**Ebola Virus Nucleocapsid-Like Structures Utilize Arp2**/**3 Signaling for Intracellular Long-Distance Transport**

**Katharina Grikscheit 1,2, Olga Dolnik 1, Yuki Takamatsu 1,3, Ana Raquel Pereira 4 and Stephan Becker 1,2,\***


Received: 18 May 2020; Accepted: 16 July 2020; Published: 19 July 2020

**Abstract:** The intracellular transport of nucleocapsids of the highly pathogenic Marburg, as well as Ebola virus (MARV, EBOV), represents a critical step during the viral life cycle. Intriguingly, a population of these nucleocapsids is distributed over long distances in a directed and polar fashion. Recently, it has been demonstrated that the intracellular transport of filoviral nucleocapsids depends on actin polymerization. While it was shown that EBOV requires Arp2/3-dependent actin dynamics, the details of how the virus exploits host actin signaling during intracellular transport are largely unknown. Here, we apply a minimalistic transfection system to follow the nucleocapsid-like structures (NCLS) in living cells, which can be used to robustly quantify NCLS transport in live cell imaging experiments. Furthermore, in cells co-expressing LifeAct, a marker for actin dynamics, NCLS transport is accompanied by pulsative actin tails appearing on the rear end of NCLS. These actin tails can also be preserved in fixed cells, and can be visualized via high resolution imaging using STORM in transfected, as well as EBOV infected, cells. The application of inhibitory drugs and siRNA depletion against actin regulators indicated that EBOV NCLS utilize the canonical Arp2/3-Wave1-Rac1 pathway for long-distance transport in cells. These findings highlight the relevance of the regulation of actin polymerization during directed EBOV nucleocapsid transport in human cells.

**Keywords:** Ebola virus; actin cytoskeleton; nucleocapsid transport; Arp2/3 complex
