The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription.
Ebola virus (EBOV) is a zoonotic pathogen causing severe hemorrhagic fevers in humans and non-human primates with high case fatality rates. In recent years, the number and extent of outbreaks has increased, highlighting the importance of better understanding the molecular aspects of EBOV infection and host cell interactions to control this virus more efficiently. Many viruses, including EBOV, have been shown to recruit host proteins for different viral processes. Based on a genome-wide siRNA screen, we recently identified the cellular host factor carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being involved in EBOV RNA synthesis. However, mechanistic details of how this host factor plays a role in the EBOV life cycle remain elusive. In this study, we analyzed the functional and molecular interactions between EBOV and CAD. To this end, we used siRNA knockdowns in combination with various reverse genetics-based life cycle modelling systems and additionally performed co-immunoprecipitation and co-immunofluorescence assays to investigate the influence of CAD on individual aspects of the EBOV life cycle and to characterize the interactions of CAD with viral proteins. Following this approach, we could demonstrate that CAD directly interacts with the EBOV nucleoprotein NP, and that NP is sufficient to recruit CAD into inclusion bodies dependent on the glutaminase (GLN) domain of CAD. Further, siRNA knockdown experiments indicated that CAD is important for both viral genome replication and transcription, while substrate rescue experiments showed that the function of CAD in pyrimidine synthesis is indeed required for those processes. Together, this suggests that NP recruits CAD into inclusion bodies via its GLN domain in order to provide pyrimidines for EBOV genome replication and transcription. These results define a novel mechanism by which EBOV hijacks host cell pathways in order to facilitate genome replication and transcription and provide a further basis for the development of host-directed broad-spectrum antivirals.
['Brandt|Janine|J|', 'Wendt|Lisa|L|', 'Bodmer|Bianca S|BS|', 'Mettenleiter|Thomas C|TC|', 'Hoenen|Thomas|T|0000-0002-5829-6305']
D000818:Animals D001221:Aspartate Carbamoyltransferase D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) D002460:Cell Line D004080:Dihydroorotase D029043:Ebolavirus D055785:Gene Knockdown Techniques D016679:Genome, Viral D006801:Humans D007181:Inclusion Bodies, Viral D009698:Nucleoproteins D011485:Protein Binding D000072417:Protein Domains D011743:Pyrimidines D012313:RNA D014158:Transcription, Genetic D014764:Viral Proteins D014779:Virus Replication
CAD Ebola virus inclusion bodies pyrimidine synthesis filovirus