Biology subjects

Walther, L.

Publications and source records attributed to Walther, L..

3 recordsLinked to original sources

bioRxiv2025

Therapeutic poxviruses induce the secretion of immunostimulating and anti-tumoral extracellular vesicles

Poxvirus-based vectors provide a versatile cancer immunotherapy platform, enabling the expression of immunostimulatory molecules and cancer-specific antigens. While infections with pathogenic viruses are well known to modulate extracellular vesicle (EV) biogenesis and function, the extent to which therapeutic poxviral vectors influence EV secretion by immune cells and thereby affect therapeutic efficacy remains underexplored. In this study, we showed that poxviruses, including the clinically relevant Modified Vaccinia Ankara (MVA), stimulate the secretion of small EVs (sEVs) containing viral proteins and immune-related signatures from peripheral blood mononuclear cells (PBMCs). Using an engineered MVA vector, we demonstrated the transfer of virus-encoded therapeutic payloads to sEVs, including the model ovalbumin (OVA)-derived peptide SIINFEKL presented by the class I major histocompatibility complex (MHC I) and the immune activators interleukin-12 (IL-12) and CD40 ligand (CD40L). Depending on the isolation method, these sEVs stimulated SIINFEKL-specific CD8 T cells with varying efficiencies in vitro. Remarkably, intravenous injection of these sEVs into E.G7-OVA lymphoma-bearing mice reduced tumor growth to an extent comparable to the virus itself. Taken together, our findings indicate that EVs released from immune cells infected with engineered therapeutic poxviruses exert potent antitumor activity. These vesicles represent actionable mediators whose secretion and functionalization can be harnessed to improve viral vector-based immunotherapies, as well as being considered as therapeutic vectors in their own. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/677320v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1eae18borg.highwire.dtl.DTLVardef@17d6fb5org.highwire.dtl.DTLVardef@311577org.highwire.dtl.DTLVardef@7839c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Specialised RNA decay fine-tunes monogenic antigen expression in African trypanosomes

Antigenic variation is a sophisticated immune evasion strategy employed by many pathogens. Trypanosoma brucei expresses a single Variant-Surface-Glycoprotein (VSG) from a large genetic repertoire, which they periodically switch throughout an infection. Co-transcribed with the active-VSG within a specialised nuclear body are expression-site-associated-genes (ESAGs), involved in important host-parasite interactions, including protecting the parasite from human serum lytic effects, modulating the hosts innate immune response and uptake of essential nutrients. Despite expression within the same polycistron, there is a significant differential expression between ESAGs and VSGs (>140-fold), however, the regulatory mechanism has remained elusive for decades. Here, using a combination of genetic tools, super resolution microscopy, proteomics and transcriptomics analyses, we identified three novel proteins, which are recruited in a hierarchical manner, forming discreet sub-nuclear condensates that are developmentally regulated and negatively regulate ESAG transcripts. Among them, Expression-Site-Body-specific-protein-2 (ESB2) contains a nuclease domain that shares structural similarity to the endonuclease domain found in SMG6, a critical component of nonsense mediated decay in mammals. Mutation of key residues required for the nuclease activity impaired ESB2 localisation and function. Overall, our findings reveal a novel mechanism of post-transcriptional regulation and shed light on how specialised RNA decay can regulate expression of specific genes.

molecular biology↗

bioRxiv2023

Structural basis for guide RNA selection by the RESC1-RESC2 complex

Kinetoplastid parasites, such as trypanosomes or leishmania, rely on RNA-templated RNA editing to mature mitochondrial cryptic pre-mRNAs into functional protein-coding transcripts. Processive pan-editing of multiple editing blocks within a single transcript is dependent on the 20-subunit RNA editing substrate binding complex (RESC) that serves as a platform to orchestrate the interactions between pre-mRNA, guide RNAs (gRNAs), the catalytic RNA editing complex (RECC), and a set of RNA helicases. Due to the lack of molecular structures and biochemical studies with purified components, neither the spacio-temporal interplay of these factors nor the selection mechanism for the different RNA components is understood. Here we report the cryo-EM structure of Trypanosoma brucei RESC1-RESC2, a central hub module of the RESC complex. The structure reveals that RESC1 and RESC2 form an obligatory domain-swapped dimer. Although the tertiary structures of both subunits closely resemble each other, only RESC2 selectively binds 5-triphosphate-nucleosides, a defining characteristic of gRNAs. We therefore propose RESC2 as the protective 5-end binding site for gRNAs within the RESC complex. Overall, our structure provides a starting point for the study of the assembly and function of larger RNA-bound kinetoplast RNA editing modules and might aid in the design of anti-parasite drugs. Key findingsO_LIThe kinetoplastid mitochondrial RNA editing factors RESC1 and RESC2 resemble a group of capping enzymes that are only found in protozoans, fungi and viruses. C_LIO_LIRESC1 and RESC2 lack the typical catalytic residues, and only RESC2 can bind a triphosphate-nucleoside. C_LIO_LIWe propose that the RESC1-RESC2 dimer selects guide RNAs based on their 5-triphosphate and serves as a protective 5-end binding complex. C_LI

molecular biology↗