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Role of the host proteins NXF1, UAP56, and CAD in the life cycle of ebolaviruses
Project
Project code: FLI-IMVZ-08-Ri-0581, 389002253
Contract period: 01.11.2017
- 30.01.2021
Purpose of research: Experimental development
Ebolaviruses are zoonotic pathogens that cause severe hemorrhagic fevers with high case fatality rates in both humans and non-human primates. While endemic in Africa, the recent massive outbreak of ebolavirus in West Africa has demonstrated the global impact these viruses can have. Until now no approved antiviral therapies are available, and treatment of ebolavirus-infected patients is usually limited to supportive measures. The targeting of host factors for the development of antivirals has numerous advantages over the traditional approach of targeting viral factors. Particularly, the occurrence of viral escape mutants should be less frequent, and even distantly related viruses often use the same host factors to accomplish similar functions in their life cycle. Thus, targeting of host factors offers the potential that the identified antivirals will be active against a broad range of viruses. This not only has implications for the availability of these antivirals against newly emerging viruses, but also makes their development more feasible with respect to economic as well as practical considerations, particularly with respect to safety and efficacy testing. However, for such an approach to be feasible, we need an increased understanding of the virus-host interface, particularly with respect to the involvement of host proteins in fundamental aspects of the virus life cycle, such as genome replication, transcription, and viral mRNA translation. To address this issue, we have recently performed a genome-wide siRNA screen for host factors important for these processes. This screen identified the proteins NXF1 and UAP56, which are RNA-associated proteins, and CAD, which is involved in nucleotide metabolism. We now propose to further investigate the involvement of these host factors in the ebolavirus life cycle. To this end we will take advantage of a number of life cycle modelling system that we have developed over the last decade, to allow us to model parts of or even the whole ebolavirus life cycle in a biosafety level (BSL) 1 laboratory. These studies will be complemented by BSL4 work with recombinant and wild-type ebolaviruses, in order to further characterize the functional and biochemical interactions of the identified host factors with ebolaviruses. Further, we will address these host factors with respect to their relationship to inclusion bodies, which are important structures induced in infected cells that we have recently identified as being sites of ebolavirus genome replication, analogous to what has been shown for several other virus families. These studies will address an urgent need to increase our knowledge regarding the virus-host interface for ebolaviruses, and hopefully also negative sense RNA viruses in general, in a way that may in future be leveraged to aid the development of antiviral against this and other important but neglected emerging infectious diseases.
Results
Virus host interactions of ebolaviruses with cellular proteins were investigated using a multipronged approach involving coimmunoprecipitation, RNA-coimmunoprecipitation, coimmunofluorescence analysis, reverse genetics-based life cycle modelling systems, and infection experiments under biosafety level 4 conditions. Using this approach, for CAD and NXF1 we could decipher the precise role these host factors play in the ebolavirus life cycle.
CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) catalyzes the first reaction in the de novo pyrimidine synthesis in the cell. Using both siRNA knockdown experiments and studies involving chemical inhibitors of this pathway synthesis we could demonstrate that de novo pyrimidine synthesis is essential for the ebolavirus life cycle (Martin et al., 2018). We could further show that CAD interacts with the ebolavirus nucleoprotein NP, and is recruited into inclusion bodies, which are viral structures induced in infected cells, and which have been shown to be sites of viral RNA synthesis (Brandt et al., 2020). Finally, using a combination of life cycle modelling systems and substrate rescue experiments we could show that it indeed is the function of CAD in the de novo pyrimidine synthesis that is essential for both virus genome replication and genome transcription.
NXF1 (nuclear export factor 1) is the major export factor for mRNAs out of the nucleus of cells. However, since the ebolavirus replication cycle takes place entirely outside of the cell nucleus, the precise function of NXF1 in the ebolavirus replication cycle as demonstrated in a genome wide siRNA screen (Martin et al., 2018) was enigmatic. Using a similar approach as for CAD, we could show that NXF1 interacts with the ebolavirus nucleoprotein NP (Wendt et al., 2020). Interestingly, NXF1 is also able to interact with viral mRNAs, but not with genomic RNAs, and there is a competition between RNA and NP in their binding of NXF1. More detailed studies revealed that the RNA-binding site and the RNA-binding site of NXF1 interact with each other. Coimmunofluorescence analysis showed that NXF1 is recruited into inclusion bodies, but appears to be rapidly exported from these structures again upon RNA binding. Finally, using life cycle modelling systems we could demonstrate that while NXF1 is required for viral protein expression, but not for genome replication, it must be involved in a step downstream of viral mRNA synthesis but upstream of viral mRNA translation. Together, these findings allow us to postulate a model in which NXF1 is recruited into inclusion bodies, where it interacts with NP and takes over nascent mRNAs from NP, resulting in a rapid export of NXF1:viral mRNA complexes from these structures for efficient viral protein expression. This defines a completely novel function of NXF1 that might also have implications for other viruses replicating in inclusion bodies. Details of this process and a possible role of NXF1 for other viruses are now being studied in a follow-up project.
Brandt J, Wendt L, Bodmer BS, Mettenleiter TC, Hoenen T. 2020. The cellular protein CAD is recruited into Ebola virus inclusion bodies by the nucleoprotein NP to facilitate genome replication and transcription. Cells 9(5):E1126.
Martin S, Chiramel AI, Schmidt ML, Chen YC, Whitt N, Watt A, Dunham EC, Shifflett K, Traeger S, Leske A, Buehler E, Martellaro C, Brandt J, Wendt L, Müller A, Peitsch S, Best SM, Stech J, Finke S, Römer-Oberdörfer A, Groseth A, Feldmann H, Hoenen T. 2018. A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle. Genome Med 10(1):58.
Wendt L, Brandt J, Bodmer BS, Reiche S, Schmidt ML, Traeger S, Hoenen T. 2020. The Ebola virus nucleoprotein recruits the nuclear RNA export factor NXF1 into inclusion bodies to facilitate viral protein expression. Cells 9(1):187.
Section overview
Subjects
- Animal health
- Biotechnology
Framework programme
Funding programme
Excutive institution
FLI - Institute of Molecular Virology and Cell Biology (FLI - IMVZ)
