Elucidation of Viral Replication Mechanisms in an Animal Model for Multiple Sclerosis
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1 Elucidation of Viral Replication Mechanisms in an Animal Model for Multiple Sclerosis Introduction It is often difficult to research human diseases due to obvious ethical implications. However, when an acceptable animal model of a disease is found, the medical world is given a rare window of opportunity to conduct crucial research at low risk. At the University of Tampa, I have been given the opportunity to work with my research mentor, Dr. Eric Freundt on a murine virus that causes similar disease pathology to human multiple sclerosis. Theiler s murine encephalomyelitis virus (TMEV), is a member of the Picornaviridae family and Cardiovirus genus. This virus was initially isolated from mice by virologist Max Theiler in 1937 at the Rockefeller Institute [1], and has since been described as one of the best models for multiple sclerosis. Once infected with TMEV, mice are susceptible to a chronic infection, which causes progressive demyelination throughout the central nervous system [2]. This pathology is an excellent model for multiple sclerosis, a disease known to be associated with axonal demyelination within the central nervous system. Multiple sclerosis has been recognized to cause severe cognitive impairments, as well as muscle weakness and other physical deficits in human patients, but remains incurable [3]. Although much is known about the effects of multiple sclerosis, what actually causes the demyelination within the human central nervous system remains unanswered, making the TMEV model a highly coveted research tool. Being in the same family as poliovirus, a highly studied and documented disease-causing virus, insight has been given to the replication of TMEV. As documented with poliovirus, TMEV replicates at intracellular vesicles that are used as docking sites for viral replication [4]. However, unlike poliovirus, the composition and origin of these replication vesicles is unknown. The primary motivation of my research is to elucidate the origin and of these vesicles at which TMEV replicates. By solving the question of how these viruses replicate, it may also be possible to trace the route of TMEV-induced demyelination and pathogenesis. If this pathological pathway of demyelination could be hindered in mice, and then applied to the human condition of multiple sclerosis, it may be possible to develop a cure or treatment. With any research project, it is necessary to identify a logical starting point. After completing an in-depth analysis of primary research on TMEV, my research mentor and I identified that there is a relationship between viral replication proteins and proteins of the trans- Golgi apparatus [5]. This finding opens up many questions about the nature of TMEV replication because the trans-golgi apparatus is responsible for sending secretory vesicles to intracellular organelles, and the cellular plasma membrane for exocytosis. Serving as a factory of modification and packaging for protein, the Golgi apparatus is a highly important part of cellular metabolism, and thus interplay with viral replication is a point of interest. The Golgi apparatus is a common target for cytopathic damage, or damage to the cell caused by viral infiltration, by numerous viruses, but has an unknown response to TMEV [6]. My initial experiments were targeted at answering how the Golgi apparatus is actually affected by infection with TMEV. Previous research points to the trans-golgi as providing vesicles for replication, but does not rule out other locations of the Golgi as vesicle origins. Thus, along with answering the issue of Golgi
2 apparatus insult, it was necessary to establish if only the trans-golgi apparatus, or if other locations within the Golgi are involved in co-localization with viral replication proteins. Thanks to the generosity of Dr. Freundt and the University of Tampa Department of Biology, I was able develop a protocol to answer my aforementioned questions using technical equipment. Materials and Methods There was no risk of harming live murine species in experimentation, as a continuous cell line of baby hamster kidney cells (BHKs) was passaged and used for infection, rather than live animals. The first question of how infection with TMEV affects the Golgi apparatus was answered through the process of indirect immunofluorescence staining, a technique commonly used to detect the presence and location of specific proteins within a cell. BHK cells were seeded onto coverslips in a 24-well plate, and allowed 24 hours to reach a point of 60-70% confluence. At this point, 16 of the wells were infected with the GDVII strain of TMEV at a multiplicity of infection (MOI) of 10, while 8 wells were left as uninfected controls. To allow determination of the rate of Golgi apparatus insult, the infection was stopped at different time intervals using 4% paraformaldehyde (PFA), a reagent that cross links cellular proteins. Specifically, the infection was halted at 6, 9, 12, and 24 hours, while uninfected cells were halted at 6 and 24 hours to generate a baseline of normal uninfected Golgi formation at different time points post seeding. After the full time plot, cells were made permeable with 0.1% Triton-X 100 in PBS. Once the cells were made permeable, a common immunofluorescence protocol [7] was used to indirectly label all cover slips with DAPI, a marker specific for the nucleus, and GM130 a marker specific for the cis-golgi [8]. Half of the wells were labeled with anti-tmev capsid antibodies, a specific marker for viral capsid proteins. The remaining 12 wells were labeled with anti-3d antibodies, a marker for the viral 3D polymerase, which is a necessary viral replication protein, that docks with the replication vesicle. All coverslips were mounted onto glass slides using Vectashield mounting media to prevent photobleaching by the fluorescence microscope. The next step was to view the cells with fluorescence microscopy and quantify the amount of Golgi fragmentation at different times post infection. Results Quantification of presence or absence of Golgi insult was collected in 10 randomly selected fields for each coverslip at 60x magnification. A total of 597 nuclei were examined for Golgi insult from the 12 coverslips stained with DAPI, GM130, and anti-capsid, and presented in Figure 1, with calculated standard error of the mean. The graph depicts two bars for each time point from quantification of two different cover slips per condition. Images of infected and uninfected cells at different time points are presented in Figure 2. The 12 wells treated with 3D polymerase were extensively examined at all-time points. However, as depicted in Figure 3, there were no examples of co-localization between the cis-golgi marker (GM130), and the viral polymerase (anti-3d).
