Saturday, February 20, 2016

Mechanics of Influenza

The more we learn, the more amazing this tiny virus seems.

One of the few regular and severe diseases still prevalent in the developed world among people of all ages is influenza. It causes misery to millions and significant mortality, and can occasionally mutate to pandemic strains that kill millions; 2% of the human population in 1918. Nor is the current vaccination system very effective. We need something better.

What a simple organism it is, though! Only nine genes, in a genome of less than 15,000 bases, enveloped in a membrane coat carrying a couple of proteins on its surface. How does it cause all this misery? Well, each of its genes is intricately designed and multifunctional. The principal outer coat protein, for instance, called hemagglutinin or HA, is specially shaped to bind to the sialic acid on the surface of our respiratory mucosa cells. Then, when ingested into the endomsome/lysosome of the poor cell, it acrobatically flips its structure to thrust out a hydrophobic spear that punctures the lysozomal membrane. This same protein then refolds its shape once again to bring the viral membrane and the lysozome membrane into close enough contact that they fuse, releasing the viral contents into the cell. Not only that, but the HA protein plays a special role in mutating all the time in different viral strains so as to retain function on the exterior of the envelope, but evade the host immune system.

Sequence of refolding and membrane fusion events, by the surface HA protein. It takes only about three HA molecules to carry out membrane fusion between the surface of the virion and the endosomal membrane.

There are many other molecular functions encoded in this tiny parasite, such as a polymerase that both replicates itself and copies itself to message RNAs, an enzyme on the surface that allows the virus to eat through the mucus in our respiratory tract, structural proteins that assemble the new viral particles, an enzyme that steals the caps of cellular mRNAs and plants them on viral RNAs, and even a proton channel protein that allows the virus to equilibrate with the low pH of the endosome/lysozome and trigger disassembly of the virion, so that, given that the membrane fusion has taken place at the same time, its genome can get out to take over the cell.

Electron micrograph of two influenza particles inside an endosome, waiting for their chance to escape and take over the cell.
The inhibition of the host immune system deserves special mention, for its deviousness. The interferon response is one of the most powerful immune and especially antiviral responses, which influenza takes special care to counter-attack. This is a rapid and non-adaptive ("innate") part of the immune system, where once a virus is detected, a program of hundreds of genes is activated that renders a cell strongly primed to shut down viral replication and commit suicide. While the virus eventually succumbs to the much slower adaptive immune response, (B cells, antibodies, killer T cells, etc.), that only happens after it has already replicated and been sneezed back out to other targets. Evading the more prompt early immune response is absolutely essential, and is partly mediated by a viral protein called non-structural protein 1 (NS1). So-called because at the time it was discovered, researchers had no idea what it did.

Now, they know that it has at least four, and possibly more, functions.
  1. It binds to a shuts down cellular TRIM25, a key part of the detection system that turns the interferon response on. 
  2. It also binds to and shuts down cellular PKR, which is one of the prime components of the interferon response downstream, which shuts down most translation, including viral translation. PKR also promotes cell suicide and amplifies the interferon response through NFkB. 
  3. Thirdly, NS1 binds and shuts down cellular CPSF4, which processes and tags cellular mRNAs for export from the nucleus. So while the interferon system is trying to shut down viral and cellular translation, the virus, through this single protein, is specifically shutting down cellular translation, and also shutting down the interferon response. 
  4. Some researchers claim a fourth function is most important, which is the ability of NS1 to bind to double-stranded RNA, like the genome of the virus and its replication and transcription structures. This binding seems to hide the RNA from OAS, an interferon-induced protein that would otherwise bind the double-stranded RNA and initiate an RNAse L response to destroy it. 
  5. Fifth, NS1 binds to and inhibits another cellular protein, PAF1, which is a regulator of transcription that has a central role in antiviral gene expression. 
  6. Sixth, NS1 binds to and activates PI3K, a kinase that promotes cell survival, which is helpful in the face of attempts by the host cell to commit suicide
  7. Seventh, a small portion of NS1 from some strains of influenza binds to PDZ domain-containing proteins DLG1 and LIN7C, thereby disrupting tight junctions between cells, and possibly helping the virus spread to other cells and tissues. 
It is simply astonishing that a small protein of 230 amino acids could do so much, and wreak such havoc.

Complete structure of NS1, colored by position in the protein, from the N terminal to the C terminal.

Portion of NS1 that binds to RNA, bound to RNA. The tiny stick molecules are  glycerol molecules from the crystallization mixture.

That is just a taste of the complexities that are known, and there is surely much else as yet unknown. But what to do about it? The current system of annual viral surveillance and specific vaccine production is routinely outsmarted by this tiny speck of a virus. We probably need a more serious program of vaccine research and testing from all parts of the virus, under the assumption that there may be epitopes not in the usually studied surface antigens that might be helpful in preventing infection, assuming that some cells manage to kill themselves and display the internal viral proteins to the adaptive immune system.

Likewise, study of all the virus products and screening of small molecule drugs against them in all their facets would be a significant research program directed to treat an infection once it has begun. This needs a broad approach that is possible with current technology, since it is hard to predict what all the functions of the virus are or which ones are most vulnerable in the body. The interfaces between the NS1 protein and its various targets are examples presenting many possible forms of therapy. Current drugs like Tamiflu are only modestly effective, if at all. This kind of commitment is something we should be doing much more actively as part of the NIH system. It would have higher and quicker payoffs than the war on cancer, while still at much lower cost.

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