Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and RNA replication, housing key steps in the virus life cycle that warrant further investigation. During infection IBs display dynamic properties regarding their size and location. Also, the contents of IBs must transition prior to further viral maturation, assembly and release, implying additional steps in IB function. Interestingly, expression of the viral nucleoprotein (NP) alone is sufficient for generation of IBs, indicating that it plays an important role in IB formation during infection. In addition to NP, other components of the nucleocapsid localize to IBs, including VP35, VP24, VP30 and the RNA polymerase L. Previously we defined and solved the crystal structure of the C-terminal domain of NP (NP-Ct), but its role in virus replication remained unclear. Here we show that NP-Ct is necessary for IB formation when NP is expressed alone. Interestingly, we find that NP-Ct is also required for production of infectious virus-like particles, and that defective VLPs with NP-Ct deletions are significantly reduced in viral RNA content. Furthermore, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation, demonstrating a functional interaction between the two proteins. Of all the EBOV proteins only VP35 is able t3o overcome the defect in IB formation caused by deletion of NP-Ct. This effect is mediated by a novel protein-protein interaction between VP35 and NP that controls both regulation of IB formation and RNA replication itself, and which is mediated by a newly identified functional domain of NP, the “central domain” (CD). IMPORTANCE Inclusion bodies (IBs) are cytoplasmic sites of RNA synthesis for a variety of negative sense RNA viruses including Ebola virus. In addition to housing important steps in the viral life cycle, IBs protect new viral RNA from innate immune attack and contain specific host proteins whose function is under study. A key viral factor in Ebola virus IB formation is the nucleoprotein, NP, which also is important in RNA encapsidation and synthesis. In this study, we have identified two domains of NP that control inclusion body formation. One of these, the central domain (CD), interacts with viral protein VP35 to control both inclusion body formation and RNA synthesis. The other is the NP C-terminal domain (NP-Ct), whose function has not previously been reported. These findings contribute to a model in which NP and its interactions with VP35 link the establishment of IBs to the synthesis of viral RNA.

word count: 249 Abstract 23 Ebola virus (EBOV) inclusion bodies (IBs) are cytoplasmic sites of nucleocapsid formation and 24 RNA replication, housing key steps in the virus life cycle that warrant further investigation. 25 88 component of the viral nucleocapsid, and in addition to triggering IB formation, its roles include 89RNA packaging, acting as a co-factor for RNA synthesis carried out by the viral polymerase L, 90 and nucleocapsid assembly (32). A second viral protein, VP35, is also a required co-factor for 91 EBOV and MARV RNA synthesis and is important in nucleocapsid function and assembly (40, 92 41). VP35 associates with NP and L, is found in IBs when co-expressed with NP, and one of its 93 functions is to act as a bridge between NP and L in the formation of productive replication 94 complexes (34, 35, 42). Physical interactions between VP35 and NP have been observed that 95 involve both the N-terminal and C-terminal regions of NP (34, 41,(43)(44)(45)(46), and these have been 96 directly implicated in supporting viral RNA synthesis. Recently VP35 was shown to possess 97 NTPase and helicase-like activities, which are proposed to support RNA remodeling during 98 synthesis (47). VP35 also has well-documented anti-interferon (IFN) activity (48, 49). 99Previously we reported the crystal structure of the C-terminal domain of NP (NP-Ct) 100 from EBOV and the corresponding proteins from Taï Forest virus (TAFV) and Bundibugyo virus 101 (BDBV) (50-52). NP-Ct is highly conserved across filoviruses and assumes a novel tertiary fold 102 structure (50-53). Whereas activities carried out by the N-terminal domain of NP (aa 1-412; see 103 Figure 1 and legend) have been well characterized and include RNA binding, NP-104 oligomerization, and physical association with VP35 and L, the activities of NP-Ct have 105remained a mystery. In this report, we demonstrate two novel and redundant functions of NP 106 that control IB formation. One of these is carried out by NP-Ct, which we observe also plays a 107 separate novel role in production of infectious transcription and replication-competent virus-like 108 particles (trVLPs). The other IB-controlling function of NP is located within a previously 109 uncharacterized region of the protein that spans amino acid positions 481-500, and is responsible 110 for binding to the interferon inhibitory domain (IID) of VP35. Importantly, we find this region 111 of NP (the "central domain"; CD) to be crucially important not only for IB formation, but also 112 for viral RNA synthesis. Together these findings reveal new activities for NP in several key 113 viral replication steps and add to the complexity of viral RNA synthesis and IB dynamics that 114 may potentially be exploited for small molecule inhibitor discovery. 115 116 Results 117 118 NP-Ct is required for production of infectious VLPs but not for transcription or RNA 119 replication 120 As illustrated in Figure 1A, NP-Ct spans amino acids 641-739, which corresponds to a region of 121 high sequence conservation among ebolavirus spe...