Titel Deelnemers "Recombinant influenza virus carrying the respiratory syncytial virus (RSV) F85-93 CTL epitope reduces RSV replication in mice" "Sarah De Baets, Michael Schotsaert, Pieter Bogaert, Walther Fiers" "Effect of receptor specificity of A/Hong Kong/1/68 (H3N2) influenza virus variants on replication and transmission in pigs" "Sjouke Van Poucke, Jennifer Uhlendorff, Zhongfang Wang, Veerle Billiau, John Nicholls, Mikhail Matrosovich, Kristien Van Reeth" "Emerging options for interfering with influenza virus entry and replication" "Evelien Vanderlinden" "Influenza viruses are highly contagious respiratory pathogens with considerable medical and socio-economical burden, whether in the context of the seasonal influenza epidemics or the unpredictable, yet potentially, severe pandemics. The current influenza vaccines require annual updating and provide only partial protection. Hence, antiviral drugs are an essential component in the prevention and treatment of influenza virus infections. At the moment, two classes of antivirals are available: the M2 proton channel blockers, amantadine and rimantadine, and the neuraminidase inhibitors, oseltamivir and zanamivir. Due to the global spread of amantadine- and oseltamivir-resistant viruses, even among untreated patients, there is an urgent need for novel antiviral agents with an original mechanism of action. To address this issue, an antiviral screening program for influenza virus was set up in our laboratory. This led to the identification of a new class of influenza virus inhibitors with an N-(1-thia-4-azaspiro[4,5]decan-4-yl)carboxamide structure and a narrow and defined structure-activity relationship. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza A/H3N2 virus, the lead compound 4c displayed a 50% effective concentration of 3 to 23 µM, and an average selectivity index of 10, whereas no activity was noted for influenza A/H1N1, A/H5N1, A/H7N2 and B viruses. In time-of-addition studies, the activity of 4c was considerably reduced when added 30 min post infection or later, indicating that 4c interferes with an early step in influenza virus replication. The influenza virus entry process is largely governed by the viral hemagglutinin (HA) spike protein. First, HA mediates the binding of the virus to the host cell receptors, and this is followed by endocytosis of the virus particle. Second, due to the low pH inside the endosome, the HA protein undergoes extensive rearrangements to adopt its fusogenic conformation and provoke fusion of the viral and endosomal membranes. 4c was proven to be an inhibitor of HA-mediated membrane fusion, given its inhibitory effect on A/H3N2 virus-induced erythrocyte hemolysis at low pH and on syncytium formation in HA-transfected cells at low pH. Its protective effect against the proteolytic digestion of HA when exposed to acidic pH, demonstrates that 4c stabilizes the prefusogenic conformation of HA. 4c-resistant virus mutants were isolated after only three passages in MDCK cells. The mutant viruses showed uncompromised fitness, with similar efficiency for binding and replication as wild-type virus, and displayed an increased pH of erythrocyte hemolysis. Starting from the amino acid substitutions detected in 4c-resistant viruses, we performed HA docking experiments, and these identified the HA binding cavity for 4c, involving Arg54 and Glu57 in the HA2 subunit. The subtype-dependent activity of 4c and rapid emergence of resistant mutants indicate that its direct clinical potential may be limited. However, our studies with 4c confirm the importance of the HA stem region around residue Lys582 in the development of influenza virus fusion inhibitors, which agrees with other reports on the small molecule inhibitor TBHQ (tert-butyl hydroquinone) and some of the broad acting anti-HA antibodies. A second series of compounds with potent anti-influenza virus activity are the lipophilic glycopeptide derivatives. The lead compound SA-19 consists of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In MDCK cells, SA-19 displayed a 50% antivirally effective concentration of 0.6 µM, which was consistent among influenza A/H1N1, A/H3N2 and B viruses, and a selectivity index of 112. Virus yield at 72 h post infection was reduced by 3.6 logs at 0.8 µM of SA-19. Time-of-addition studies showed that SA-19 inhibits an early step in influenza virus replication. The compound was found not to act on binding of HA to its cell receptors, nor on HA-mediated membrane fusion or the low-pH-induced refolding of HA. