De rol van receptor tyrosine kinasen en geassocieerde gangliosiden in de replicatie van het influenza virus

Influenza infections produce significant medical and socio-economic burden. Every year, human influenza A and B viruses cause millions of cases of severe respiratory illness and death. Every so often, a pandemic arises when a zoonotic influenza A virus with sustained human transmissibility enters the human population. Antivirals are crucial for influenza treatment and prevention, especially in fragile populations such as elderly. At the moment, neuraminidase (NA) inhibitors are the only drug class that is globally available, however resistance against these agents is a serious concern. New inhibitors with an entirely different mode of action are absolutely required. Besides targeting the virus itself, it is imperative to consider host cell factors that are less prone to resistance. Among the multitude of possibilities, we here focused on receptor tyrosine kinases (RTK), a class of protein kinases for which several therapeutics have been developed during the past decade. In Chapter 2, we first evaluated a library of 276 protein kinase inhibitors for anti-influenza virus activity in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 emerged as a robust inhibitor of influenza A and B virus replication, producing up to 3-log10 reduction in viral load at non-toxic concentrations. Detailed mechanistic investigations revealed that Ki8751 interferes with PDGFRβ-mediated influenza virus internalization, indicating that the virus matches with this specific RTK. By using two related CHO cell lines, we demonstrated that this route of virus uptake depends on gangliosides present on the cell membrane. Entering virus was shown to prefer GM3- over GM1-positive lipid rafts, consistent with a stimulating effect of GM3 on PDGFRβ signaling and virus uptake via this pathway. Our intriguing observation that the phosphorylated PDGFRβ undergoes desialylation by the viral NA, could indicate an as yet unknown function of NA in virus entry. PDGFRβ-mediated virus uptake coincided with activation of the Raf/MEK/Erk signaling pathway, but not of PI3K or phospholipase C-γ. In Chapter 3, we elaborated on the role of RTKs at later replicative stages, after observing that a VEGFR2-transfectant CHO cell line synthesized higher levels of viral RNA, an effect that was independent of cell surface-associated VEGFR2. The stimulating effect of VEGFR2 on viral RNA synthesis was strongest at 6 h p.i. and coincided with enhanced nuclear export of viral ribonucleoprotein (vRNP) complexes. We propose that nuclear VEGFR2 might alter the phosphorylation status of the viral nucleoprotein (NP), for instance at residues Tyr296/Ser297, and thereby prime the vRNPs for nuclear export. However, since the CHO cell line was found to express an incomplete form of VEGFR2 (i.e. lacking the kinase domain), the cooperation of another RTK appears required to alter the NP phosphorylation status and enhance influenza virus replication. In conclusion, our study indicates that cellular RTKs contribute to influenza virus replication at two stages: entry and nuclear export. To enter into the host cell, the virus efficiently exploits the PDGFRβ/GM3-mediated signaling pathway in which not only the viral HA but also its NA are playing a role. This tight association with PDGFRβ creates an opportunity to suppress virus replication with PDGFRβ inhibitors like Ki8751 or more advanced clinical candidates. To achieve robust activity against influenza A and B viruses, in combination with a favorable therapeutic window, inhaled formulations of PDGFRβ blockers deserve further attention. Besides, our findings indicate that viral nuclear export is regulated by RTK pathways acting in the nucleus, like VEGFR2-associated kinases or phosphatases. This plausibly involves altering the NP phosphorylation status. Since several RTK inhibitors are already approved or in clinical development, combining these therapeutics with direct acting antiviral drugs may be a valuable strategy to make influenza therapy more effective or address the antiviral drug resistance problem.

Publication year:2020

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