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

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.

Limited anti-HIV neutralizing antibody breadth and potency before and after HIV superinfection in Danish men who have sex with men.

BACKGROUND: The role of the anti-HIV neutralizing antibody response in protecting against HIV superinfection, and changes in neutralizing antibody potency and breadth after HIV superinfection have not been fully elucidated. This study examined the rate of HIV superinfection in men who have sex with men (MSM) also diagnosed with syphilis in Denmark, and the anti-HIV neutralizing antibody response in men who became superinfected. MATERIALS AND METHODS: MSM enrolled in the Danish HIV cohort who acquired syphilis were examined longitudinally for HIV superinfection using a validated next-generation sequencing assay. HIV superinfection cases were matched 3:1 to controls, and neutralizing antibody responses before (cases/controls) and after (cases) HIV superinfection were determined using a 20-pseudovirus panel. RESULTS: Four cases of HIV superinfection were identified from 95 MSM screened for a rate of HIV superinfection of 1.56/100 pys (95% CI = 0.43-4.01). Prior to HIV superinfection neutralizing antibody responses were low in breadth and potency, and did not differ between cases and controls (p = 1.0). In cases, neutralizing antibody responses increased modestly after HIV superinfection. CONCLUSIONS: These data support the theory that the natural neutralizing antibody response to HIV infection may not be the controlling factor in protecting against a subsequent HIV challenge.

A genome-wide siRNA screen identifies a druggable host pathway essential for the Ebola virus life cycle.

BACKGROUND: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4). METHODS: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory. RESULTS: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses. CONCLUSIONS: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.