FISCALIZACION Y LEGISLACION

Many viruses eject their genetic material through a unique vertex. The vertex contains specialized proteins that participate in the ejection process. In tailed phages, a

dodecameric portal structure occupies their procapsid’s unique vertex (1, 2). The genome is packaged through the portal by the viral terminase, an enzymatic complex that couples ATP hydrolysis to genome packaging (3). The phage tail attaches to the portal once packaging is completed. Thus the genome is ejected through the portal structure to initiate an infection (4, 5).

Portals are not limited to phage systems. Herpesviruses, for example, also utilize these structures (6, 7). Their portals are structurally similar to those of phage, and they also package and eject their genomes through them (8, 9). During infection, the enveloped herpes virion fuses with the cytoplasmic or endocytic membrane, releasing the capsid into host cell’s cytoplasm (10). The capsid is then trafficked to the nucleus. The portal docks with a nuclear pore and the viral genome is ejected into the nucleus (11–13). The unique vertex is preordained in all of the above systems. It is found in the procapsid and remains in place throughout the assembly process. However, some viruses appear to lack a preordained unique vertex.

Poliovirus, for example, appears to utilize a single vertex during genome delivery, yet all of the virion’s vertices are identical. To initiate an infection, poliovirus first binds to a host cell’s surface receptors, triggering its endocytic pathway (14, 15). The virus becomes internalized within an endosome where it undergoes structural changes,

releasing its internal protein VP4 (16, 17). VP4, together with the VP1 capsid proteins at the open vertex, become membrane associated and form a channel, releasing the ssRNA genome into the host cell’s cytoplasm (18–20). It appears that poliovirus does not need a unique vertex, as the viral proteins are contained within an endosome and its capsid contains 60 copies of both VP4 and VP1 (21). The proteins are icosahedrally ordered. They are present at every 5-fold symmetry axis; therefore, any vertex is capable of pore formation.

FX174, by contrast, carries twelve copies of its DNA piloting protein H. During an infection event, ten H proteins exit the capsid and form a cell wall spanning conduit. The cryo-electron microscopy (Cryo-EM) reconstructions of eclipsing jX174 particles, both DNA-filled and empty, suggest that the H proteins appear to emerge from a single 5-fold axis of symmetry. (22). In the DNA-filled reconstruction, a single, unopened vertex is attached to the lipid bilayer (Figure 4.1A and D). There is a cylindrical region of lower density above the attached vertex. The region extends 110 Å into the virion and is 80 Å wide. This region likely contains the H protein oligomer as proteins are typically less dense than DNA. In the second structure, the contents of the virion have exited through a 30 Å wide opening at the attached vertex (Figure 4.1B and E). Thus, jX174 also utilizes

a unique vertex. However, it is not known if the unique vertex is preordained during virion morphogenesis or induced at the infection site.

An H protein oligomer would likely be found at a preordained vertex, if one exists. However, the jX174 procapsid is constructed from twelve identical, 12S*, assembly intermediates, each containing a monomeric H protein (Figure 4.2). The H proteins would need to congregate at a single vertex after procapsid assembly. Two procapsid structures were determined, a 26 Å resolution cryo-EM reconstruction and a 3.5 Å resolution X-ray crystal structure (23, 24). In both structures, under each 5-fold axis of symmetry, there is a region of protein-like density that has been attributed to H protein, but the density could not be confidently assigned. This region is surrounded by density belonging to the internal scaffolding protein. If this is correct, H appears to remain monomeric within the procapsid and the internal scaffolding protein may hold it in place.

H oligomerization could occur during genome packaging or at the subsequent infection site. During packaging, the internal scaffolding proteins are displaced by the DNA binding protein. H protein may be freed and allowed to congregate at a single vertex during this process. The structure of the jX174 virion was determined to 3.5 Å

resolution. Like the procapsid structures, electron density could not be unambiguously assigned to H proteins. Again, there is diffuse density under the 5-fold axes. It may be produced by H protein or the ssDNA genome, thus the status of a unique vertex is still ambiguous. Cryo-EM was also used to examine virions for any asymmetric features. Thousands of particles were examined and no asymmetry was detected. However,

simulated micrographs were produced of an H-tube containing jX174 virion, i.e. the H- tube X-ray structure was placed inside the virion X-ray structure (Figure 5.3, row a). Increasing amounts of noise were added to the simulated micrographs. Nearly all

structural details were washed out when this level of noise reached that of actual jX174 virion micrographs.

A second approach was taken to detect asymmetry within jX174 virions: bubblegram imaging. During electron microscopy, samples are bombarded with an electron beam. Repeated bombardment of biological samples causes radiolytic damage to the sample’s proteins and nucleic acids, producing H2. Samples are embedded in vitreous ice in cryo- EM preparations. This traps the H2, producing a bubble that is visible in the resulting micrographs. For reasons that are not understood, the different biological components undergo radiolysis at different rates. The rate is also influenced by the component’s local environment. For example, proteins embedded in DNA bubble faster than surface

exposed proteins, while surface proteins bubble faster than DNA. Thus, this technique can be used to determine if a virion contains proteinaceous structures embedded within its packaged genome (25, 26). If the bubbles’ locations are consistent, they can be used to generate an asymmetric reconstruction of the virion based on its inner features.

A bubblegram analysis was performed on the microvirus ST-1 to determine if an H protein core exists within the virion. Purified ST-1 virions were flash frozen on cryo-EM grids. The virions were repeatedly dosed with electrons and monitored for bubble

indicate unpackaged virus-like particles in panel 1 and 8. It appears that proteins, most likely protein H, are embedded within the ST-1 genome. However, the bubble locations were not consistent enough to guide image reconstruction. Thus, it is still unknown if the H proteins form a core within the virion and if so, where it is located.

A third approach could be designed; however, it relies on an unproven assumption. Many results suggest, but do not rigorously demonstrate, that a portion of H protrudes from the virion. Two studies have shown that H protein may participate in host attachment. In the first, his-tagged H was purified and tested for LPS binding specificity (27, 28). The protein only bound LPS purified from jX174-susceptible host strains. This evidence falls short of compelling, as H protein evolved alongside its host’s LPS. The protein may bind poorly to foreign LPS because it has been optimized to the host’s LPS structure. In the second study, mutations conferring changes near H protein’s N-terminus inhibited attachment (29). Again, this is not entirely compelling; mutant DNA binding proteins, which are completely internalized, can affect attachment kinetics (30). Thus, anything could possibly affect virion structure could affect attachment. Lastly, it was reported that monoclonal antibodies that bind to denatured H protein can cross-react with virions and procapsids. However, this data was only mentioned as a personal communication within another manuscript (23).

Each finding falls short of compelling when considered individually. Yet, the results tend to suggest that a portion of H protein may protrude from the virion. If this is true, then it may be possible to raise an antibody against or tag the exposed portion of H protein.

Once marked, cryo-EM could be used to visualize the location(s) of the exposed H- protein segment. If a single vertex is marked, then this would be a strong indication that a preordained unique vertex exists. The tag or fab fragments could then be used to reveal unique vertex’s structure after building an asymmetric or 5-fold averaged reconstruction of the virion. Alternatively, if all vertices were marked, efforts could be focused on understanding how ten H proteins relocate and oligomerize at a single vertex during the infection.