**1. Introduction**

Ebola virus (EBOV) is a zoonotic pathogen belonging to the genus *Ebolavirus* within the order *Filoviridae*, and is the causative agen<sup>t</sup> of severe hemorrhagic fevers in humans and non-human primates with high case fatality rates [1,2]. Increasing numbers of EBOV outbreaks in Africa highlight the importance of understanding the molecular mechanisms of the EBOV life cycle and virus-host cell interactions better in order to develop new countermeasures against this virus. EBOV possesses a non-segmented single-stranded RNA genome of negative polarity that forms a helical nucleocapsid in the center of virions together with the ribonucleoprotein (RNP) complex proteins. During assembly of the nucleocapsid, the RNA genome is tightly coated with the viral nucleoprotein (NP), which protects

it from degradation and recognition by the cellular immune response [3]. During EBOV infection, NP-associated RNA genomes serve as templates for mRNA transcription and genome replication [4]. For viral replication, NP interacts with the polymerase cofactor VP35, which acts as a linker between NP and the RNA-dependent RNA polymerase L [5]. NP, VP35, and L are sufficient to facilitate EBOV genome replication, while for viral transcription the transcriptional activator VP30 is additionally required [6,7]. EBOV replication and transcription takes place in cytoplasmic inclusion bodies, which represent a characteristic feature of EBOV infections in cells [8,9]. Their formation can be driven by the expression of NP alone [5,10,11]. Due to the limited number of viral genes, successful genome replication and transcription is highly dependent on host cell factors, which play an important role during the EBOV life cycle. For instance, the host factor STAU1 has been shown to interact with multiple EBOV RNP components, and to redistribute into NP-induced or virus-induced inclusion bodies, suggesting that STAU1 plays a crucial role during viral RNA synthesis by facilitating the interaction between the viral genome and RNP proteins [12]. EBOV has also been shown to recruit SMYD3 into inclusion bodies, which modulates NP-VP30 interaction and enhances mRNA transcription [13]. Similarly, RBBP6 was found to influence EBOV replication by disrupting the interaction between NP and VP30 [14]. Importin-α7 was described as being required for the efficient formation of inclusion bodies [15]. Furthermore, several cellular kinases and phosphatases are known to localize in inclusion bodies to support EBOV replication and transcription [16–18]. Finally, we previously showed that EBOV NP recruits the nuclear RNA export factor 1 (NXF1) into inclusion bodies to facilitate viral mRNA export from these structures into the cytoplasm [19]. Despite this recent progress in our understanding of the interplay between host factors and EBOV, there remains a considerable need to identify and, more importantly, characterize further host factors required for EBOV replication to identify novel targets for antiviral drug development.

We previously performed a genome-wide siRNA screen using a minigenome system to identify potential host-directed targets [20]. In this system, a minigenome, i.e., a truncated version of the EBOV genome lacking all viral open reading frames (ORF) and consisting of a reporter gene (e.g., a luciferase or green fluorescent protein) flanked by the viral non-coding terminal leader and trailer regions, is expressed from a plasmid in mammalian cells together with the plasmids encoding the viral RNP proteins [6]. For initial transcription of the minigenome RNAs from the minigenome-encoding plasmids most existing EBOV minigenome systems use a T7 RNA polymerase (T7) promoter, and therefore require expression of T7 polymerase, which is usually provided via a T7-expressing plasmid that is cotransfected with the plasmids encoding the RNP proteins [6,21]. However, recently, an EBOV minigenome system using the cellular RNA polymerase II (Pol-II) for initial minigenome RNA transcription has also been established and shown to be more efficient at least in some cell types [22]. After initial transcription and encapsidation by RNP proteins, minigenome RNAs are recognized as authentic templates by the viral polymerase due to their leader and trailer regions, and are replicated and transcribed into mRNAs, which results in expression of the reporter protein. Thus, minigenome assays allow us to study viral genome replication and transcription, as well as viral protein expression, outside of maximum containment laboratories, simplifying the identification of host factors involved in these processes. By using this system, we recently identified the trifunctional protein carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) as being important for the EBOV life cycle [20].

