Biology subjects

Hoenen, T.

Publications and source records attributed to Hoenen, T..

7 recordsLinked to original sources

bioRxiv2026

CD81 is an Ebola virus inhibiting factor that is antagonized by GP and VP40

Viruses manipulate the host cell membrane of infected cells for evasion of antiviral immunity, prevention of superinfection and optimization of viral replication and spread. The Ebola virus glycoprotein (EBOV GP) mediates virus entry, but is also known as important factor for subversion of the hosts antiviral immune response. We characterized the dysregulation of cell surface-residing proteins by EBOV GP and found that among several membrane proteins GP interferes with the tetraspanins CD81, CD63 and CD9. This was a conserved function of several filoviral GPs and not observable for viral glycoproteins of other virus families. While CD63 and CD9 were largely dispensable for EBOV replication, CD81 suppressed virus-like particle entry and replication at multiple steps. This phenotype might be explainable by sustained suppression of NF{kappa}B by CD81, that is otherwise activated by VP40 and EBOV trVLP replication. We further demonstrate that not only GP but also VP40 interferes with CD81 functionality and that antibody-mediated clustering of CD81 suppresses EBOV infection. Altogether, the tetraspanin CD81 emerges as druggable NF{kappa}B and EBOV-inhibiting factor, supporting an important role of NF{kappa}B in EBOV replication and potentially virus-induced immunopathogenesis. HighlightsO_LIEBOV GP and VP40 interfere with CD81 cell surface expression C_LIO_LICD81 suppresses NF{kappa}B signaling which is activated by VP40 C_LIO_LICD81 restricts EBOV VLP uptake and replication C_LIO_LITargeting CD81 by a crosslinking antibody inhibits EBOV infection C_LI

microbiology↗

bioRxiv2024

Ebola virus infection of Flt3-dependent, conventional dendritic cells and antigen cross-presentation leads to high levels of T-cell activation

Severe Ebola virus disease (EVD) is characterized by excess, dysregulated T-cell activation and high levels of inflammation. Previous studies have described that in vitro EBOV infection of monocyte-derived DCs (moDCs) inhibits DC maturation resulting in suppression of T-cell activation. However, it is unknown how other DC subsets distinct from moDCs respond to EBOV infection. To better understand how DCs initiate T cell activation during EBOV infection, we assessed the response of FMS-like tyrosine kinase 3 (Flt3)-dependent, conventional mouse DCs (cDCs) to EBOV infection, and developed a DC-T-cell co-culture system utilizing a recombinant EBOV expressing the model antigen ovalbumin. Our findings suggested that, in contrast to moDCs, cDC2 and cDC1 were poorly infected with EBOV, although both infected and bystander cDCs displayed high levels of activation. DCs were able to activate CD8 T cells via cross-presentation of EBOV antigens obtained from cell debris of EBOV-infected cells. Of note, rather than interfering with cross-presentation, EBOV infection enhanced the cross-presentation capacity of DCs. Our findings indicate that EBOV infection of Flt3-dependent cDCs, results in activation rather than inhibition leading to high levels of CD8 T-cell activation. With that we propose a mechanistic explanation for the excess T-cell activation observed in severe human EVD.

immunology↗

bioRxiv2023

Nucleocapsid condensation drives Ebola viral factory maturation and dispersion

Replication and genome encapsidation of many negative-sense RNA viruses take place in virus-induced membrane-less organelles termed viral factories (VFs). While liquid properties of VFs are believed to control the transition from genome replication to encapsidation, the nucleocapsid assembly, VF maturation and interactions with the cellular environment remain elusive. Here we apply in situ cryo-correlative light and electron tomography to follow nucleocapsid assembly and changes in VF morphology and their liquid properties during Ebola virus infection. We show that Ebola viral nucleocapsids transition from loosely packed helical assemblies in early VFs to condensed cylinders that arrange into highly organized parallel bundles later in infection. Early VFs associate with intermediate filaments and are devoid of other host material, but become progressively accessible to cellular components. Our data suggest that this process is coupled to VF solidification and dispersion, and that changes in liquid properties of VFs promote nucleocapsid transport to budding sites. Highlights- Cryo-ET reveals the molecular architecture of Ebola virus replication compartments - Loosely coiled nucleocapsids transition to condensed cylinders forming bundles - Nucleocapsid condensation drives dispersion of viral factories promoting viral egress - Intermediate filaments associate with and are critical for virus factory formation

microbiology↗

bioRxiv2022

The Ebola virus VP40 matrix undergoes endosomal disassembly essential for membrane fusion