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 caused a marked inhibitory effect on the transduction exerted by pseudotyped retroviral particles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein, providing evidence that SA-19 interacts with a cellular factor that is commonly involved in influenza and VSV entry. Confocal microscopy experiments using anti-nucleoprotein (NP) staining revealed that SA-19 arrests the influenza virus replication by the formation of NP-containing aggregates in the cytoplasm, resulting in a complete block of the influenza virus nuclear entry. This compound apparently interacts with cell membrane component(s), thereby disturbing the endocytic uptake of the virus and trapping it in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential therapeutic relevance. In the final part of our thesis project, we embarked on nucleoside inhibitors of the influenza virus polymerase. Evaluation of diverse base- and ribose-modified UTP and GTP analogues in an enzymatic assay with influenza virus polymerase, revealed the following potent inhibitors displaying 50% inhibitory concentrations in the range of 4-14 µM: 5-bromo-UTP, 2’-fluoro-5-methyl-UTP, 2’-fluoro-2’-deoxy-UTP, 7-deaza-GTP and 2’-fluoro-2’-deoxy-GTP. These data provided a rationale to design nucleoside inhibitors targeted towards influenza virus. In order to improve the intracellular disposition of the modified nucleoside 5’-monophosphate and achieve higher levels of the nucleoside 5’-triphosphate, the nucleoside analogues were synthesized under their phosphoramidate prodrug (ProTide) form. In influenza virus-infected cells, two 2’-fluoro-2’-deoxyuridine ProTides were found to exert moderate antiviral activity, while the parent nucleoside analogue was inactive. 2’-Fluoro-2’-deoxyguanosine was subjected to a double prodrug approach, that combines a ProTide motif and a lipophilic 6-O-substitution to further enhance the cellular uptake of the ProTide. In a PCR-based virus yield assay, four 6-O-substituted ProTides derived from 2’-fluoro-2’-deoxyguanosine displayed favorable anti-influenza virus activity with an EC99 value (i.e. concentration causing 100-fold reduction in virus titer) of about 12 µM, whereas the corresponding nucleoside analogues were inactive. Using the influenza virus polymerase assay, we demonstrated that 5-methyl-UTP, 5-bromo-UTP and 7-deaza-GTP act as efficient alternative substrates for the natural UTP or GTP, and support viral RNA elongation when incorporated. The consequences of their incorporation on viral RNA transcription and translation are still unclear. Our results confirm the viability of the ProTide approach for nucleoside inhibitors of influenza virus and propose some structural modifications (i.e. 5-halogen-substituted pyrimidine, 2’-substituted ribose and 7-deaza-purine) which may lead to optimized ProTides with potential clinical relevance." "In Vitro Characterization of the Carbohydrate-Binding Agents HHA, GNA, and UDA as Inhibitors of Influenza A and B Virus Replication" "Evelien Vanderlinden, Lieve Naesens, Dominique Schols" "Here, we report on the anti-influenza virus activity of the mannose-binding agents Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA) and the (N-acetylglucosamine) n -specific Urtica dioica agglutinin (UDA). These carbohydrate-binding agents (CBA) strongly inhibited various influenza A(H1N1), A(H3N2), and B viruses in vitro, with 50% effective concentration values ranging from 0.016 to 83 nM, generating selectivity indexes up to 125,000. Somewhat less activity was observed against A/Puerto Rico/8/34 and an A(H1N1)pdm09 strain. In time-of-addition experiments, these CBA lost their inhibitory activity when added 30 min postinfection (p.i.). Interference with virus entry processes was also evident from strong inhibition of virus-induced hemolysis at low pH. However, a direct effect on acid-induced refolding of the viral hemagglutinin (HA) was excluded by the tryptic digestion assay. Instead, HHA treatment of HA-expressing cells led to a significant reduction of plasma membrane