Function of the Viral Matrix Proteins VP40 and VP24 for the Life Cycle of Ebola Virus
Ebola virus (EBOV), a member of the family Filoviridae in the order Mononegavirales, is the causative agent of a severe haemorrhagic fever. Due to its high case fatality rate of up to 90% and to the fact that no approved vaccination or treatment is available for EBOV infection, it is classified as a...
Hygiene u. Med. Mikrobiologie mit Medizinaluntersuchungsamt
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|Summary:||Ebola virus (EBOV), a member of the family Filoviridae in the order Mononegavirales, is the causative agent of a severe haemorrhagic fever. Due to its high case fatality rate of up to 90% and to the fact that no approved vaccination or treatment is available for EBOV infection, it is classified as a biosafety level 4 (BSL4) agent, which restricts reasearch on it to a few facilities worldwide. Systems that model individual aspects of the viral life cycle under BSL2 conditions are, therefore, highly desirable. Based on available reverse genetics systems we have developed several new systems that allow the analysis of viral genome transcription, replication and packaging, as well as nucleocapsid morphogenesis, particle formation, budding, entry and initial transcription in target cells under BSL2 conditions. We were able to model two of these steps, morphogenesis of a fully functional nucleocapsid and initital transcription in target cells, for the first time for a negative strand RNA-virus, which is a significant advantage in reverse genetics systems for these viruses. The established systems were then used to analyze the role of EBOV proteins, particularly the matrix proteins VP24 and VP40, in the viral life cycle. The role of VP24, the minor matrix protein of EBOV, has long been enigmatic. Recently, it has been shown to be involved in interferon antagonism; however, data regarding a possible involvement of VP24 in nucleocapsid morphogenesis and particle formation have remained controversial. Using a newly developed infectious virus-like particle assay with na¨ıve target cells we were able to show that VP24 is not necessary for budding of particles or genome packaging, but that it is indispensable for the formation of functional nucleocapsids. This is the first functional evidence for a role of VP24 in nucleocapsid formation. Although the role of the major matrix protein, VP40, is much better understood, virtually nothing is known about the function of the different oligomeric forms of VP40, namely dimers, hexamers and octamers. Previously, we have been able to show that VP40 octamerization is indispensable for the viral life cycle. As part of this work we have further analyzed the role of VP40 octamerization. Also, based on the available crystal structures for VP40 we designed and characterized a dimerization incompetent VP40 mutant and included this mutant in our studies. We were able to show that VP40 dimerization is a prerequisite for budding, while octamerization does not play a role in this process. Also, VP40 octamerization is not important for packaging or the formation of a functional nucleocapsid. However, VP40 octamers seem to influence transcription and/or replication of viral genomes, a phenomenon that has been previously described for the matrix protein of Rabies virus, another member of Mononegavirales. Also, our data suggest that VP40 is involved in inhibition of cellular transcription and/or translation, a phenomenon widely known for matrix proteins of Mononegavirales, and that VP40 dimerization is important for this function. Finally, we analyzed the interactions of the nucleocapsid protein NP with VP40. We were, for the first time, able to directly show an interaction between these two proteins, and have mapped the interaction domain on VP40 to two β-strands in the N-terminal domain. Based on the crystal structure of VP40 we have identified two residues in this region that may be crucial for the interaction with NP. This work has increased our understanding of the role of EBOV matrix proteins in the viral life cycle, and has revealed several new functions for these proteins. The obtained results will allow us to specifically target individual aspects of the viral life cycle in order to develop new countermeasures against EBOV, but also to further investigate molecular details of these processes.|