As we have previously discussed, there is no specific or effective treatment for West Nile Virus at this time. WNV is treated medically through supportive care that can help manage pain and other symptoms. This is a particularly concerning obstacle for the more serious forms of West Nile Virus, such as the West Nile neuroinvasive diseases, which can result in severe and life-threatening symptoms. Lack of specific treatment options to target West Nile Virus contributes to morbidity and mortality rates, and increases the risk of infected people suffering through extensive recovery periods and persistent side-effects post recovery. There is currently a lot of scientific research being done to better understand the pathology of West Nile Virus. This research aims to better understand how West Nile Virus causes and maintains infections inside humans, in hopes of figuring out what antiviral medications would best target the virus and its mechanisms of infection.
One scientific study published in 2014 made important contributions to this field of research by improving our current understanding of how West Nile Virus and other flaviviruses are able to evade host immune responses in humans. This breakthrough study found evidence to support the belief that West Nile Virus evades host immune responses by preventing interferon production and signaling in infected cells. Not only that, the results of the study suggested that a specific human protein plays a vital role in preventing and combating WNV infection, and found a way to hamper the spread of WNV virions by re-targeting this protein to the endoplasmic reticulum during WNV replication.
This experimental study focused on the human MxA protein, which is induced by interferon production and localizes in the cytoplasm of cells. Previous studies have shown that MxA proteins have the ability to self-assemble into highly ordered oligomers and prevent active replication and infection of invading viral cells (Hoenen et al., 2014). Studies have also suggested that MxA’s antiviral properties had to do with their ability to oligomerize into ring-like structures that form around viral nucleocapsids and inhibit their replicative function (Hoenen et al., 2014). To study the effects of MxA protein in viral WNV cells, researchers used laboratory methods to express the human MxA protein into a WNV replicon, and then transfected the replicon into a WNV packaging cell line. Replicons were transfected using in vitro transcription and electroporation techniques. Subsequently, the researchers analyzed supernatants by performing titer tests to determine the concentrations of WNV particles in the solution. Concentrations of viral particles were performed using plaque assays. Interestingly, analysis showed that the titers recovered from the transfected WNV cell line were significantly lower than titers recovered from WNV replicons that were not transfected with human MxA protein (Hoenen et al., 2014). This lead the researchers to believe that human MxA protein may indeed play a role in inhibiting WNV cell replication.
Figure 1. Graph A displays data suggesting that the WNV particle concentrations (in infectious particles/mL) were lower for WNV cells transfected with human MxA protein than for regular WNV cells (Hoenen et al., 2014).
To study how human MxA protein might inhibit WNV cell replication, researchers also performed immunofluorescence analysis of the WNV cells transfected with human MxA protein. These immunofluorescence analysis involved cryofixation, preparations of cryosections, and immunolabeling with appropriate antibodies and proteins. Images were then developed by contrasting these immunolabeled mediums were viewed under a transmission electron microscope. The researchers looked at the effects of MxA protein on structural characteristics of the viral cells. After finding that MxA proteins aggregated in the cytoplasm of cells, they conducted more immunofluorescence tests and confocal microscopy investigations that looked at how MxA protein aggregates affected the distribution of viral proteins that comprised the West Nile Virus envelope. This investigated the claim that antiviral proteins like MxA inhibited West Nile Virus infection by disrupting the flavivirus envelope and thereby inhibiting replication abilities (Hoenen et al., 2014).The results of immunofluorescence assays showed that WNV cells that were transfected with human MxA protein, exhibited re-distribution of viral envelope proteins. The distribution of these proteins was also different from that of normal WNV cells, whose envelope proteins were locally diffused within the cytoplasm and nucleolus (Hoenen et al., 2014). Further analysis of viral envelope proteins in MxA transfected WNV cells was conducted using electron tomography. Electron tomography showed that aggregation of human MxA protein within WNV cells resembled organized bundles of hollow tubes located adjacent to membranes and vesicles involved in WNV replication. These results suggest that the co-localization of human MxA protein with viral replication structures may interfere with the ability of viral capsid proteins to assemble during the replication process; this could result in restricted production of infectious viral particles by WNV (Hoenen et al., 2014).
Researchers also wanted to better understand the antiviral properties of human MxA protein on WNV, so they cloned WNV cells and genetically transfected them with different genetically altered versions of human MxA protein. One type of human MxA protein that was MxA protein that contained a genetically altered targeting sequence that signaled for the protein to be retained in the endoplasmic reticulum (ER). Immunofluorescence assay showed that the during replication, transfected WNV cells exhibited localization of protein disulfide isomerase(PDI)-positive ER membranes in the ER with MxA (Hoenen et al., 2014). PDI-positive membranes have previously shown to play a role in WNV replication and assembly; localization of these membranes in the ER would therefore inhibit viral production (Hoenen et al., 2014). These results were significant because they suggested that human MxA protein could directing MxA protein to appropriate cellular locations would allow it to target its antiviral mechanisms to normally inaccessible viral components. The results of this study contribute greatly to the field of WNV research by providing evidence of promising antiviral effects of human MxA protein on West Nile Virus. Not only does the data suggest MxA has intrinsic abilities to inhibit viral particles, but it also suggests that MxA can also have antiviral effects on WNV when targeted to specific regions like the ER (Hoenen et al., 2014). In addition, the inhibition of viral particle production as a result of localization of viral replicative components to specific regions of the ER, implies that these are regions that flaviviruses might assemble within infected host cells.
Figure 2. Panels A and B reveal the presence of oligomerization of hollow tubes by human MxA protein in WNV cells and shows their location adjacent to viral membranes and vesicles related to replication. Panels C and D show the localization of MxA protein oligomer bundles near the rough ER. Panels E and F show the co-localization of MxA proteins with WNV virals in the ER together (Hoenen et al., 2014).
There are a number of additional research experiments that could be conducted in the future to follow up on the discoveries made by this 2014 study. First and foremost, it would be beneficial for this study to be replicated to verify the antiviral properties of human MxA protein on WNV or on different strains of WNV. It would also be beneficial to conduct similar studies using different interferon-induced human proteins to see if they may also have antiviral properties. Further studies that test different hypotheses on how human MxA proteins inhibit the production of new WNV particles would also be helpful in understanding what viral mechanisms should be targeted by antiviral compounds. One of the important questions to investigate asks whether or not human MxA protein has the potential for application as antiviral therapy for West Nile Virus. In order for a potential antiviral therapy to be developed, it would be important for future research how to stimulate interferon-induced production of human MxA proteins in the presence of WNV infection. At the same time, it would be valuable for researchers to begin studies on how human MxA proteins of people with WNV could be re-targeted to cells in certain regions of the body like the ER.
Hoenen, A., Gillespie, L., Morgan, G., van der Heide, P., Khromykh, Al, & Mackenzie, J. (2014). The West Nile virus assembly process evades the conserved antiviral mechanism of the interferon-induced MxA protein. Virology, 448, 104-116. doi: 10.1016/j.virol.2013.10.005
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