Periodic Reporting for period 1 - FlaviRNA-HMT (Neurotropic flavivirus dsRNA-protein interface in humans, mosquitoes and ticks)
Période du rapport: 2023-09-01 au 2025-08-31
Zoonotic viruses represent a major global health challenge. Climate change, growing populations, and increased travel bring humans into closer contact with animal vectors that carry infectious diseases. In Europe, this is especially evident with arthropod-borne (arbo)viruses. Warmer Mediterranean climates have allowed tropical mosquito-borne viruses to become endemic and they now threaten to spread further north. At the same time, milder climates in Northern Europe extend the season of tick activity, creating more opportunities for tick-borne viruses to circulate. To address these growing risks, new strategies against arboviruses are needed, beginning with a better understanding of how viruses such as tick-borne encephalitis virus (TBEV) replicate in their hosts.
The interactions between the RNA that carries the genetic information of RNA viruses and their hosts have long been underestimated. Yet a central question remains: how can a molecule so fragile as viral RNA adapt to the constantly changing environments in which it replicates—shifts in temperature and cellular conditions—throughout infection? For neurotropic Orthoflaviviruses such as TBEV, this challenge is especially striking. The virus must adapt to dramatic changes: from a dormant tick that overwinters and becomes active at mild temperatures, to about 34 °C during tick feeding, and finally to 37 °C in humans. Within this cycle, TBEV first replicates in the tick midgut, then spreads to the salivary glands for transmission. Once the female tick bites, TBEV infects skin cells at the feeding site, spreads through the body via immune cells, and eventually reaches the central nervous system, where it can cause severe disease.
To investigate these processes, we studied TBEV RNA from three complementary perspectives in relevant models: (1) its interactome, to identify interacting host proteins involved in viral replication, (2) its structure, to understand its shape and interactions with the host, and (3) its sequence adaptation to pinpoint regions under different selective pressures. We compared two immune cell models in which TBEV replication may be restricted: one monocytic cell line derived from circulating blood cells and one microglial cell line derived from the central nervous system, and also included a neuronal cell line, in which TBEV replicates efficiently. For comparison to human cells, we used whole female ticks collected in the wild.
The interactions between the RNA that carries the genetic information of RNA viruses and their hosts have long been underestimated. Yet a central question remains: how can a molecule so fragile as viral RNA adapt to the constantly changing environments in which it replicates—shifts in temperature and cellular conditions—throughout infection? For neurotropic Orthoflaviviruses such as TBEV, this challenge is especially striking. The virus must adapt to dramatic changes: from a dormant tick that overwinters and becomes active at mild temperatures, to about 34 °C during tick feeding, and finally to 37 °C in humans. Within this cycle, TBEV first replicates in the tick midgut, then spreads to the salivary glands for transmission. Once the female tick bites, TBEV infects skin cells at the feeding site, spreads through the body via immune cells, and eventually reaches the central nervous system, where it can cause severe disease.
To investigate these processes, we studied TBEV RNA from three complementary perspectives in relevant models: (1) its interactome, to identify interacting host proteins involved in viral replication, (2) its structure, to understand its shape and interactions with the host, and (3) its sequence adaptation to pinpoint regions under different selective pressures. We compared two immune cell models in which TBEV replication may be restricted: one monocytic cell line derived from circulating blood cells and one microglial cell line derived from the central nervous system, and also included a neuronal cell line, in which TBEV replicates efficiently. For comparison to human cells, we used whole female ticks collected in the wild.
Recombinant TBEV development.
To facilitate the readout of viral replication, we generated two recombinant TBEV using reverse-genetics: a fluorescent reporter for high-content imaging and a bioluminescent reporter for plate-reader quantification. Benchmarked against wild-type virus, a reference antiviral or siRNA, we highlight the importance of the choice of reporter and genome design when building a reporter virus.
TBEV RNA interactome and validation.
We optimized an RNA pull-down and mass spectrometry workflow with TBEV genomic and subgenomic RNAs as baits, and adapted the protocol for whole tick material. An analysis pipeline combined fold-change statistics and hit scoring, compared conditions across models and integrated public resources (expression in cell line of interest, available datasets on RNA–protein binding, known interactions from related viruses) to rank candidates. Robust interactome datasets were obtained in two human cell models; limited material prevented conclusive datasets from one neuronal line and from ticks. From the ranked list, we curated a panel of ~250 host genes screened in a high-content functional assay to prioritize host co-factors for mechanistic validation. Using public RNA–protein binding data and GraphProt, we also predicted likely RNA–protein interaction (RPI) sites to guide targeted validation.
