Saturday, March 16, 2013

Invasion of the stone cold killers

How one virus docks and enters.

These are wonderful times in biology. Advances in technology make routine what was before extremely arduous or impossible. An example is the study of structures at atomic and near-atomic scales. The data bank of biological atomic structures- of proteins, mostly, but also of DNA and RNAs- has ~90,000 entries, including increasingly complicated and large structures.

Combining atomic structures (deduced from X-ray crystallography, typically) with more gross-level imaging by electron microscopy has become possible as well. A recent paper described the large-scale structure of a bacterial virus as it docks and then injects its payload of DNA. Quite reminiscent of a space landing craft, really!

Example images of viral docking, in three stages. Stage A, the virus is positioned on the surface. Stage B, it has inserted its syringe-like channel. Stage C, its head is empty, after all its DNA has entered the cell. Note that this is not to scale- these cells are not full-size E. coli, but mini-ized vesicles derived from them.

This paper clarified the first parts of this docking story, showing where the tail fibers are at different stages of infection, at reasonably high resolution. The method was a lot of electron microscopy at the highest possible resolution, of hundreds of viruses, which were then averaged together to form smoothed-out pictures of higher resolution that any single one alone.

Averaged image of T7 viruses, showing its tail, internal core, and tail fibers around the outside (C is the view from bottom). In A, a viral mutant was used that has no tail fibers.

This virus (the T7 bacteriophage, well-studied in molecular terms) first detects and binds to a bacterial cell (the usual lab specimen of E. coli) using its tail fibers, shown in yellow:

Schematic interpretation of the data, with tail fibers in tucked position, and the detailed structure of the end of the tail fiber, which binds specifically to bacterial surface LPS, superimposed at scale.

These yellow spokes are the landing gear, nicely tucked up against the body of the virus, which is a sort of ordered crystal of coat proteins enclosing a DNA cargo (of only 39937 nucleotides). One oddity is that, while there are six tail fibers, the body of the virus is an icosahedron with five-fold symmetry. So the fibers can't be very tightly bound to the body. The ends of these fibers seem free enough to bind their receptors whether tucked against the virus body or extended out. They bind a common and large molecule on the surface of bacteria like E. coli, called lipopolysaccharide, or LPS. This binding is very loose, however, so the virus can roll around a bit before settling down on its target.

Once a fiber binds, it flips out into an extended shape, and other fibers can then bind and flip out as well. When all six have bound, the virus is well-attached to the ill-fated bacterium, and is also properly positioned for the next step, which is the descent of the central (red) channel and injection of the viral DNA.

Data and schematics of T7 in the docked position, before and after tail insertion. OM is the bacterial outer membrane, IM is the bacterial inner membrane. PG is the peptidoglycan cell wall layer between the two membranes.

What happens next is a little more mysterious. The binding of the tail fibers to the bacterial surface- even all six- does not seem to signal the viral core to fire its payload. The central red tail does not seem to have a specific receptor that it binds to or recognizes either, as far as I can read. Rather, it seems to expose enzymes perhaps like lysozyme, that begin to degrade the bacterial surface. This results in a little indentation of the surface (see the image above). At some point, a large core complex of proteins inside the virus bodies is signalled to come out, forming a pore that spans both the outer and inner bacterial envelopes / membranes (parts C & D, above).

Author's interpretation of the docking and insertion sequence. The DNA enters quite slowly, actually, taking several minutes and happening in three stages, pulled successively by viral proteins/internal pressure, then the host RNA polymerase, and lastly the RNA polymerase encoded by T7 itself.

An interesting aspect of what happens next is that the viral DNA doesn't just shoot into to the bacterial cell. It carries sequences that would be susceptible to one of the bacteria's defense mechanisms- the so-called "restriction" enzymes that cut foreign DNA. So the virus feeds in just a little of its DNA, (850 nucleotides), protecting it with special proteins. That DNA holds three promoters, which attract the resident bacterial RNA polymerase, which through its action of transcription physically pulls more of the viral DNA into the cell. One of these genes is a specific inhibitor of bacterial restriction enzymes. Later on, after T7's own RNA polymerase is transcribed and synthesized, (and after many host proteins have been destroyed), it comes back to pull the rest of the viral DNA into the cell at a speedier rate. The whole cycle of infection can be done within half an hour.

A diabolical mechanism, to be sure. But viruses like this one are being considered as next-generation antibiotics, to take up the slack after our penicillin-related antibiotics wear out due to overuse and evolved resistance. So don't be surprised if you end up swallowing some of these viruses as medicine someday.

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