02 Apr 2018
Arenaviruses have been shown to be rodent-borne agents of various diseases, which include the potentially lethal human hemorrhagic fevers (238). From Chapter 1, it was revealed that the arenaviruses have an enveloped, encapsidate, bisegmented ambisense single-stranded RNA genome, that can be packaged in variable copy numbers (106). The electron cryomicroscopy and image analysis of New World Pichinde and Tacaribe arenaviruses and Old World lymphocytic choriomeningitis virus has revealed pleomorphic enveloped particles ranging in diameter from ∼400 to ∼2,000 (angstroms) and are occasionally filamentous, reaching more than 20μm in length (76). The virions are covered with numerous membrane-spanning glycoproteins (GP spacing), (which can be described and referred to within this thesis as Viral Spikes) (131, 239). The distinctive surface spikes are spaced ∼100 Å apart and extended ∼90 Å from the maximum phospholipid head group density of the outer bilayer leaflet (240). In this thesis, electron cryo-microscopy (cryo-EM) and image analysis were used to examine the supramolecular architecture of the Pichinde (PICV), Junin, Tacaribe (TCRV) and Lymphocytic choriomeningitis (LCMV) arenaviruses. Cryo-EM has also revealed that they are pleomorphic enveloped viruses tend to have a spherical or ellipsoidal appearance, characterised by the studded feature that is clearly evident on the membrane surface, these studs are in fact the viral spike projections that correspond to oligomers (receptors) of the attachment and fusion proteins of a host cell. (See Figure 3.1).
From Figure 3.1 the concept of infectivity is shown with the virion attaching its viral spikes to the receptors which then fuse directly with the plasma membrane releasing the viral genome into the cytoplasm of the host cell. Alternatively the virus particle is internalized by adsorptive or receptor mediated endocytosis and delivered to an endosome. The acidic pH triggers fusion of the viral membrane with the endosome membrane, which permits the viral genome to be discharged into the host cell cytoplasm. Within the virion can be seen small clusters of dots, these are the free ribosomes and give the arenavirus its name as the latin name arena means sandy. Also there are two objects that look like beads labelled L and S these are the encapsulated viral RNA genomes, the beads are in fact nucleoproteins that are wraped around the RNA genomes and protect them from host cell degradation attack.
Pleomorphism means shape and size variation, it occurs in many viruses and manifests itself by causing deformations an the shape of a virion e.g. the virion can vary between spherical to ellipsoidal and even a dumbel shape. The forces causing this shape variation are as yet not fully defined.
3.3 Quality Control
3.3.1 Cryo-EM and image analysis.
Cryo-EM of purified LCM, Tacaribe or Pichinde at a concentration of _1 mg/ml in TNE was performed as described by Neuman and Milligan (240). Micrographs of each preparation displaying minimal drift and astigmatism were digitized by using a Zeiss SCAI scanner with Phodis software. Images were now scanned at 7 m per pixel and twofold pixel averaging was applied, corresponding to 4 Å per pixel at the level of the specimen (See Figure 3.2) (241). The histogram for a representative portion of the image containing vitreous ice and protein was normalized by adjustment of the densitometer settings until the mean image intensity was cantered as nearly as possible at a gray value of 127 on a scale of 0 to 255. Image processing was performed with the EMAN software suite (242).
From Figure 3.2; the membrane-spanning glycoproteins (GP spacing), can be seen on the virion surface and the pleomorphic shape and size observed (85). The surface spikes are spaced around 100 Å apart and extended up to 90 Å from the maximum phospholipid headgroup density of the outer bilayer leaflet with a distinctive stalk and head regions extended radially _30 and _60 Å from the outer bilayer leaflet, respectively (240). The frozen, vitrified pleomorphic arenavirus virion’s varied exstensively in diameter 400–2000 angstroms (Å) (240, 243). The mechanism used in causing pleomorphic virion shape has not been fully defined, with the best studied examples of vesicle-forming proteins appearing to initiate membrane curvature by inserting amphipathic protein domains into one side of the membrane, and so displacing lipid molecules and effectively stretching one membrane face more than the other (244) (245). While there is considerable evidence for this mechanism, it has proved difficult to directly demonstrate the lipid molecule displacment after proteins are inserted. Therefore showing how a lipid membrane becomes deformed so as to produce a shape change in the lipid bilayer membrane (85). With this membrane protein-protein interaction remaining difficult to demonstrate due to the technical difficulty of directly being able to investigate the processes that occur within the membrane, it was therefore the intention of this thesis to investigate with applied bioinformatics. This was done by using the physical and analytical measurements, along with factual based evidence, with supporting size related phenomena due to the Glycoproteins (spikes). Therefore develop an hypothesis, that addresses the issue of shape pleomorphism (See Figure 3.1) (240). Side views were obtained by masking the edges of virion images. This action permitted the visulation of the distribution density for viral spikes adjacent to the lipid bilayer. The face projections obtained were now measured for distance between each spike.by masking the central region of each projected virion, approximately the area covered by a concentric circle with half of the virion radius. (Such images represent the superposition of densities on the near and far sides of the virions, as well as the internal contents). Boxed images included approximately 4,000 side and 2,500 face views of Pichindevirus, 7,500 side and 5,000 face views of Tacaribevirus, and 10,000 side and 6,500 face views of LCMvirus, also control images of vitreous ice or copurified empty vesicles were processed in parallel with the arenavirus images. The optical density histograms for each boxed image were normalized to a common mean and standard deviation (SD) to correct for any remaining variations in optical density between individual boxed images (A schematic of a virion showing the concept of viral spikes protruding from the virion is shown in Figure 3.3).