mobility. Crosslinking of membrane glycoproteins, through interaction with HA, could also explain the inhibitory effect on the release of newly formed virions when HHA was added at 6 h p.i. These CBA presumably interact with one or more N-glycans on the globular head of HA, since their absence led to reduced activity against mutant influenza B viruses and HHA-resistant A(H1N1) viruses. The latter condition emerged only after 33 cell culture passages in the continuous presence of HHA, and the A(H3N2) virus retained full sensitivity even after 50 passages. Thus, these CBA qualify as potent inhibitors of influenza A and B viruses in vitro with a pleiotropic mechanism of action and a high barrier for viral resistance." "Distinct effects of T-705 (favipiravir) and ribavirin on influenza virus replication and viral RNA synthesis" "Evelien Vanderlinden, Bram Vrancken, Jeroen Van Houdt, Graciela Andrei, Philippe Lemey, Lieve Naesens" "T-705 (favipiravir) is a new antiviral agent in advanced clinical development for influenza therapy. It is supposed to act as an alternative substrate for the viral polymerase, causing inhibition of viral RNA synthesis or virus mutagenesis. These mechanisms were also proposed for ribavirin, an established and broad antiviral drug that shares structural similarity with T-705. We here performed a comparative analysis of the effects of T-705 and ribavirin on influenza virus and host cell functions. Influenza virus-infected cell cultures were exposed to T-705 or ribavirin during single or serial virus passaging. The effects on viral RNA synthesis and infectious virus yield were determined and mutations appearing in the viral genome were detected by whole-genome virus sequencing. Besides, the cellular nucleotide pools were quantified as well as direct inhibition of the viral polymerase enzyme. We demonstrate that the anti-influenza effect of ribavirin is based on IMP dehydrogenase inhibition, which results in fast and profound GTP depletion and an imbalance in the nucleotide pools. In contrast, T-705 acts as a potent and GTP-competitive inhibitor of the viral polymerase. In infected cells, viral RNA synthesis is completely inhibited by T-705 or ribavirin at ≥50 μM, whereas exposure to lower drug concentrations induces formation of non-infectious particles and accumulation of random point mutations in the viral genome. This mutagenic effect is two-fold higher for T-705 than for ribavirin. Hence, T-705 and ribavirin both act as purine pseudobases, but profoundly differ with regard to the mechanism behind their antiviral and mutagenic effects on influenza virus." "Hemagglutinin Cleavability, Acid Stability, and Temperature Dependence Optimize Influenza B Virus for Replication in Human Airways" "Manon Laporte, Annelies Stevaert, Mohammed Benkheil, Bart Vanaudenaerde, Lieve Naesens" "Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses." "Antibodies directed towards neuraminidase restrict influenza virus replication in primary human bronchial epithelial cells." "Catani João Paulo Portela Catani João, Goncalves Amanda, Xavier Saelens, Emma Job" "Structures of influenza A virus RNA polymerase offer insight into viral genome replication." "Els Pardon, Jan Steyaert" "De rol van receptor tyrosine kinasen en geassocieerde gangliosiden in de replicatie van het influenza virus" "Pieter Vrijens" "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." "4-Octyl itaconate reduces influenza A replication by targeting the nuclear export protein CRM1" "Dirk Daelemans" "Itaconate derivates, as well as the naturally produced metabolite, have been proposed as antivirals against influenza virus. Here, the mechanism behind the antiviral effects of exogenous 4-octyl itaconate (4-OI), a derivative of itaconate, against the influenza A virus replication is demonstrated. The data indicate that 4-OI targets the cysteine at position 528 of the CRM1 protein, resulting in inhibition of the nuclear export of viral ribonucleoprotein complexes in a similar manner as previously described for other selective inhibitors of nuclear export. These results postulate a mechanism not observed before for this immuno-metabolite derivative. This knowledge is helpful for the development of derivatives of 4-OI as potential antiviral and anti-inflammatory therapeutics."