Jessica Morel

The influenza A virus hijacks host cell machinery by “snatching” molecular caps from cellular RNAs to prime its own transcription. While this process is typically associated with the beginning of genes, this study reveals that the viral RNA polymerase exerts a far-reaching influence on host gene expression. Using genome-wide mapping and comparative pathway analyses, we show that viral polymerase (FluPol) recruitment is decoupled from general host transcriptional activity; instead, FluPol is markedly enriched at a distinct subset of promoters and enhancers associated with defense-related pathways. Beyond the initiation site, FluPol remains associated with host transcripts throughout elongation, extending into downstream-of-gene (DoG) regions. It further accumulates at gene ends, potentially contributing to widespread defects in transcription termination. By remaining linked to host transcripts across the entire gene body, FluPol could extend its opportunity for cap-snatching and impair the recycling of cellular transcription machinery. Together, these findings demonstrate how FluPol accumulation across key cellular defense loci spans from initiation through to termination, driving host transcriptional attenuation.

L’objectif général de ce travail de thèse a été de mieux comprendre le fonctionnement de l’ARN-polymérase des virus Influenza (FluPol) qui assure transcription et réplication du génome viral dans le noyau de la cellule hôte. Les virus influenza ne disposant pas de la capacité à synthétiser une coiffe à l’extrémité 5’ des ARNm viraux, ils utilisent les ARNm cellulaires coiffés pour amorcer la transcription virale par un mécanisme appelé vol de coiffe. Ainsi, FluPol se fixe sur la coiffe d’un ARNm cellulaire pour le cliver 10-15 nucléotides en aval et générer l’amorce permettant de transcrire l’ARN génomique en ARNm. Par ailleurs, FluPol interagit avec le domaine non structuré de l’ARN-polymérase II cellulaire (Pol II) constitué de 52 répétitions du motif YSPTSPS. La phosphorylation de la sérine5 de ce motif, marque de l’initiation de la transcription cellulaire, apparait être un prérequis à l’interaction FluPol - Pol II. Cette interaction permet vraisemblablement à FluPol de cibler les étapes d’initiation de la transcription. Le premier objectif de ce travail de thèse a été d’identifier des interactions additionnelles entre FluPol et Pol II potentiellement impliquées dans le vol de coiffe. Une interaction entre le domaine N-terminal de lasous-unité PB2 de FluPol et la sous-unité RPB4 de Pol II a ainsi été identifiée. Cette interaction est conservée chez les virus de type A, B et C. Dans une seconde partie, ce travail a permis de mettre en évidence par des approches de séquençage à haut-débit que FluPol reste fixée à Pol II tout au long de la transcription cellulaire et non uniquement à son initiation. Ces approches ont également permis de mettre en évidence un ciblage préférentiel de FluPol pour les gènes ayant un rôle dans l’immunité innée, participant ainsi au contrôle de la réponse innée de l’hôte. Enfin, afin de mieux comprendre le fonctionnement de FluPol, des nanoligands de deux types (des VHH et des protéines artificielles) spécifiques de FluPol ont été sélectionnés. Un VHH s’est avéré bloquer efficacement l’activité enzymatique de FluPol et reconnaitre les sous-unités PA et PB1 de FluPol. A terme, ces nouveaux outils d’étude devraient permettre d’élucider la dynamique des mécanismes moléculaires associés à la transcription et la réplication virale par la caractérisation des fonctions altérées de FluPol et des domaines ciblés à l’échelle atomique.

Indoor air quality (IAQ) monitoring in hospital settings is critical to limit airborne transmission of pathogens to vulnerable patients and healthcare workers. However, bioaerosol sampling remains challenging because of the diversity of devices and methodologies and the very low airborne concentration of pathogens in real‐world settings, especially when infectivity‐based measurements, are required for assessing transmission risk. In the absence of harmonized evaluation procedures, it remains difficult to select sampling strategies that are both reliable and fit for purpose. In this study, we developed a controlled and reproducible experimental framework for the evaluation of bioaerosol samplers under conditions relevant to hospital air monitoring. Two widely used cyclonic bioaerosol samplers, Coriolis+ and Coriolis Compact, were assessed in a calibrated 20 m ³ test chamber generating stable atmospheres containing infectious SARS‐CoV‐2, Staphylococcus aureus , or Aspergillus fumigatus at different concentration levels. Samplers′ performance was evaluated using complementary molecular and viability‐based endpoints and was combined with computational fluid dynamics (CFD) and experimental airflow measurements to characterize airflow dynamics. Our results demonstrated that samplers′ performance strongly depended on the biological endpoint and microorganism considered. For SARS‐CoV‐2, the two devices showed comparable recovery of infectious virus, at intermediate and high concentrations, but neither detected infectious particles at the lowest level. In contrast, Coriolis+ achieved near‐complete recovery of viral genomes, whereas Coriolis Compact recovered only a fraction of the expected signal. Regarding S. aureus and A. fumigatus , Coriolis+ achieved superior recovery of viable microorganisms, whereas Coriolis Compact generally yielded higher genomic recovery at moderate and high concentrations. CFD analyses further showed that local airflow conditions and sampler‐specific flow patterns, particularly for the higher flow Coriolis+, are critical for interpreting biological recovery and for defining appropriate placement and operating modes. Overall, this study demonstrates that bioaerosol sampler evaluation must jointly consider molecular, microbiological, and aerodynamic dimensions. The proposed framework offers a basis for more standardized and reproducible performance assessment of bioaerosol samplers and supports evidence‐based selection of air monitoring strategies in healthcare environments.

