your office might look like some kind of complex organism. It has various subunits, like the boss's office, the lunchroom and the mail room and these organelles use physical and chemical mechanisms like telephones, computers and xerox machines to do routine operations. As your office goes about its daily business it uses energy and it continually changes and develops, as goods and materials and people go in and out. It may grow and occupy new space, or more likely in this economy, shrink. The various operations of your office and the outside world have to be coordinated, too, and that requires a communication system. Our visitor from space might be tempted to say the office was alive. Let's put you inside this organism.

Suppose your job is to xerox things for distribution and one day the mail room brings you an 8 x 10 business envelope addressed to "Resident, Your Town." You open it up and inside there is a set of instructions marked Urgent: do this first. There are four instructions. (1) Make a copy of the instructions. (2) For each copy go to the supply cabinet and get an 8 x 10 business envelope just like the one the instructions came in and put the copy into it. (3) Put that new copy-containing envelope in Outgoing Mail with the address "Resident, Your Town" written on it. (4) Do it again.. So you start to do this but since there is no stop command, you keep doing it and meanwhile all the rest of the necessary work of the office gets backed up and things start to go badly wrong. You use up all the paper and envelopes so there is nothing left for things the office really needs to do.

What I've just described is both what a virus does, and pretty much what a virus is: genetic material with instructions inside a protein envelope. The virus isn't alive, at least in the usual sense. It just does one thing. Hijack the host cell's xerox machine and tell it to make a copy and put it in an envelope in the outgoing mail where it will wind up in some other office and create the same kind of problem there. And so on.

While I described things in very broad outline, in reality all the steps require substeps that are so familiar we tend not to mention them. You have to go to the mail room (that means traveling from one place to another and knowing where to go by using landmarks), you have to have permission to pick up the mail, you have to literally pick it up, carry it back, open the envelope, take out its contents, move it to the xerox machine, open the flap on top, place the instructions on it, push the button, etc., etc., then go to the supply cabinet and get a new 8 x 10 envelope and put the copy in it. You get the idea. Lots and lots of little sub steps. If you interfere with some of those substeps (for example, you don't have an opener for the envelope) you interfere with the attempt to hijack the office xerox machine.

What's this got to do with the Nature paper? Researchers in Germany (Berlin, Hannover, Würzburg) were thinking about a different way to attack the influenza virus. Instead opf going after the virus itself, they decided to interfering with the "office tools" in your cell. Offhand that sounds like a dangerous thing, because it is attacking the host, but there are a lot of things your cells do that it can get along without for a while, either because they aren't needed all the time or there are other tools that a handy office worker can substitute or adapt for the job. There's a lot of redundancy. In other words, the strategy was to alter the host rather than the virus. If you think about it, that's just what we do when we go to bed when we are sick or put a cast or splint on an injured limb. When we are healed, we get up or take it off.

The trick here is to recognize what office tools the virus needs. There could be tens or hundreds of them. That's where genome-wide RNAi (RNA interference, the more general name for what siRNA does) comes in. The genome-wide part means that they were screening tens of thousands of genes from all over your genetic blueprint. They weren't targeting one gene or even one chromosome. They were in effect sampling a large proportion of the whole shebang. The other part of the technique, the small interfering RNAs, are just what their name implies: small lengths of RNA that interfere with the operation of making new protein in the cell. If you remember your elementary biology, your cell's DNA (in the chromosomes located in the nucleus) transcribes its information into messenger RNA which in turn goes out into the cell and operates the levers of the protein making machinery according to the DNA instructions. There are tens of thousands of genes that are making protein and they need to be coordinated and controlled. siRNA is part of the apparatus for doing that, stopping messenger RNA function even after it has been made in the nucleus but isn't needed at a particular moment or place.

Scientist have made huge "libraries" of siRNA that interfere with the RNA made by tens of thousands of different genes, some of whose functions we understand some which we don't. What the German scientists did was take cells from the human lung and incubate them with individual items from this huge library of siRNAs. This stops the function of the RNA made by whatever gene the siRNA was matched with (I'm leaving out a lot of technical details that aren't important for the main idea). After a suitable lapse of time they then inoculated the cells with influenza virus (a regular lab strain, H1N1/PR8; pandemic H1N1 swine flu; a seasonal flu like H1N1; and bird flu, H5N1, all in various experiments). They were looking for which genes (which "office tools" like the envelope opener) the flu virus needed to make copies of itself and they found several hundred that prevented one or another of the flu viruses from replicating. They also found a number that prevented all the influenza A viruses they tried (human, avian, swine) from replicating well yet where temporary interference didn't seem to harm the cell.

When they checked the genes involved, they mostly made sense from what was known about influenza biology, although this was a much broader net than previous studies. The value of this work is two fold. The first is that it is a new tool to tease apart the delicate mechanism of flu virus replication. The second is that it identifies the bottle necks and reveals new targets for drugs that might work against a wider range of flu viruses and perhaps other non-flu viruses.

It's nifty work, but one suggestion the authors make I don't quite understand:

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. (Karlas et al., "Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication", Nature advance online publication 17 January 2010 | doi:10.1038/nature08760 [cites omitted])

It's not obvious to me why the virus can't as easily mutate in ways to adapt to a missing "office tool" as to a drug that affects an important viral function. Whether that's the case or not, this is still extremely interesting work and is another powerful item in our tool box for figuring out how this wily adversary does its mischief.

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It's not obvious to me why the virus can't as easily mutate in ways to adapt to a missing "office tool" as to a drug that affects an important viral function.

I think the fundamental difference is that in the latter case, the virus needs just modify an already-present function; but if the tool is missing altogether, the virus would have to develop a whole new function from scratch, an altogether more difficult task.

That's not to say that a virus could not do it -- we see examples of this all the time, with viruses that have co-opted host functions and in some cases even host genes. But the process is usually much slower; we tend to recognize those events in hindsight, whereas we can often watch viral genes adapting to drugs in real time.

iayork: Could be. Thanks for the suggestion. I really didn't know why they said this and your explanation seems plausible to me.

An interesting antiviral host-cell approach was published in the Dec 2008 Nature Medicine. The target: exposed phosphatidylserine (PS). Normally sequestered to the inside of a cell's plasma membrane, PS becomes exposed on virus-infected cells, and on enveloped viruses themselves. Further, exposed PS is now understood to be a fundamental initiator of non-inflammatory phagocytosis and upregulation of anti-inflammatory cytokines (primarily TGF-beta and IL-10). Could viruses be exploiting the ancient and silent clean-up of apoptotic cells?

the paper:
Targeting Inside-Out Phosphatidylserine as a Therapeutic Strategy For Viral Diseases, Nat Med. 2008 December; 14(12): 1357â1362.

MT: sounds interesting. I'll take a look when I come up for air from grant writing.

this approach has been used to study multiple other cell processes (like cell cycle regulation, apoptosis, cell signaling etc.) as well as other viruses (HIV, HCV Dengue virus, and others), including flu. if you're interested, you should check out the Dec 24 issue of Cell. there are two papers in there that use the same approach to identify host factors utilized by influenza.