3.3.2 Radial Density
The circular virions were selected initially for clarity in the determination of one-dimensional radial density profiles, also called transects (246). The characteristic density minimum in the centre of the bilayer was used as a reference mark for alignment and averaging of the radial density profiles from particles with various diameters (246). Statistical operations were performed with Microsoft Excel. Radial density profiles were used to calculate the relative image intensities of Z, GP, NP, core, ice and membrane from images recorded at similar defocus levels. Two interior layers of density opposed to the inner leaflet of the viral lipid bilayer were assigned as protein Z and nucleoprotein (NP) molecules on the basis of their appearance, spacing, and projected volume (See Figure 3.3) (247).
3.3.3 Virion Glycoprotein (GP) spacing & Total number of GP’s
The viral spike density is difficult to determine by cryo-microscopy, this is due to the fact that the pictures taken are all in two dimensions (This fact introduces an error into the research). Figure 3.2 (A) illustrates the problem. It can be seen that if a projected diameter is photographed then it can show spikes; overlapping each other and so therefore a true identification of the spike density in the projected plane cannot be fully produced. Figures 3.2 (B) and 3.2 (C) further illustrate this problem. The schematic in Figure 3.3 shows the spikes as a neat equidistant layer around the circumference of the projected plane which is what would be expected if they were dispersed in uniform density around the circumference in all directioos’. Figure 3.2 (B) shows the spikes as forming clusters as it might happen in real life, due to the probable removal of the overlapping spikes. Therefore measurement of spike density is not an exact science at present. However, theoretical measurements can be realised when the outside surface area of the spike influence is evaluated and this value then, when divided into the total surface area of the virus particle will give the theoretical maximum number of spikes possible on a given virion.
Image processing as previously stated was performed with the EMAN software suite using images between _1.2 and _3.1 _µm under focus. These were then selected for single particle analysis with a Gaussian low-pass filter that was used to truncate frequencies beyond the first node of the contrast transfer function, which ranged from 18 to 26 Å_1 for individual images, so that there were no phase reversals of the amplitudes. Side views were obtained by masking the edges of virion images in order to examine the distribution of density adjacent to the lipid bilayer. The optical density histograms for each boxed image were normalized to a common mean and standard deviation (SD) to correct for any remaining variations in optical density between individual boxed images. The estimated value of the average spacing distance between each GP was manually measured from the cryo-microscopic photographs by making use of the “boxer” module of the computer program EMAN and a calibrated pixel width of 4.0 Å per pixel. From the analyzed results it was possible to extract the values for each GP density with respect to the specific diameter of a virion. GPs were now counted by eye using the EMAN module “boxer” for image display purposes. Each visible GP on visible outer edge of the virion was counted. This method was used for counting on all of virions of LCMV, TCRV, PICV and JUNV as well. By comparing the observed GP count to the theoretical number of GPs on a virion of a given size, it was possible to express GP coverage as a percent of the theoretical maximum (See Figure 3.4).
Fig 3.4: Shows the concept of Glycoprotein (GP) spacing and the method of measurement used to establish the distance between each successive GP spike, which also allowed for counting of the GPs. Each peak in the graph as well as representing a specific protein is also a density marker. The actual spacing was done by using the image slides and using the cryo-EM software package so as to facilitate manual measurement and visual counting.
3.4.1 Virus growth and Preparation of Materials
“Baby hamster kidney (BHK) cells were maintained in Dulbecco’s minimum essential medium supplemented with 8% fetal bovine serum, 2 mM L-glutamine, and antibiotics. The Pichinde-AN3739 (Pic), Tacaribe-TRVL 11573 (Tac), and lymphocytic choriomeningitis virus-Arm4 (LCM) strains were propagated in 850-cm2 roller bottles at 37°C with 5% CO2. Semiconfluent BHK cells were inoculated at a low multiplicity of infection. Virus-containing cell culture medium was collected 48 h after inoculation, and virions were isolated by polyethylene glycol precipitation and Renografin density gradient centrifugation (244). Protein concentrations were determined by the method of Bradford with bovine serum albumin as the standard (255). For radiolabeled virus, Tran35S-label (ICN, Costa Mesa, Calif.) was added at 24 h postinfection to a final concentration of 15 Ci/ml. The virus titer was determined by plaque assay on Vero-E6 cells (256). Samples of Pic, Tac, and LCM possessed infectious titers in excess of 109 PFU/mg of total protein. These methods as described were carried out by Dr John Burns and Dr B. W. Neuman”.
Due to the small size of a virion it has not been possible to carry out direct physical research into the actual rates of change that take place when a virion is being generated. However some experiments have shown nthe effect of protein – protein interaction, as those carried out in trying to establish the mechanisim by which a viral spike assemble is able to penetrate and remain in a seemingly fixed position within the virion membrane (246). To develop a hypothesis for the reason causing pleomorphisim it was necessary to use a system of applied bioinformatics and seek to deduce a mathematical biophysics equation that would describe the result in terms of energy usage. The first step was to describe a force system using classical mechanics that causes similar drformations to that which exists in virions. The system was from inorganic structures and is the one that describes deformation in thin shells (257), (258). The second step was to use the results from Optical Stretching on Giant Unilamellar Vesicles (GUV) (259). The third step was the syetem used for actual spike penetration into and through the virion membrane (246). With these major systems now in place it was possible to in the first instance draw a clear analogy between an inorganic and an organic system and compare the effects in both cases. The next step was to look at the biological optical stretching system for GUVs and do qa comparison between the force systems of the inorganic/organic structures. After doing a comparative analysis it was seen that a hypothesis could be developed as can now be seen in the results obtained from research done within this thesis.
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