3 Percentage of Cells with Fragmented Golgi Compared to a Popula@on of Uninfected Cells 100 % of Cells with Fragmented Golgi Hour Control 6 HPI 9 HPI 12 HPI 24 HPI 24 HPI Figure 1: Percentage of cells with insulted Golgi at different time points. (HPI = hours post infection) A) 6 hour control B) 6 hour post infection C) 9 hour post infection D) 24 hour post infection E) 12 hour post infection F) 24 hour control Figures 2. TMEV infection correlates with Golgi fragmentation. Images are shown that represent staining with anti- GM130 (shown in green), DAPI (shown in blue), and anti- TMEV capsid (shown in red).
4 A) 6 hour control B) 6 hour post infection C) 9 hour post infection D) 12 hour post infection E) 24 hour post infection F) 24 hour control Figures 3. TMEV 3D polymerase does not colocalize with cis- Golgi protein GM130. Images are shown that represent staining with anti- GM130 (shown in green), DAPI (shown in blue), and anti- 3D polymerase (shown in red). Discussion The uninfected controls at both 6 and 24 hours in this experiment have established a baseline of approximately 10% of uninfected cells have fragmented Golgi, and this may be caused by natural cell death or mitotic events. The most noticeable cytopathic effect of infected cells is Golgi fragmentation. Figure 1 depicts a direct correlation between time post infection and amount of fragmented Golgi within infected cells. There is not a substantial increase in fragmentation after 12 hours, indicating that at a high MOI, 12 hours is sufficient time to cause Golgi fragmentation within infected cells. This result points strongly to the conclusion that infection with GDVII strain of TMEV causes fragmentation of the Golgi apparatus. Additionally, Figure 2 shows an increase in distance of fragmented Golgi protein spread throughout the cell as time post infection increases. Data from the coverslips exposed to anti-3d polymerase (Figure 3) strongly supports that GM130 cis-golgi protein marker does not co-localize with the replication vesicles. This data introduces the possibility that TMEV selectively utilizes trans-golgi proteins for replication vesicles, and may even suggest that TMEV has evolved to utilize vesicles sent from the trans-golgi to be secreted from the cell. If this hypothetical mechanism of replication is correct, it will identify a novel mechanism of viral replication, and potentially, provide a more efficient release pathway for the virus, requiring further research on viral release. Disruption of the trans-golgi by TMEV may also provide insight into the pathology of demyelination. Myelination of neurons in the central nervous system (CNS) is not a fully understood process. However, it is known that neuronal cells have the capacity to communicate with Schwann cells and oligodendrocytes, which are responsible for neuronal myelination, through axonal connections and cues [9]. If TMEV replication blocks the normal neuronal exocytosis and secretory pathway used for communication between neurons in the CNS and oligodendrocytes, a demyelination mechanism may result. Additionally, disruption of the Golgi of oligodendrocytes could prevent trafficking of myelin proteins and lead to oligodendrocyte cell
5 death. My next steps are to experimentally test this hypothesis of pathology with immunofluorescence staining specifically for secretory vesicle protein, and viral 3D polymerase. High co-localization of secretory vesicle proteins, and 3D polymerase in an immunofluorescence stain will strongly support my proposed mechanism of viral replication on sequestered post- Golgi vesicles in the secretory pathway. Reference List Theiler, M., (1937). Spontaneous Encephalomyelitis of Mice, a New Virus Disease. The Journal of Experimental Medicine, 65, Mecha, M., Carrillo-Salinas, F.J., Mestre, L., Feliú, A., & Guaza, C., (2013). Viral Models of Multiple Sclerosis: Neurodegeneration and Demyelination in Mice Infected with Theiler's Virus. Progress in Neurobiology, 101, doi: /j.pneurobio Moreno, M.J., Garcia, C.M., Gonzales, P.A., & Benito, A.Y., (2013). Neuropsychological Syndromes in Multiple Sclerosis. Psicothema, 25, doi: /psicothema Richards, A.L., & Jackson W.T., (2012). Intracellular Vesicle Acidification Promotes Maturation of Infectious Poliovirus Particles. PLoS, 9. doi: /journal.ppat Jauka, T., Mutsvunguma, L., Boshoff, L., Edkins, A.L., & Knox, C., (2010). Localisation of Theiler's Murine Encephalomyelitis Virus Protein 2C to the Golgi Apparatus Using Antibodies Generated against a Peptide Region. Journal of Virology Methods, 168, doi: /j.jviromet Beske, O., Reichelt, M., Taylor, M.P., Kirkegaard, K., & Andino, R. (2007). Poliovirus infection blocks ERGIC-to-Golgi trafficking and induces microtubule-dependent disruption of the Golgi complex. Journal of Cell Science, 120, doi: / jcs Najm, F.J., Zaremba, A., Caprariello, A.V., Nayak, S., Freundt, E.C., Scacheri, P.C., Miller, R.H., & Tesar, P.J. (2011). Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells. Nature Methods, doi: /nmeth.1712 Nakamura, N., Rabouille, C., Watson, R., Nilsson, T., Hui, N., Slusarewicz, P., Kreis, T.E., & Warren, G., (1995). Characterization of a cis-golgi matrix protein, GM130. Journal of Cell Biology 131, doi: /jcb Simons, M., & Trotter, J., (2007). Wrapping it up: the cell biology of myelination. Current Opinion in Neurobiology, 17,
Nature Medicine: doi: /nm.4322
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