Background: The COVID‐19 pandemic has underscored the critical role of indoor air quality and ventilation in the transmission of respiratory viruses. Although portable air cleaners (PACs) have proliferated in recent years, most performance evaluations rely on indirect measurement approaches or surrogate microorganisms, leaving uncertainty regarding their true efficacy against infectious human bioaerosols. Objectives: We aimed to develop and validate a highly controlled test bench for comprehensive evaluation of PACs in accordance with evolving standards, incorporating infectious respiratory human pathogens, spectrometric analysis under realistic environmental conditions, and computational fluid dynamics (CFD) supporting experimental design and interpretation of airflow patterns. Methods: The experimental platform consists of a 20 m ³ BSL‐3 chamber equipped for precise control of airflow, humidity, and temperature, allowing nebulization of infectious viruses (SARS‐CoV‐2, influenza A/H1N1pdm09, and adenovirus), bacteria ( Staphylococcus aureus ), and fungi ( Aspergillus fumigatus ). Natural decay rates and stabilities were characterized for each microorganism in homogeneous, highly contaminated atmospheres. The performance of a HEPA H14 PAC (Camfil City M) was evaluated under varying positions (center/corner) and airflow scenarios, with additional interference simulated by open‐window ventilation. Air samples were analyzed by TCID50 assays and particle spectrometry. Results: Stable contaminated atmospheres were reproducibly generated, with airborne concentrations remaining stable for at least 1 h. S. aureus exhibited the greatest stability, while adenovirus and influenza A decayed more rapidly. PAC efficacy depended on device placement and ventilation conditions, with synergistic effects observed when combining air filtration and ventilation. Experimental decay rates for infectious virus and aerosols consistently exceeded CFD predictions, likely due to modelling limitations. Conclusions: This study presents an advanced methodology for evaluating PAC performance against infectious airborne pathogens under realistic, variable environmental conditions. The findings underscore the importance of considering infectious bioaerosol dynamics, device placement, and airflow characteristics when assessing PAC effectiveness and highlight the limitations of solely modelling inert particles in CFD analyses.

The influenza A virus (IAV) RNA polymerase (FluPol) primes the viral transcription by using capped 5 prime-ends snatched from host nascent RNAs. However, the exact localization of the FluPol on the genome or the timing of its snatching activity remains poorly characterized. Here, we have monitored IAV infection from the perspective of the FluPol interaction with the host chromatin template. Quantification of chromatin-bound RNAs shows a significant perturbation in host transcription that correlates with a relocalization of RNA polymerase II (RNAPII) from the gene bodies to downstream intergenic regions. This extended transcription leading to the production of RNA downstream of genes (DoGs) was previously linked to the NS1-mediated inhibition of the transcriptional termination. However, immunoprecipitation of FluPol-bound RNA in the chromatin fraction revealed that FluPol remains linked to nascent host transcripts during phases of transcriptional elongation and termination, thereby extending the window of opportunity in which cap-snatching may occur. In addition, chromatin-associated FluPol was enriched at transcription termination sites, suggesting that it may participate in the virus-induced termination defects. Finally, we observed that, rather than targeting just highly expressed genes, the FluPol was preferentially recruited to promoters activated by the viral infection and enhancers. Together, these observations suggest the FluPol uses the early immune response to target regulatory elements of defense-related genes at which it interferes with the fate of the transcripts, and possibly also limits RNAPII re-initiation by impairing the termination process.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3′-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5′-end of nascent host mRNA.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3’-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and found to occur with the homologous domains of influenza B and C FluPols. Residues [1-72] of RPB4 were found critical in this interaction. Numerous punctual mutants generated at conserved positions between influenza A, B and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defect. These results suggest that FluPol interacts with several domains/subunits of Pol II, the CTD to bind Pol II initiating host transcription and a second on RPB4 to locate FluPol at the proximity of the 5’-end of nascent host mRNA.