Our innate immunity is our first line of defence against pathogens. We require this immunity to control the numerous viral infections we are challenged with during our lives. However, little is known about the exact molecular mechanisms of our innate immunity, particularly components that have specific antiviral potential. One potent mediator of this antiviral activity is the interferon system. Activation of the interferon system leads to the production of several interferon-induced proteins, which inhibit the multiplication of viruses by distinct mechanisms. A key example of one of these mediators is the human MxA protein. Human MxA has the capacity to inhibit many different viruses from diverse families. In many cases it is proposed that MxA interferes with key viral components, such as incoming or newly formed nucleocapsids. It is speculated that MxA traps and missorts these viral components so they are no longer available for virus production and virus dissemination is inhibited. West Nile virus belongs to Flaviviridae family of viruses and was involved in the outbreak of virus-associated encephalitis in New York City in 1999. In this thesis I show that West Nile virus is insensitive to antiviral activity of MxA and describe how West Nile virus has developed a replication strategy that avoids MxA recognition and activation. I show that virus-induced changes of cytoplasmic membranes provide a protective microenvironment for viral replication and the viral components essential for viral replication. This hypothesis was proven by preventing the formation of these membrane structures with the fungal chemical Brefeldin A. Under these conditions I observed that stable expression of MxA could partially restrict West Nile virus RNA replication. Subsequently, I showed that the assembly mechanism of West Nile virus prevents interaction between the MxA protein and the viral capsid proteins. This was achieved by the use of a trans-packaging cell line whereby the West Nile virus structural proteins are expressed stably in trans instead of in cis from the polyprotein. When this cell line was transfected with a West Nile virus replicon expressing the human MxA protein distinct co-localisation and redistribution of the MxA with West Nile virus capsid proteins into large tubular structures within the cytoplasm of transfected cells was observed. Strikingly, these tubular aggregates are visually analogous to structures observed during infection of MxA expressing cells infected with members of the Bunyaviridae, particularly La Crosse virus. Moreover, retargeting MxA to specific sites of the endoplasmic reticulum in cells transfected with the West Nile virus infectious clone resulted in co-localisation between MxA and the West Nile virus capsid proteins and significantly inhibited the production of infectious particles. These results suggest that the sequestering of viral capsids within cytoplasmic inclusions maybe a conserved mechanism for antiviral activity of the MxA protein across the viruses families and highlight the innate ability of such molecules to recognise key molecular patterns within pathogens. Finally, I sought to exploit the antiviral potential of MxA as a therapeutic agent against infection with Influenza A viruses; viruses that have a very high sensitivity for the antiviral activity of MxA. By expressing MxA from the West Nile virus replicon, infection with the highly pathogenic Influenza virus H5N1 strain could be inhibited in vitro. Furthermore, in vivo studies in Mx-negative mice indicated that intranasal inoculation with MxA expressed from the West Nile virus replicon can protect these mice against an otherwise lethal infection with a low pathogenic Influenza A virus. Taken all together, in this thesis I provide evidence that strongly supports the existence of an evolutionary working mechanism of a significant mediator of our immune system, the antiviral MxA protein. Furthermore, I show how an important human pathogen, such as West Nile virus has evolved a replication strategy to evade this antiviral protein. These results will open new pathways for the development of a new type of antiviral therapies that utilize the potent antiviral activity of the MxA protein.
| Identifer | oai:union.ndltd.org:ADTP/279306 |
| Creators | Antje Hoenen |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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