Biology subjects

Mehnert, A.-K.

Publications and source records attributed to Mehnert, A.-K..

5 recordsLinked to original sources

bioRxiv2025

The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes

Hepatitis E virus (HEV) is a significant human pathogen causing both acute and chronic infections worldwide. The cell-intrinsic antiviral response serves as the initial defense against viruses and has been shown to be activated upon HEV infection. HEV can replicate in the presence of this response, but the underlying mechanisms remain poorly understood. Here, we investigated the role of the HEV structural proteins ORF2 and ORF3 in immunocompetent cells. Mechanistically, we validated that ectopic ORF2, but not ORF3, interfered with antiviral and inflammatory signaling downstream of pattern recognition receptors, in part through interaction with the central adaptor protein TANK binding kinase 1. In the full-length viral context, ORF2 contributed to a reduced antiviral response and consequently, more efficient viral replication. In addition, we discovered a protective mechanism mediated by ORF2 that shielded viral replication from antiviral effectors. Using single-cell RNA-sequencing, we confirmed that the presence of ORF2 in infected cells dampened antiviral responses in both actively infected cells and bystanders. As a consequence, we found that early in the infection process, the progression of authentic HEV infection relied on the presence of ORF2, facilitating a balance between viral replication and the antiviral response within immunocompetent cells. Altogether, our findings shed new light on the multifaceted role of ORF2 in the HEV life cycle and improve our understanding of the determinants that may contribute to HEV persistence. Significance statementHepatitis E virus (HEV) is an important yet often underestimated pathogen. Depending on the genotype, HEV infections can progress to chronicity, but the underlying mechanisms remain poorly understood. To gain insight into potential determinants, we investigated how HEV evades the bodys first line of defense, the cell-intrinsic antiviral response. We discovered that the HEV capsid protein ORF2 is crucial in limiting this response by interfering with antiviral signaling pathways and shielding viral replication from immune effectors. This balance between viral replication and the antiviral response contributes to persistent HEV infection in immunocompetent cells. Our findings reveal a new role for the HEV capsid protein in the viral life cycle and highlight it as an important target for novel therapeutic approaches.

microbiology↗

bioRxiv2023

RBM39 shapes innate immunity through transcriptional and splicing control of IRF3 and other key factors

RNA-binding motif protein 39 (RBM39) is an RNA-binding protein involved in tumorigenesis, cell metabolism, and development. Here, we performed a genome-wide CRISPR/Cas9 screen in two liver-derived cell lines and identified RBM39 as a regulator of cell intrinsic innate immune responses. The knockdown of RBM39 or the treatment with Indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections upon sensing by toll-like receptor 3 (TLR3) or cytosolic RIG-I-like receptors. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion. RBM39 knockdown further restrained type I and type III IFN pathways, by reducing expression of the type I IFN receptor subunit interferon alpha and beta receptor subunit 2 (IFNAR2), type III IFN receptor subunit interleukin 10 receptor subunit beta (IL-10RB) and transcription factor signal transducer and activator of transcription (STAT) 1 and 2. RBM39 overall orchestrates innate immunity by regulating basal expression of key factors of the interferon response via transcription and/or alternative splicing. SignificanceThe function of RBM39 in tumorigenesis has been investigated intensively in the last decade, but its immunological role is still largely unknown. In our study, we identified RBM39 as a regulatory factor of cell intrinsic signaling via a CRISPR/Cas9 screen. Depletion of RBM39 impairs TLR3, RIG-I/MDA5, and IFN pathways, and thus attenuates innate immune responses. Our omics analysis revealed that RBM39 governs the basal expression of several key factors within these pathways, such as RNA sensors RIG-I and MDA5, type I/III receptors, transcription factors IRF3, STAT1 and STAT2, via its transcriptional and splicing function. Therefore, RBM39 might be a therapeutic target to modulate innate immunity, e.g. in the context of autoimmune disorders.

immunology↗

bioRxiv2021

DNA double strand break position leads to distinct gene expression changes and regulates VSG switching pathway choice.

Antigenic variation is an immune evasion strategy used by Trypanosoma brucei that results in the periodic exchange of the surface protein coat. Underlying this process is the movement of variant surface glycoprotein genes in or out of a specialized locus known as bloodstream form expression site by homologous recombination, facilitated by blocks of repetitive sequence known as the 70-bp repeats, that provide homology for gene conversion events. DNA double strand breaks are potent drivers of antigenic variation, however where these breaks must fall to elicit a switch is not well understood. To understand how the position of a break influences antigenic variation we established a series of cell lines to study the effect of an I-SceI meganuclease break in the active expression site. We found that a DNA break within repetitive regions is not productive for VSG switching, and show that the break position leads to a distinct gene expression profile and DNA repair response which dictates how antigenic variation proceeds in African trypanosomes.

molecular biology↗

bioRxiv2020

RAD50 promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 is required for RAD51-dependent homologous recombination, phosphorylation of histone H2A and controlled resection following a DNA double strand break (DSB). Perhaps surprisingly, DSB resection in the rad50 nulls was not impaired and appeared to peak earlier than in the parental strains. Finally, we show that RAD50 suppresses DNA repair using donors with short stretches of homology at a subtelomeric locus, with null strains producing a greater diversity of expressed VSG variants following DSB repair. We conclude that RAD50 promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.

molecular biology↗

bioRxiv2018

Improved immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells

Immunofluorescence staining is the key technique for visualizing organization of endogenous cellular structures in single cells. Labeling and imaging of blood stage Plasmodium falciparum has always been challenging since it is a small intracellular parasite. The gold standard for parasite immunofluorescence is fixation in suspension with addition of minute amounts of glutaraldehyde to the paraformaldehyde-based solution. While this maintains red blood cell integrity, it has been postulated that antigenicity of the parasite proteins was, if at all, only slightly reduced. Here we show the deleterious effect that even these small quantities of glutaraldehyde can have on immunofluorescence staining quality and present an alternative cell seeding protocol that allows fixation with only paraformaldehyde. The highly improved signal intensity and staining efficiency enabled us to carry out RescueSTED nanoscopy on microtubules and nuclear pores and describe their organization in greater detail throughout the blood stage cycle.\n\nHighlights- Omitting glutaraldehyde from fixative improves immunofluorescence staining\n\n- STED nanoscopy is readily applicable to infected red blood cells\n\n- Intranuclear microtubules are nucleated from distinct sites

cell biology↗