**4. Discussion**

In this work, we identified CAD, an essential component of the de novo pyrimidine synthesis pathway, to be important for both EBOV genome replication and transcription, and demonstrated that the function of CAD in pyrimidine synthesis is responsible for this effect. Knockdown of CAD was also shown to affect replication and transcription of other viruses, e.g., hepatitis C viruses [38]. Furthermore, inhibitors of CAD, e.g., the antinucleoside N-phosphonacetyl-l-aspartate (PALA), which transiently inhibits the aspartate transcarbamylase activity of CAD, were effective in vitro against various viruses, including vaccinia virus and arenaviruses [39,40]. The fact that these compounds exhibit antiviral activity against a broad range of viruses qualifies CAD as a promising indirect antiviral target. However, whether PALA shows antiviral efficiency against EBOV remains to be investigated. Further, whether targeting viral RNA synthesis by inhibition of CAD will be synergistic with other inhibitors of EBOV RNA synthesis, such as remdesivir [41], will have to be addressed in future studies.

Our results are consistent with the fact that several pyrimidine synthesis inhibitors are effective against EBOV in vitro, underlining the importance of the pyrimidine pathway for these viruses [20,42]. Examples are the FDA-approved drug leflunomide and its active metabolite teriflunomide, as well as SW835, a racemic version of GSK983, which has been described to exhibit a broad-spectrum antiviral activity [20,42,43]. These compounds all impair de novo pyrimidine biosynthesis through inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme downstream of CAD in the pyrimidine pathway. Interestingly, treatment with these inhibitors seems to have similar inhibitory effects on EBOV minigenome assays compared to the effect we observe for CAD knockdown, although CAD activity is not directly affected [20,42]. Provision of pyrimidines or upstream metabolites, e.g., orotic acid, reversed antiviral activity of all pyrimidine pathway inhibitors in EBOV minigenome assays, which is consistent with our observation that supplementation with pyrimidines restores reporter activity after CAD knockdown. Interestingly, inhibition of DHODH by using SW835 not only showed pyrimidine depletion, but also stimulated ISG (interferon-stimulated gene) expression, which contributes to the innate immune response [42]. However, currently, the mechanism behind this stimulation of the innate immune response by DHODH inhibitors remains incompletely understood and needs to be further analyzed. Further supporting the importance of CAD for the EBOV lifecycle is the fact that the de novo pyrimidine synthesis activity of CAD is a prerequisite for cell division, which has been suggested to be necessary for productive infection of cells with EBOV [44].

We were further able to show that CAD is recruited to EBOV inclusion bodies, which represent the site of EBOV replication and transcription [8,9]. Since we observed CAD recruitment into NP-induced inclusion bodies during expression of NP alone and detected an interaction of CAD with NP using CoIP studies, we sugges<sup>t</sup> that this recruitment is mediated via an interaction of CAD with NP. So far, knowledge regarding direct interactions between CAD and the proteins of other viruses is limited, but Angeletti et al., showed that CAD recruits the preterminal protein (pTP) of adenoviruses to the site of adenovirus replication in the nuclear matrix via direct interaction. This interaction is believed to be required for anchorage of the adenovirus replication complex at the nuclear matrix in close proximity of the cellular factors required to segregate replicated and genomic viral DNA [45,46].

In the context of its cellular function, CAD has been shown to localize primarily in the cytoplasm, although small amounts can also be detected in the nucleus of dividing cells. Redistribution of CAD into nuclear compartments during cell growth and proliferation is believed to be in response to phosphorylation by MAP kinases at position Thr-456, which results in upregulation of the enzymatic activity of CAD [30]. Since NP is known to recruit a number of factors, including kinases and phosphatases, into inclusion bodies [16–18], it is possible that recruited CAD is activated in inclusion bodies in order to provide pyrimidines for EBOV replication and transcription. However, CAD lacking the CPS domain, which contains Thr-456, was still recruited into NP-induced inclusion bodies, excluding selective recruitment of Thr-456-phosphorylated and thus activated CAD into inclusion bodies.

Overall, we have shown that CAD is recruited into NP-induced and virus-induced inclusion bodies to provide sufficient amounts of pyrimidines for EBOV genome replication and transcription. Furthermore, we demonstrated that the GLN domain of CAD is required for recruitment into inclusion bodies. These findings increase our understanding of EBOV and its host cell interactions, and provide a basis for future identification of molecular targets for the development of novel therapies against this virus.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/5/1126/s1: Figure S1. Comparison of T7 and Pol-II-driven replication-deficient minigenomes.

**Author Contributions:** Conceptualization, J.B., L.W., T.H.; investigation, J.B., L.W., B.S.B., T.H.; supervision, T.C.M., T.H.; visualization, J.B., T.H.; funding acquisition, T.H.; writing—original draft preparation, J.B., T.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** Funding was provided by the Deutsche Forschungsgemeinschaft (DFG), gran<sup>t</sup> number 389002253, (J.B., L.W.), and by the Friedrich-Loeffler-Institut, intramural funding (T.H.), and funding as part of the VISION consortium (B.S.B.).

**Acknowledgments:** The authors would like to thank Logan Banadyga (Public Health Agency of Canada) for his help in establishing the coIP procedure and Stephan Becker (Philipps University Marburg) for providing cell lines. We further thank Luca Zaeck (Friedrich-Loeffler-Institut) for technical assistance with the confocal microscope, as well as Allison Groseth and Sandra Diederich (Friedrich-Loeffler-Institut) for technical assistance with the BSL4 work.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of the data; in the writing of the manuscript, or in the decision to publish the results.
