**1. Introduction**

Ebola virus (EBOV) belongs to the genus *Ebolavirus* within the family *Filoviridae* and causes a severe hemorrhagic fever, called Ebola virus disease, in humans with high case fatality rates of about 40–60% [1,2]. Ongoing and past outbreaks of Ebola virus disease in Africa highlight the importance of a better understanding of the EBOV life cycle in order to develop new therapeutic approaches. During the viral life cycle the EBOV nucleoprotein (NP) encapsidates the negative-stranded RNA genome and is essential for viral replication and transcription [3]. NP interacts with the transcriptional activator viral protein 30 (VP30), which bridges NP and the RNA-dependent RNA polymerase L [4–6]. Furthermore, NP interacts with the polymerase cofactor VP35 [5,6]. This interaction regulates the oligomerization and RNA-binding of NP, and also bridges NP to L [5–9]. NP, VP35, VP30, and L, together with the RNA genome, form the ribonucleoprotein complex (RNP) and are su fficient to mediate viral replication and transcription [3], which takes place in cytoplasmic inclusion bodies [10]. The formation of these inclusion bodies is driven by expression of NP, which is localized in these structures not only during infection, but also after sole expression of this protein [5,6]. However, only limited knowledge exists regarding host factors that interact with the viral proteins and RNAs found in these structures. One such host factor that has been identified is importinα7, which seems to be involved in inclusion body formation [11]. Marburg virus, a close relative of EBOV, was shown to recruit components of the endosomal sorting complex required for transport (ESCRT) to inclusion bodies to facilitate the tra fficking of nucleocapsids to the plasma membrane for viral assembly and budding [12,13]. Kinases and phosphatases such as PP2A-B56 are also known to be recruited to inclusion bodies, and are important in regulating the activity of VP30 in viral RNA synthesis, which is dependent on its phosphorylation status [14,15]. Similarly, RBBP6 appears to regulate the balance of replication and transcription by binding to VP30, and also Staufen1 was described to influence viral RNA synthesis [16,17]. Finally, EBOV VP35 appears to sequester cellular stress granule proteins within inclusion bodies in order to prevent stress granule formation [18].

To obtain a comprehensive picture of the pro- and anti-viral factors that are important for EBOV RNA synthesis (i.e., genome replication and transcription) and/or protein expression, we recently performed a genome-wide siRNA screen [19]. As primary readout we used a minigenome assay (reviewed in [20]). In this assay RNA minigenomes, i.e., miniature versions of the EBOV genome with all viral genes removed and replaced with a reporter gene, are expressed in mammalian cells together with the viral RNP proteins. Since the minigenomes still contain the regulatory terminal leader and trailer regions of the EBOV genome that carry the replication and transcription promoters, the RNP proteins recognize these minigenomes as authentic templates. This results in their replication and transcription, and ultimately reporter protein expression reflecting these aspects of the viral life cycle. In our siRNA screen we showed that the nuclear RNA export factor 1 (NXF1) is necessary for the EBOV life cycle, and also for the life cycle of the highly pathogenic New World arenavirus Junín virus. These data sugges<sup>t</sup> a mechanism of action that may be conserved among several cytoplasmically replicating negative-stranded RNA viruses (NSVs) that share commonalties in their replication cycles, such as replication in cytoplasmic inclusion bodies [10,21–24]. Importantly, we also could show that NXF1 is important for the life cycle of EBOV in context of infectious virus [19]. However, the precise function of NXF1 in the EBOV life cycle remained unclear.

NXF1 is a crucial component of the nuclear mRNA export pathway, where it exports cellular mRNAs from the nucleus by interacting with nucleoporins [25,26]. For this interaction NXF1 forms a stable heterodimer with p15 (also called NXT1) via its NTF2-like domain (NTF2) [27,28]. The NTF2-like domain is one of the five functional domains found in NXF1, with the other ones being the RNA-binding domain (RBD), the pseudo RNA recognition motif (RRM), a region of leucine-rich repeats (LRR), and the ubiquitin-associated domain (UBA) [27,29,30]. NTF2 and UBA promote interaction with nucleoporins, while the three amino-terminal domains (RBD, RRM, and LRR) are important for mRNA binding [26,27,31]. Recently, NXF1 was shown to be loaded onto cellular mRNAs co-transcriptionally, a process that depends on several other export adapters that promote the interaction between NXF1 and mRNA, in order to enable mRNA export from the nucleus [29,31,32]. However, the NXF1-driven nuclear mRNA export pathway is also utilized by many viruses that replicate in the nucleus [33–36]. For instance, herpesviruses and influenza viruses usurp this host pathway to export their viral RNAs, and particularly their mRNAs, from the nucleus [33,34,37–39]. Furthermore, NXF1 is known to export retroviral RNAs containing constitutive transport elements and Hepatitis B virus pregenomic RNA [35,36]. Thus far, the only non-nuclear function of NXF1 has been described for gammaretroviruses, which use NXF1 for loading of gag RNA onto polysomes, rather than for mRNA export [40]. However, since EBOV replicates in cytoplasmic inclusion bodies, the function of NXF1 during an EBOV infection must di ffer from those previously described [10]. Therefore, we characterized the interaction of NXF1 with EBOV on a biochemical and functional level. Based on our results we propose a model in which NXF1 is recruited to inclusion bodies where it interacts with NP and subsequently with viral mRNAs, which it then exports from inclusion bodies.

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