Biology subjects

Hesping, E.

Publications and source records attributed to Hesping, E..

3 recordsLinked to original sources

bioRxiv2026

Proteomic mapping of the dengue virus NS1 microenvironment in infected cells identifies novel host dependency factors including TM9SF3

Dengue virus (DENV) is endemic in over 100 countries and causes approximately 100 million symptomatic infections annually, with symptoms ranging from mild febrile illness to life-threatening severe vascular leakage and haemorrhagic fever. There are currently no approved antiviral therapies available to treat DENV infections. The DENV non-structural protein 1 (NS1) is essential for viral RNA replication and infectious virus particle production, while secreted NS1 contributes to immune evasion and pathogenicity. Towards the identification of novel NS1-host protein interactions that are critical to these functions, an APEX2 proximity labelling-coupled quantitative proteomics approach was employed to map the proteomic composition of the NS1 microenvironment in live infected cells. Our analysis identified a panel of 51 NS1-proximal host proteins, including established DENV host dependency factors (HDFs) involved in NS1 folding and N-glycosylation, as well as previously unrecognised host factors. Loss-of-function approaches were used to determine the importance of these NS1-proximal host proteins to DENV infection, identifying several novel HDFs, including transmembrane 9 superfamily member 3 (TM9SF3). Importantly, the knockout of TM9SF3 was shown to impair DENV infectious virion production and intracellular NS1 abundance and secretion, consistent with the recently described roles of TM9SF3 in Golgi integrity and glycosylation fidelity. Together, this study demonstrates the successful application of APEX2 proximity labelling-coupled quantitative proteomics to the identification of functionally relevant NS1-associated host proteins that may inform the development of future antiviral therapies. IMPORTANCEThe DENV NS1 protein is a non-enzymatic, multifunctional glycoprotein that plays multiple distinct roles in viral replication organelle formation, viral RNA replication and infectious virus particle production. It is also secreted from infected cells as an oligomeric lipoparticle that participates in immune evasion and vascular damage. Many of its enigmatic roles are thought to be mediated via its interactions with other viral proteins and host proteins. Here, we have employed an infectious NS1-tagged DENV reporter virus and proximity biotinylation-coupled mass spectrometry to characterise the protein microenvironment of NS1 during viral infection. We have then employed functional genomics approaches to identify NS1-proximal host factors that contribute to the viral replication cycle. Amongst the novel host factors that were identified was TM9SF3, which has recently emerged as a Golgi-resident Golgiphagy receptor that is important for maintenance of Golgi integrity and glycosylation fidelity and may represent a future DENV antiviral drug target.

microbiology↗

bioRxiv2025

Cas9-expressing HC-04 hepatocytes facilitate CRISPR-based analysis of Plasmodium falciparum sporozoite-host interactions

Sporozoites of Plasmodium falciparum, the deadliest malaria parasite, are transmitted into the skin by infected mosquitoes and migrate to the liver to initiate infection. There, they invade hepatocytes and develop into exoerythrocytic merozoites that, eventually, enter the bloodstream and invade erythrocytes, leading to malaria. The parasite journey involves cell traversal, where sporozoites transiently enter and exit host cells beginning in the skin, lysing membranes to move deeper into tissue and evade immune cell destruction. After reaching the liver and traversing several hepatocytes, sporozoites productively invade a final hepatocyte to establish liver-stage infection. The molecular mechanisms underlying traversal, invasion, and intracellular development remain incompletely understood, particularly with respect to host determinants. To address this, we engineered human HC-04 hepatocytes, the only known cell line supporting P. falciparum liver-stage development, to express Cas9-mCherry, enabling CRISPR-based functional genomics studies. We validated Cas9 activity and demonstrated successful guide-RNA-directed gene disruption via non-homologous end joining in HC-04 Cas9+ (clone 2B3) cells. Optimized traversal and invasion assays with HC-04 2B3 cells led to a robust cytometric assay suitable for screening human genes involved in P. falciparum infection. As proof-of-concept, we performed a small screen involving disruption of 10 human genes previously implicated in infection by bacterial and viral pathogens, confirming utility of this platform. While no new host factors were identified for malaria parasites in this initial study, we have developed a tractable system for genome-wide CRISPR screens to uncover novel hepatocyte biology and host determinants of infection by liver-tropic pathogens.

microbiology↗

bioRxiv2025

Chemovaccination with a novel antimalarial targeting the late liver stage induces durable immunity against malaria

Vaccination with Plasmodium falciparum, the most lethal malaria parasite, using sporozoites that arrest during liver stage infection either by irradiation, genetic attenuation or chemotherapy have been developed, with late liver stage arrest providing very high efficacy. Such vaccines require complex manufacture, deployment and intravenous administration. Here, we report an alternative strategy of chemo-attenuation of malaria parasites at the late liver stage using first-in-class antimalarials under clinical development that target the parasite aspartyl proteases plasmepsin IX and X. A single low-dose infection with virulent Plasmodium berghei sporozoites followed by drug treatment cleared infection by producing chemo-attenuated liver merozoites (CALM) that induced sterile immunity in mice for up to 21 months. Protection arose from humoral responses to circumsporozoite protein and robust CD8+ T cell responses, including liver-resident memory cells reactive to diverse antigens including SERA1 and RPL6. Drug treatment also attenuated the human pathogen P. falciparum by preventing liver merozoites from infecting human erythrocytes in humanized chimeric liver mice, confirming that the mechanism of liver-stage merozoite attenuation (ie, CALM) via inhibition of plasmepsins IX and X is conserved, likely due to conservation of binding site amino acids of both proteases across the Plasmodium genus. Therefore, plasmepsin IX/X-targeting antimalarials offer a new approach to achieving late liver stage arrest against all circulating Plasmodium species and strains. This study establishes the basis for clinical trials assessing CALM for chemoprevention and chemovaccination against diverse Plasmodium species to advance new therapeutic strategies in malaria control. It also suggests the prospect of chemovaccination by natural exposure to mosquito-borne parasites if development of a long-acting injectable formulation of plasmepsin IX/X inhibitors proves feasible.

microbiology↗