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On Wednesday, the CDC reported that influenza A H1N1 viruses from 13 patients with confirmed diagnoses of swine flu had been tested for resistance to a variety of antiviral drugs. The good news was that all of the isolates were susceptible to the antiviral drugs oseltamivir (Tamiflu) and zanamivir (Relenza). However, all 13 were resistant to adamantane-based drugs (amantadine and rimantadine). Resistance to adamantane drugs (which were developed first) has actually become quite widespread among flu viruses in general, so oseltamivir and zanamivir are commonly the drugs of choice.
The reason for the difference is that the adamantane drugs target different viral proteins from oseltamivir and zanamivir. The two major proteins on the surface of the influenza virus are hemagglutinin (the “H” in H1N1) and neuraminidase (the “N”). Due to the evolution of the influenza virus over time, these proteins come in a variety of different forms, and we label strains of flu by the specific class of hemagglutinin and neuraminidase the virus carries (H1N1, H3N2, etc.). These are the largest proteins on the surface of the virus, and they are the ones that our immune system generally reacts to. However, they aren’t the only ones.
The surface of the influenza virus also has a much smaller protein called M2, which acts as a channel to let hydrogen ions pass across the virus’ outer membrane. M2 plays an important role in viral function (more on that below), and, not surprisingly, so do hemagglutinin and neuraminidase. Specifically, hemagglutinin allows the virus to attach to a cell in order to infect it, and after the virus has replicated within the cell, neuraminidase allows the daughter viruses to detach from that cell and infect other cells. For a more in depth description of these proteins, check out this post at Effect Measure.
Getting back to antiviral drugs, oseltamivir and zanamivir work by inhibiting neuraminidase (for details, once again check out the above post at Effect Measure or this more recent one). The adamantane-based drugs, however, target M2. In January 2008, two research groups independently published atomic-resolution structures of the M2 channel bound to an inhibitor. Using NMR, Jason Schnell (in James Chou’s lab at Harvard) solved the structure of rimantadine bound to M2 (shown above with the drug in red). Alternatively, Amanda L. Stouffer (in William DeGrado’s lab in Pennsylvania) used x-ray crystallography to solve the structure of amantadine bound to M2.
The overall structures were similar, but the two methods detected totally different drug binding sites. Stouffer et al. found the drug directly blocking the pore through which hydrogen ions pass. Schnell et al., however, found the drug bound to the outside of the protein, suggesting an allosteric or more indirect mechanism. Rimantadine and amantadine are very similar in structure, and it is not expected that the two drugs would bind in different sites, so the likely explanation of this discrepancy is that one of the structures is “wrong” (i.e. the drug was binding in a site that was not physiologically relevant or the data was improperly interpreted).
Before I go any further, I should offer a disclaimer: Jason Schnell recently joined my department. I don’t know him well, but we have met and conversed, and I have seen him present his work a few times. Largely because I am more familiar with his work, the rest of this discussion will be based on the Schnell/Chou model of M2 drug inhibition. Beyond that, though, I find the Schnell/Chou model more convincing. Although I am mostly an NMR scientist, I actually tend to believe that x-ray crystallography is a more powerful method for protein structure determination (in fact, I solved the structure of a protein complex by crystallography last summer, and my paper on that structure is currently in review). However, in this case, the NMR-based method that Schnell used to detect drug binding is much more specific and less open to interpretation than the crystallography-based method used by Stouffer. The NMR-based method is also less prone to artifacts and more physiologically relevant since it was done in solution. Schnell and Chou have also further validated their model (most recently in a paper authored by Rafal Pielak). However, Stouffer and DeGrado have also provided additional evidence for their model. I certainly cannot tell you definitively which model is correct, although I’ve told you which way I’m leaning. If you are so inclined, you should take a look at the original papers and decide for yourself. Regardless, the following discussion is based on the Schnell/Chou model.
The M2 channel is made up of four identical units. Each of these units has a central portion that crosses the viral membrane and two small portions on either side of the membrane. The pore that hydrogen ions travel down sits in the middle of the protein, surrounded by all four units. Normally, though, the M2 channel is closed. This is important for the influenza virus, because once the pore opens, it causes the pH to decrease inside the virus, initiating the disassembly of the virus, which is necessary for the virus to be able to replicate within the cell. So that this process won’t happen prematurely outside of the cell, the M2 channel only opens when the virus is exposed to low pH.
After influenza binds to the outside of the cell, it is encircled by part of the cell membrane, which then breaks off inside the cell, forming a compartment called an endosome. The low pH of the endosome then activates the M2 channel, initiating virus disassembly. Low pH opens up the M2 channel by loosening the connections between the individual units, causing the protein to become dynamic enough that hydrogen ions can squeeze through the central pore.
Adamantane-based drugs, however, bind to the outside of M2 and cause it to be locked in the closed state. This means that in the presence of these drugs, the pH inside the virus never lowers and the virus is unable to replicate.
Swine flu, however, has a single mutation (S31N) that makes it resistant to adamantane drugs. This is actually a relatively common mutation found in flu viruses in general and the main source of all flu adamantane resistance. Over time, widespread use of this relatively older class of drugs has put significant evolutionary pressure on influenza, giving viruses with this mutation a selective advantage, leading to increasing rates of adamantane resistance in recent years.
It turns out that the S31N mutation does not directly affect the part of the protein that the drug binds to, but it actually has a more indirect effect: it makes the M2 channel slightly more dynamic and the connections between the individual units slightly looser. The channel still remains closed at higher pH, so M2 still functions properly, but the mutation makes the connections between the subunits loose enough that adamantane drugs are unable to lock M2 into the closed state.
This figure from Pielak et al. illustrates this phenomenon schematically:
Overall, these findings are interesting for two reasons. Firstly, they explain an important medically-relevant phenomenon–influenza drug resistance–which has become all the more timely with the current outbreak of swine flu and the threat of pandemic. Secondly, this is a poignant illustration of the central role that protein flexibility and dynamics play in biology, a concept that scientists have only just begun to explore, but one that you’ll likely hear much more about in the future, due in part to powerful advances in the field of protein NMR
Schnell, J., & Chou, J. (2008). Structure and mechanism of the M2 proton channel of influenza A virus Nature, 451 (7178), 591-595 DOI: 10.1038/nature06531
Pielak, R., Schnell, J., & Chou, J. (2009). Mechanism of drug inhibition and drug resistance of influenza A M2 channel Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0902548106
Stouffer, A., Acharya, R., Salom, D., Levine, A., Di Costanzo, L., Soto, C., Tereshko, V., Nanda, V., Stayrook, S., & DeGrado, W. (2008). Structural basis for the function and inhibition of an influenza virus proton channel Nature, 451 (7178), 596-599 DOI: 10.1038/nature06528
Stouffer, A., Ma, C., Cristian, L., Ohigashi, Y., Lamb, R., Lear, J., Pinto, L., & DeGrado, W. (2008). The Interplay of Functional Tuning, Drug Resistance, and Thermodynamic Stability in the Evolution of the M2 Proton Channel from the Influenza A Virus Structure, 16 (7), 1067-1076 DOI: 10.1016/j.str.2008.04.011
For more on flu, check out Effect Measure and Aetiology.
Update: Check out Pharyngula, where PZ Myers uses this post to take on some creationist nonsense.
Also, check out Discovering Biology in a Digital World for a discussion about M2 channel structures and mechanism of adamantane inhibition (using the Stouffer/DeGrado model).
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