Ebola viruses (EBOVs) are filamentous particles, whose shape and stability are determined by the VP40 matrix. Virus entry into host cells occurs via membrane fusion in late endosomes; however, the mechanism of how the remarkably long virions undergo uncoating including virion disassembly and nucleocapsid release into the cytosol, remains unknown. Here, we investigate the structural architecture of EBOVs entering host cells and discover that the VP40 matrix disassembles prior to membrane fusion. We reveal that VP40 disassembly is caused by the weakening of VP40-lipid interactions driven by low endosomal pH that equilibrates passively across the viral envelope without a dedicated ion channel. We further show that viral membrane fusion depends on VP40 matrix integrity, and its disassembly reduces the energy barrier for fusion stalk formation. Thus, pH-driven structural remodeling of the VP40 matrix acts as a molecular switch coupling viral matrix uncoating to membrane fusion during EBOV entry.

microbiology↗

bioRxiv2021

TRIM25 and ZAP target the Ebola virus ribonucleoprotein complex to mediate interferon-induced restriction

Ebola virus (EBOV) causes highly pathogenic disease in primates. Through screening a library of human interferon-stimulated genes (ISGs), we identified TRIM25 as a potent inhibitor of EBOV transcription-and-replication-competent virus-like particle (trVLP) propagation. TRIM25 overexpression inhibited the accumulation of viral genomic and messenger RNAs independently of the RNA sensor RIG-I or secondary proinflammatory gene expression. Deletion of TRIM25 strongly attenuated the sensitivity of trVLPs to inhibition by type-I interferon. The antiviral activity of TRIM25 required ZAP and the effect of type-I interferon was modulated by the CpG dinucleotide content of the viral genome. We find that TRIM25 interacts with the EBOV vRNP, resulting in its autoubiquitination and ubiquitination of the viral nucleoprotein (NP). TRIM25 is recruited to incoming vRNPs shortly after cell entry, and leads to dissociation of NP from the vRNA. We propose that TRIM25 targets the EBOV vRNP, exposing CpG-rich viral RNA species to restriction by ZAP. HighlightsO_LITRIM25 and ZAP play a major role on type I IFN-mediated inhibition of EBOV trVLP replication C_LIO_LITRIM25 interacts with the EBOV NP and is recruited to vRNPs in the cytoplasm after viral entry C_LIO_LITRIM25 ubiquitinates NP and displaces it from the viral genome, facilitating ZAP interaction C_LIO_LIZAP targets CpGs in the EBOV genome to inhibit EBOV trVLP replication C_LI

microbiology↗

bioRxiv2021

Ebola virus glycoprotein domains associated with protective efficacy

Ebola virus (EBOV) is the cause of sporadic outbreaks of human hemorrhagic disease in Africa, and the best-characterized virus in the filovirus family. The West Africa epidemic accelerated the clinical development of vaccines and therapeutics leading to licensure of vaccines and antibody-based therapeutics for human use in recent years. The most widely used vaccine is based on vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP)(VSV-EBOV). Due to its favorable immune cell targeting, this vaccine has also been used as base-vector for the development of second generation VSV-based vaccines against Influenza, Nipah, and Zika viruses. However, in these situations it may be beneficial if the immunogenicity against EBOV GP is minimized to induce a better protective immune response against the other foreign immunogen. Here, we analyzed if EBOV GP can be truncated to be less immunogenic yet still able to drive replication of the vaccine vector. We found that the EBOV GP glycan cap and the mucin-like domain are both dispensable for VSV-EBOV replication. The glycan cap domain, however, appears critical for mediating a protective immune response against lethal EBOV challenge in mice.

microbiology↗

bioRxiv2020

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

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 absolutely required 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 retention of viral RNA within these particles. 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 to 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 domain of NP, the "central domain" (CD). ImportanceInclusion 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.

microbiology↗