Adenovirus infection pathway and structure

We used AFM to disrupt single Adenovirus capsids and TIRFM to observe the release of the viral genome. The human Adenovirus (Ad) causes mainly respira-tory, ocular, and gastrointestinal infections. Moreover, engineered virus versions are used as delivery vehicles in approaches for cancer gene therapy [38].

Figure 2.9: Overall structure of the Adenovirus. Adopted from [39].

The Ad is one of the largest and most complex non-enveloped double-stranded DNA viruses. The adenoviral capsid has an icosahedral shape with diameters of about 90nm [40]. The icosahedron consists of 20 triangular faces which are com-posed of hexon trimers (capsomers). In total, the virus has 240 of these capsomers (g. 2.9).

The icosahedron has also 12 vertices which are build as pentamers (pentons). From the pentons bers protrude (not shown).

During the viral infection pathway of eukaryotic

cells, the viral shell must be disassembled to release the viral genome into the cell.

Once the Adenovirus bers bind to a cellular receptor, a process is induced by which a clathrin coated pit is formed taking up the particle into the cell [41]. The modication of the viral capsid starts at the plasma membrane were the protrud-ing bers are lost [42]. The formation of the clathrin pit marks the beginnprotrud-ing of endocytosis. Thereupon, the viral particle is taken up by an endocytic vesicle. In this endosome the virus encounters an acidic pH which induces the loss of pen-tons and peripheral core proteins [43]. The low endosomal pH and the presence of Ad particles in combination with other factors (e.g. integrin) are believed to be responsible for endosome disruption [44]. After release, the partly disrupted virions travel to the nucleus mediated by microtubules and the minus-end directed motor complex dynein/dynactin. At the nuclear pore complex the capsid binds to a nuclear pore complex lament protein. The capsid is also indirectly linked to Kinesin-1 (running to the positive end of the microtuble). The Kinesin-1 mo-tor protein disrupts the viral capsid and compromises the nuclear pore complex integrity. Finally the genome enters the nucleus [45].

To develop its full infective potential, the virus requires a maturation step during assembly. In this step the viral protease cleaves several capsid and core proteins.

The cleavage leaves the proteins in a certain conformation so that a triggered change of their conformation can eect the virons structural integrity. Hence, the viral capsid is primed for the sequential disassembly procedure during infection

[43]. An Ad2 mutant called TS1 lacks the maturation step. As a result, non-cleaved protein precursors stabilize the connection between pentons and the viral core. Moreover, the virus core is more compact and stable due to condensing action of unprocessed proteins [43]. Therefore, the TS1 phenotype shows a disturbed structural disassembly and is not able to pass the endosome membrane during endocytosis [46], [47]. From these results it becomes clear that the maturation step is essential to render the Adenovirus to be fully infective.

The dierences in structural disassembly between the WT and the TS1 mutant connects infectivity to mechanical stability. The dynamics of disassembly induced by mechanical fatigue were investigated for both virus types by Ortega-Esteban et al. [48]. The AFM based experiments revealed stability dierences between WT Adenovirus and TS1. The mechanical disassembly was shown to start with a sequential loss of pentons at the vertices. This indicates their relevance as starting point for virus shell disintegration. The removal of pentons in the TS1 capsid re-quired more AFM loading cycles (pushes/energy) than in the WT case. Moreover, the TS1 core remained more compact after shell disassembly as compared to the WT core. The height of the remaining core and shell structures were measured in AFM images for the WT group to be ∼35nm while the TS1 height was with∼70 nm much higher. The later value corresponds roughly to the core diameter plus an underlying layer of the shell.

Since the mentioned experiments and ndings are based on AFM images, it is not possible to dierentiate between genome and capsid structures after dissasembly.

However, this could be realized by specic uorescence labeling. Moreover, the images were acquired in tapping mode which requires hundreds of contacts between tip and sample. To investigate the spread of the remaining viron structures in a more diusive driven way, an alternative to the ongoing AFM imaging is required.

This leads to an experimental design which allows to induce a capsid rupture (mechanically) and to observe the spread of the genome (optically) afterwards.

Here, the combined AFM-TIRFM instrument comes into play. In this setup the capsid dissasembly can be induced with a force distance curve (single push) and the genome spread can be followed by uorescence labeling. The labeling can be done by using a DNA specic dye (YoYo-1) which increases its uorescence

∼3000 times when bound to the double-stranded DNA. The DNA binding can be observed in real time when the dye gains access after capsid breaking.

2.4.3 70S ribosome

During protein synthesis the ribosome translates genetic information from messenger Ribonucleic acid (mRNA) into a polypeptide. The polypeptide is assembled from polymeric protein molecules (amino acids) whose sequence is controlled by the sequence of the mRNA. The decoding process is largely performed by transfer RNA (tRNA). The tRNA compares the mRNA information and delivers a specic amino acid to ribosome. After each added amino acid the mRNA strand has to move through the ribosome to allow the next tRNA to read the next information package (codon). For each amino acid added to the polypeptide the ribosome un-dergoes large conformational changes to facilitate translocation of the mRNA and tRNA and to recruit several translation factors. The SFB860 A4 ribosome project aims to get a deeper insight into the functioning of the ribosome. In a subproject AFM was used to measure small uctuations in height by placing the AFM tip on top of a single ribosome, g. 2.10. The preliminary results were interpretated in terms of contour changes of the ribosome which may occur during its enzymatic activity.

Figure 2.10: Fluctuations in height measured in preliminary AFM experiments on ribosomes. Data acquired by Frédéric Eghiaian.

The custom made AFM-TIRFM setup was also planned to complete the AFM observations with a method to visualize single factors (tRNAs and EFG) that are transiently bound to the 70S ribosome during translation. This should be realized by simultaneous Total Internal Reection Fluorescence (TIRF) microscopy detec-tion of the uorescently labeled factors. Combined experiments would then allow a correlation of the height uctuations with the biochemical state of the ribosome.

Therefore, the uorescence of ribosome constructs labeled with a single dye molecule was characterized. The characterization includes bleaching times, signal to noise ratios, and possible detection frame rates. Moreover, in combined AFM-TIRFM experiments, the background light from AFM cantilevers during single molecule detection needs to be determined as well as the quenching potential of the can-tilever tip.