Ebola Virus Inclusion Body Formation and RNA Synthesis Are Controlled by a Novel Domain of Nucleoprotein Interacting with VP35

Abstract

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.

Liquid–Liquid Phase Separation by Intrinsically Disordered Protein Regions of Viruses: Roles in Viral Life Cycle and Control of Virus–Host Interactions

“…In the case of Borna disease virus (BDV, another member of the Mononegavirales order), the P protein alone is sufficient to sustain the formation of IBs with a probable liquid nature [184]. Likewise, the N protein from EBOV is sufficient for IB generation [185]. By contrast, in the case of VSV, co-expression of the L protein is required [180].…”

“…This is an interesting example of crosstalk between MLOs formed by LLPS and classical intracellular organelles [156,194]. The cytosolic puncta (i.e., the small, spherical condensates) form and dissolve reversibly and react promptly to changes in the chemical environment, such as hypotonic stress and addition of 1,6-hexanediol, a reagent known to perturb LLPS [185]. The viral inclusions can form even when only one type of RNP is expressed, thus revealing that their formation does not depend on RNA-RNA interactions among distinct ribonucleoproteins, as previously thought.…”

New Perspectives on the Biogenesis of Viral Inclusion Bodies in Negative-Sense RNA Virus Infections

“…It was recently shown that the C-terminal domain of EBOV NP is necessary for IB formation and that co-expression of VP35 can rescue IB formation upon expression of a C-terminal deleted NP [166]. This experiment suggests that IB formation and other functions of NP and VP35 involved in transcription and replication of viral RNA are separated processes, since RNA synthesis could not be rescued in this setting.…”

How SARS-CoV-2 and Other Viruses Build an Invasion Route to Hijack the Host Nucleocytoplasmic Trafficking System

“…Several viruses similarly re-localize hnRNP C1/C2 to the cytoplasm, either for viral replication or IRES-dependent viral protein translation [99][100][101][102], which suggests that VP24-dependent hnRNP C1/C2 cytoplasmic accumulation is essential for EBOV replication [97]. Together, nucleoproteins (NPs) and VP35 form EBOV inclusion bodies (IBs) for viral replication [103]. A study conducted by Gabriel et al showed that importin-α7 is also involved in IB formation [104].…”