The Cellular Protein CAD is Recruited into EbolaVirus 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.
Rabbit Anti-Zaire-Ebola virus nucleoprotein (Mayinga EBOV NP) protein antiserum
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.
ebov-detection
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.
Production and Characterization of Monoclonal Antibodies Against GpProtein of EbolaVirus.
The DNA fragment encoding predicted main antigenic region, aa 1-300 on Gp protein of Ebola virus (EBOV) was cloned into the vector pGEX-KG. The recombinant GST-tagged Gp-300 was expressed in Escherichia coli BL21 (DE3) by induction with 1 mM isopropyl-1-thio-b-d-galactoside and purified by dialysis.
Four monoclonal antibodies (mAbs) named 1C4, 2A3, 2G7, and 2H9 against Gp protein were generated by fusing mouse myeloma cell line SP2/0 with spleen lymphocytes from Gp-300 protein-immunized mice.
The activity of the mAbs was then characterized by enzyme-linked immunosorbent assay, indirect immunofluorescent assays (IFA), and western blot analysis.
The results demonstrated that all the mAbs showed high specificity and sensitivity in IFA and in western blot analysis, which indicated that these mAbs against Gp protein of EBOV may be used as valuable tools for analysis of the protein functions and pathogenesis of EBOV.
Recombinant Zaire-Ebola virus nucleoprotein (Mayinga EBOV NP) protein control for Western
The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems.
Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different protein compositions.
Only three of the five EBOV nucleocapsid proteins-nucleoprotein, VP35, and VP24-were necessary and sufficient to form transport-competent nucleocapsid-like structures.
The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells.
Ebola virus Nucleoprotein Antibody
The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix protein VP40 and the viral surface glycoprotein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.
