Ebola virus inclusion body formation and RNA synthesis are controlled by a novel domain of NP interacting with VP35

Abstract

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...

Negri bodies and other virus membrane-less replication compartments

“…Co-expression of N and P proteins of paramyxoviruses and RABV after cell transfection also leads to the formation of spherical inclusions [ 25 , 30 , 37 , 38 ]. In the case of VSV, the presence of L protein is also required for such inclusions to be formed [ 31 ] whereas in the case of EBOV, NP alone is sufficient for IB generation [ 39 ]. For both RABV and MeV, the N-P inclusions formed in this minimal system have the same liquid characteristics as the viral factories [ 25 , 30 ].…”

“…However, co-expression of the nucleocapsid component VP35 overcomes deletion of NP-Ct in triggering IB formation. This effect is mediated by an interaction between VP35 and NP implicating a central domain (CD, residues 480–500) of NP enriched in acidic residues and the interferon (IFN) inhibitory domain of VP35 [ 39 ].…”

Single-cell image-based genetic screens systematically identify regulators of Ebola virus subcellular infection dynamics

“…Transcription from the negative stranded virus genome produces viral mRNAs and, in turn, production of viral proteins leads to formation of inclusion bodies (IBs), cytoplasmic foci that serve as sites for viral RNA synthesis (Hoenen et al, 2012(Hoenen et al, , 2019Nanbo et al, 2013). The EBOV nucleocapsid protein (NP) induces formation of these IBs (Miyake et al, 2020;Wu et al, 2023) and the viral polymerase cofactor VP35 interacts with NP to regulate IB formation (Leung et al, 2015;Miyake et al, 2020). At later stages of infection, these proteins exhibit a diffuse cytoplasmic localization pattern and, finally, localize to the cell periphery during virus budding (Nanbo et al, 2013).…”

A protein-proximity screen reveals Ebola virus co-opts the mRNA decapping complex through the scaffold protein EDC4

“…EBOV replica4on depends on the mul4func4onality of its own proteins as well as virus-host protein interac4ons to successfully replicate and evade host an4viral responses. Through interac4ons with NP, VP35 influences the assembly of viral inclusion bodies 4,7 . VP35 caps the blunt end of viral dsRNA, preven4ng detec4on by RIG-I sensing and concurrent IFNβ produc4on 8 .…”