The Ebola Virus Nucleoprotein Recruits the Nuclear RNA Export Factor NXF1 into Inclusion Bodies to Facilitate Viral Protein Expression

Abstract

Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.

Negri bodies and other virus membrane-less replication compartments

“…The phosphorylated mitogen-activated protein kinase p38, a key regulator of cellular inflammatory and stress responses, and the O-linked N -acetylglucosamine (OGN) transferase, an enzyme that catalyzes the posttranslational addition of OGN to protein and is also involved in stress response, are sequestered in RSV IBs [ 99 ]. Finally, EBOV NP recruits several proteins into the IBs, which include the nuclear RNA export factor NFX1 to drive viral protein synthesis [ 100 ] and the histone-lysine-methyltransferase SMYD3 [ 101 ] as well as the CAD protein [ 102 ] to facilitate mRNA transcription and replication.…”

Lipidome is lipids regulator in gastrointestinal tract and it is a life collar in COVID-19: A review

“…Consequently, the NXF1 promotes the export of viral mRNA:NXF1 complexes from inclusion bodies. This provides a basis for new therapeutic approaches for viruses[ 84 ]. The ribosomal proteins (RPs) contain 60S subunit control translation of specific mRNAs.…”

The Cellular Protein CAD is Recruited into Ebola Virus Inclusion Bodies by the Nucleoprotein NP to Facilitate Genome Replication and Transcription

“…CoIPs were performed as previously described [19]. Briefly, 293T cells were seeded in 6-well plates and transfected with expression plasmids encoding FLAG/HA-tagged CAD and EBOV-NP using Transit LT-1 (Mirus Bio LLC) following the manufacturer's instructions.…”

“…Furthermore, several cellular kinases and phosphatases are known to localize in inclusion bodies to support EBOV replication and transcription [16][17][18]. Finally, we previously showed that EBOV NP recruits the nuclear RNA export factor 1 (NXF1) into inclusion bodies to facilitate viral mRNA export from these structures into the cytoplasm [19]. Despite this recent progress in our understanding of the interplay between host factors and EBOV, there remains a considerable need to identify and, more importantly, characterize further host factors required for EBOV replication to identify novel targets for antiviral drug development.…”