ABSTRACT
Emerging viruses are currently a burden with the recent SARS-CoV-2 pandemic resulting in more than 6 million death worldwide. Other viruses such as Arboviruses, transmitted by mosquitoes, can also emerge easily and represent a threat for humans and animals. These viruses are often RNA enveloped viruses and require to be studied in a BSL-3 (Bio-Safety- Laboratory of level 3 security), underlying the need to develop simple and rapid antiviral screenings. In recent years, the zebrafish has become a powerful tool in the biomedical sector to study viral infection and immunity. The optical transparency of the zebrafish embryo offers a major advantage for real time imaging of the infection process, and study hostpathogen interactions at subcellular levels in living systems using fluorescently labelled pathogens. While zebrafish embryos have been shown to be a successful vertebrate model to study a large panel of human disease, this model has been very little explored to study BSL-3 pathogen infection and propagation. This project was created to develop the zebrafish infection model for emerging BSL-3 viruses, and evolve towards high-content screening methods for antiviral molecules. Thanks to the setup of a microinjection system under a laminar flow hood in the BSL-3, we were able to inject in zebrafish embryos several emerging viruses, including the Chikungunya virus (CHIKV), Dengue virus (DENV) and SARS-CoV-2. Using fluorescently tagged viruses, infection was monitored in real time in vivo and confirmed with classical virology methods (RT-qPCR, plaque assays, TCID50). We then tested several nucleoside analogues described for their antiviral activity in vitro for these different viruses and we were able to validate the antiviral effect of some of these molecules in infected embryos. Altogether, our zebrafish infection model will provide us a better understanding of in vivo infection and propagation of these emergent BSL-3 viruses, and will be used as an intermediate model between in vitro antiviral screens and in vivo screens in mammals.
ABSTRACT
Background: Human immunodeficiency virus (HIV) and Influenza A virus (IAV) remain a global health concern. Further, emergence of novel coronavirus SARS-CoV-2, which rapidly became global pandemic, increases the concern in biomedical research field for antiviral treatment. To develop new antiviral therapy, we must need to understand the molecular and cellular mechanisms involved in assembly and replication. It is known for some viruses (HIV and IAV) that the host actin cytoskeleton has been involved in various stages of the virus life cycle. Regulation of actin cytoskeleton requires several actin binding proteins, which organize the actin filaments (F-actin) into higher order structures such as actin bundles, branches, filopodia and microvilli, for further assistance in viral particle production. Thus, our objective for this work is to understand the role of these actin regulator proteins, like cofilin and one of its cofactor WDR1, in viral particle assembly and release. Methods: Here we used a combination of different experimental methods like RNA interference, immunoblot, immunoprecipitation, immunofluorescence coupled to confocal and STED fluorescence microscopy. In order to study only virus release, and bypass viral entry, we set up a minimal system for virus-like particles production in transfected cells, giving HIV-1 Gag-VLP, Influenza M1-VLP and SARS-CoV-2 MNE-VLP (developed by D. Muriaux lab). For image analysis, we used Image J software. Statistical analysis was performed with non-parametric t-tests or one-way Anova test. Results: Using siRNA strategy, we have shown that upon knock down of actin protein cofilin or WDR1, HIV-1 and IAV particles production increases in contrario to SARS-CoV-2 VLP release. Further, using immunoprecipitation, we report that HIV-1 Gag is able to form an intracellular complex with WDR1 and cofilin. Similarly, IAV-M1, which like HIV Gag-MA binds with plasma membrane phospholipids, is able to form an intracellular complex with cofilin. These results suggested that virus budding from the host cell plasma membrane seemed restricted by the cofilin/WDR1 complex. Finally, using confocal/STED microscopy on cell producing VLP, we observed actin fibers rearrangement with cell protrusions, suggesting a role for actin in viral particles assembly and release. Conclusion: In conclusion, regulators of actin dynamic are involved in HIV-1 Gag, IAV-M1 and SARS-CoV-2 VLP production but play a differential role in assembly and release of these RNA enveloped viruses.