**4. Discussion**

In this study, we have identified the Rac1/Wave1/Arp2/3 pathway as being involved in the actin-dependent transport of EBOV NCLS in human cell culture. Arp2/3 activity is also essential to inducing propulsive and polar-localized actin tails at the rear ends of NCLS, which can be robustly visualized in live cell imaging. Furthermore, the association of actin tails with EBOV NCLS and nucleocapsids can also be observed via super resolution in fixed samples.

Actin comet tails were initially characterized in *Listeria monocytogenes* in the late 1980s [40]. Since then, diverse types of intracellular pathogens have been identified as inducing actin polymerization at their surface. For instance, vaccinia virus protein A36 is able to recruit NCK and GRB2, which in turn recruit N-WASP to stimulate the Arp2/3 complex, or virus protein p78/83 mimics N-WASP and directly activates Arp2/3-induced actin polymerization [30,41]. Here, we showed that EBOV NCLS induce actin tails at one end, and actin tail formation is sensitive to the inhibition of Arp2/3 using CK666, indicating that the hijacking of the highly abundant and conserved Arp2/3 complex for induction of actin tails is a common mechanism in diverse types of pathogens without a common origin [30].

We further show that directed long-distance transport is regulated via Arp2/3 activity, ye<sup>t</sup> the siRNA depletion of Arp3 in cells forming NCLS does not entirely block intracellular transport. Furthermore, the e ffects of siRNA treatment on track straightness appear more pronounced after Rac1 knockdown, when compared to the depletion of Arp3 or Wave1. These findings also sugges<sup>t</sup> that other actin regulators downstream of Rac1 either compensate Arp2/3 activity, or synergistically regulate actin polymerization. One candidate could be the sca ffolding protein IQGAP1 that interconnects multiple pathways of actin dynamics and interacts with Rac1. In cells infected with MARV, IQGAP1 was recruited to inclusion and to the read end of nucleocapsids, and the down-regulation of IQGAP1 resulted in the impaired release of MARV, suggesting a role for other major actin regulators in this process [14].

In addition, we also gained unprecedented evidence that Wave1 upstream of Arp2/3 is involved in regulating the long-distance transport of NCLS in Huh7 cells. Given that nucleocapsid transport in EBOV-infected cells does not depend on *N*-WASP [3], we concluded that other NFPs could be involved in the upstream regulation of Arp2/3. Further supporting this notion, N-WASP is typically involved in endocytotic events, where it regulates actin polymerization in a manner reminiscent of actin tails that propel pathogens through cells [42]. Consequently, enveloped viruses, such as vaccinia and EBOV, might profit from mimicking or recruiting N-WASP during their life cycle.

In recent years, additional NPFs, like WASH, WHAMM and JMY, have been described to promote actin tail formations at intracellular membranes like endosomes and autophagosomes [43–45]. In contrast, filoviral nucleocapsids travel in the cytosol without a membrane, and are highly structured protein complexes that probably utilize endocytosis-independent pathways for their transport, thereby likely avoiding membranous structures prior to their arrival at the plasma membrane. Wave1 and Rac1 signaling is considered primarily relevant to actin polymerization in lamellipodial cell protrusions [43], thus it is not surprising that long-distance NCLS transport is best observed in areas with a high activity of this pathway in Huh7 cells. Future studies using high resolution microscopy or electron microscopic approaches shall reveal whether and how actin regulators are actively recruited to EBOV nucleocapsids. It could be that at di fferent stages of the nucleocapsid transport, from inclusion bodies to the budding sites, di fferent actin tail-inducing machineries are exploited by the virus.

As described for other viruses, the long-distance transport of EBOV NCLS is accompanied by the induction of polar actin polymerization, likely resulting in the directionality of movement also observed in other actin tail-inducing pathogens and in in vitro reconstructions [41,46,47]. How this polar induction of actin polymerization is initiated is not understood in detail. One hypothesis derives from studies in *Listeria*, where it was shown that the surface protein ActA accumulates locally, likely during cell wall growth, which subsequently results in polar interactions with actin regulators [48]. In contrast, filoviral nucleocapsids are not enveloped when they leave inclusion bodies, and only encounter the viral proteins GP and VP40, which are transported independently, when they reach the plasma membrane to form infectious particles [49–52]. Thus, it remains unclear how this polar actin polymerization is induced in filoviral nucleocapsids, and which viral protein might be relevant for the actin polymerization. One explanation derives from a study investigating the structure of MARV capsids using cryo-electron microscopy, revealing that nucleocapsid assembly itself results in polar structures [10]. Here, it was demonstrated that MARV nucleocapsids are highly oriented towards the plasma membrane, with the pointed end of the nucleocapsid directed towards the plasma membrane prior to budding [11,53]. Thus, these findings support the notion that polar nucleocapsid assembly itself might result in specific protein conformations, thereby exposing the binding sites for cellular proteins that induce actin polymerization.

Taken together, our minimalistic transfection-based system reveals new opportunities to study the cellular transport of filoviral nucleocapsids, and to reliably quantify the dynamics of subviral structures. Future studies have to further characterize how filoviral proteins modulate highly spatial–temporal RhoGTPase signaling, and identify the direct interaction partners and structural changes that are required for long-distance transport during the EBOV life cycle.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/7/1728/s1, Figure S1: Analysis of tracks in cells treated with the Arp2/3 inhibitor CK666 in subgroups, Movie S1: NCLS induce actin tails during long-distance movement. Huh7 cells expressing EBOV proteins NP, VP35, VP24 and VP30-GFP (NCLS) and LifeAct-CLIP were monitored by time-lapse microscopy. Here, a single NCLS structure is followed showing a pulsative actin tail, Movie S2: NCLS movement and actin tails depend on Arp2/3 activity. A Huh7 cell expressing EBOV proteins NP, VP35, VP24 and VP30-GFP (NCLS) and LifeAct-CLIP was monitored by time lapse confocal microscopy. Note that NCLS capsids transported over long distances show actin tails (white arrow). The same cells were then incubated with the Arp2/3 inhibitor CK666 and reimaged. All long–distance movement is abolished while actin structures like filopodia and stress fibers remain una ffected.

**Author Contributions:** Conceptualization, K.G. and S.B.; methodology, K.G., Y.T., O.D., A.R.P.; formal analysis, K.G.; investigation, K.G., O.D., A.R.P.; writing—original draft preparation, K.G., S.B.; writing—review and editing, K.G., S.B., O.D.; visualization, K.G.; supervision, S.B.; funding acquisition, S.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by German Centre for Infectious Research (DZIF; number FKZ8009801908) and by the Deutsche Forschungsgemeinschaft (DFG) through the Sonderforschungsbereich 1021 project B03.

**Acknowledgments:** We thank Astrid Herwig and Martina Weik for their technical support. We also wish to thank Katrin Roth (Marburg Imaging Facility) and Andreas Rausch (THM Giessen) for their support and helpful discussion.

**Conflicts of Interest:** The authors declare no conflict of interest.