TBEV RNA structure, adaptation.
We used chemical tagging and mutational profiling to map full-length TBEV RNA structure in viral particles, three human cell types, and tick tissue culture system. We also passaged TBEV in human cells and in ticks and analyzed each passage by nanopore sequencing to track adaptation. The structure maps reveal cell- and species-specific features, which are enriched in the 3′-untranslated regions. Serial passaging revealed context-dependent replication gains and model-specific amino-acid changes.
In short, the interactome plus functional screen nominates the most relevant host proteins; RNA structure and adaptation help refine how and where these interactions occur; and in silico RPI predictions connect the different layers together into testable models. Final integration and experimental validation, enabled by the reporter viruses, are in progress.
To facilitate the readout of viral replication, we generated two recombinant TBEV using reverse-genetics: a fluorescent reporter for high-content imaging and a bioluminescent reporter for plate-reader quantification. Benchmarked against wild-type virus, a reference antiviral or siRNA, we highlight the importance of the choice of reporter and genome design when building a reporter virus.
TBEV RNA interactome and validation.
We optimized an RNA pull-down and mass spectrometry workflow with TBEV genomic and subgenomic RNAs as baits, and adapted the protocol for whole tick material. An analysis pipeline combined fold-change statistics and hit scoring, compared conditions across models and integrated public resources (expression in cell line of interest, available datasets on RNA–protein binding, known interactions from related viruses) to rank candidates. Robust interactome datasets were obtained in two human cell models; limited material prevented conclusive datasets from one neuronal line and from ticks. From the ranked list, we curated a panel of ~250 host genes screened in a high-content functional assay to prioritize host co-factors for mechanistic validation. Using public RNA–protein binding data and GraphProt, we also predicted likely RNA–protein interaction (RPI) sites to guide targeted validation.
TBEV RNA structure, adaptation.
We used chemical tagging and mutational profiling to map full-length TBEV RNA structure in viral particles, three human cell types, and tick tissue culture system. We also passaged TBEV in human cells and in ticks and analyzed each passage by nanopore sequencing to track adaptation. The structure maps reveal cell- and species-specific features, which are enriched in the 3′-untranslated regions. Serial passaging revealed context-dependent replication gains and model-specific amino-acid changes.
In short, the interactome plus functional screen nominates the most relevant host proteins; RNA structure and adaptation help refine how and where these interactions occur; and in silico RPI predictions connect the different layers together into testable models. Final integration and experimental validation, enabled by the reporter viruses, are in progress.
The project provides: (i) extended TBEV RNA-protein interaction datasets in relevant human cells, supported by a transparent scoring pipeline; (ii) full-length RNA structure maps across human cells, viral particles and tick tissue; (iii) comparative adaptation trajectories across hosts; and (iv) two validated reporter viruses that enable reproducible, scalable assays. Together, these resources form a coherent toolkit to study TBEV and related flaviviruses.
Potential impacts:
- Actionable evidence: host factors and constrained RNA regions (from interactome, structure and in-silico RPI predictions) as starting points for host-directed or RNA-targeted studies.
- Ready-to-use tools: GFP/Nluc reporter systems for quantitative replication and screening.
Key needs to ensure uptake and success: finalize integration of interactome, functional, structure and adaptation layers with the RPI predictions to produce ranked, testable interaction models; validate top RNA–protein contacts and targeted perturbations; broaden testing to primary human cells and tick compartments; and release datasets, code and minimal reporting standards (FAIR) to maximize reuse. No translatable elements are available or anticipated in this period; any potential application (RNA-modalities: aptamers, antisense) would depend on substantial future validation and is outside the current scope. For wider access, availability of alternatives to infectious virus (replicon systems) will support community adoption without compromising biosafety.
Potential impacts:
- Actionable evidence: host factors and constrained RNA regions (from interactome, structure and in-silico RPI predictions) as starting points for host-directed or RNA-targeted studies.
- Ready-to-use tools: GFP/Nluc reporter systems for quantitative replication and screening.
Key needs to ensure uptake and success: finalize integration of interactome, functional, structure and adaptation layers with the RPI predictions to produce ranked, testable interaction models; validate top RNA–protein contacts and targeted perturbations; broaden testing to primary human cells and tick compartments; and release datasets, code and minimal reporting standards (FAIR) to maximize reuse. No translatable elements are available or anticipated in this period; any potential application (RNA-modalities: aptamers, antisense) would depend on substantial future validation and is outside the current scope. For wider access, availability of alternatives to infectious virus (replicon systems) will support community adoption without compromising biosafety.