CAD is an important component of the pyrimidine pathway that catalyzes the first three steps during the de novo biosynthesis of pyrimidine nucleotides using its four distinct enzymatic domains [23–25]. The first domain, glutaminase (GLN), initiates the pathway by catalyzing the hydrolysis of glutamine. This is followed by the synthesis of carbamoyl phosphate facilitated by the carbamoyl-phosphate synthetase (CPS). Carbamoyl phosphate is in turn the substrate for the aspartate transcarbamylase (ATC), which catalyzes the reaction of aspartate with carbamoyl phosphate to carbamoyl aspartate [26,27]. Finally, carbamoyl aspartate is converted to dihydroorotate by dihydroorotase (DHO) [28]. In response to cell growth and proliferation, CAD activity is upregulated

by phosphorylation through MAP kinases at position Thr-456, while in resting cells Thr-456 is dephosphorylated [29]. Furthermore, CAD is known to primarily localize in the cytoplasm of resting cells, but in response to cell growth and Thr-456 phosphorylation a small fraction is translocated into nuclear compartments, suggesting a cellular function of CAD in the nucleus [30,31]. However, little is known about the role of CAD during virus infection, and particularly the role of CAD in the EBOV life cycle still needed to be further analyzed. Therefore, we wanted to characterize the interaction of CAD with EBOV on both a biochemical and functional level. Based on our results, we sugges<sup>t</sup> that CAD is important for both genome replication and transcription due to its function in pyrimidine synthesis and that it is recruited into NP-induced and virus-induced inclusion bodies to facilitate the de novo biosynthesis of pyrimidine nucleotides.

### **2. Materials and Methods**

### *2.1. Cell Lines*

Human embryonic kidney cells (HEK 293T, Collection of Cell Lines in Veterinary Medicine CCLV-RIE 1018), African green monkey kidney cells (Vero E6, kindly provided by Stephan Becker, Philipps University Marburg), and human hepatocellular carcinoma cells (Huh7, kindly provided by Stephan Becker, Philipps University Marburg) were cultured in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 μg/mL streptomycin (PS; Thermo Fisher Scientific), and 1× GlutaMAX (Thermo Fisher Scientific). All cells were incubated at 37◦C and 5% CO2.

### *2.2. Plasmids and Cloning*

Minigenome assay components, including expression plasmids coding for the EBOV RNP proteins, T7 polymerase, firefly luciferase, and a classical T7-driven monocistronic minigenome (pT7-1cis-EBOV-vRNA-nLuc) have been previously described [20,32]. A NanoLuc luciferase-expressing T7-driven replication-deficient minigenome was cloned from a classical minigenome expressing NanoLuc luciferase as a reporter by deletion of 55 nucleotides (nt) in the antigenomic replication promoter as previously described [32]. Based on this, a Pol-II-driven replication-deficient minigenome was generated by PCR to amplify a linear version of the replication-deficient minigenome flanked by hammerhead and hepatitis delta virus ribozymes using primers #4571 (5-AGC TTA CGT GAC TAC TTC CTT CGG ATG CCC AGG TCG GAC CGC G-3) and #4572 (5-GAC CGG TAG AAA ACT GAT GAG TCC GTG AGG ACG AAA CGG AGT CTA GAC TCC GTC TTT TCC AGG AAT CCT TTT TGC AAC GTT TAT TCT G-3). The linearized construct was subsequently inserted into pCAGGS. The CAD gene was cloned from 293T cells into pCAGGS, and deletion mutants and domains of CAD were then generated using PCR-based approaches. All constructs were first cloned into pCAGGS, followed by subcloning into a pCAGGS plasmid encoding an N-terminal FLAG/HA-tag (DYKDDDDKLDGGYPYDVPDYA) immediately upstream of a BsmBI cloning site, allowing a seamless insertion of the open reading frame of interest. The expression plasmid for N-terminally myc-tagged VP35 was constructed by cloning a myc-tag (EQKLISEEDL) immediately before the VP35 ORF. Detailed cloning strategies are available on request.
