Abstract
Influenza A viruses (IAVs) are respiratory pathogens that threaten global health through seasonal epidemics and periodic pandemics. As IAVs rely on host factors to replicate, understanding the virus-host interplay can provide essential insights to gauge zoonotic potential and design effective antivirals. IAV entry is a critical step in the virus lifecycle that is ideal for intervention and can affect host range but remains understudied. While attachment of most IAVs is mediated by binding of viral hemagglutinin (HA) to cellular sialic acid (Sia)(1), the identities of host factors that mediate virus internalization through multiple endocytic routes is unclear. In addition, entry of a subset of IAVs is facilitated by major histocompatibility class II (MHC II) proteins rather than Sia (2-5), yet the repertoire of strains that possess this ability and whether IAVs can recognize both receptor species is unknown. In the second chapter of this thesis, we assessed whether IAVs can possess dual receptor specificity and found that human and certain avian H2N2 IAVs can enter cells via MHC II proteins independently of Sia. At the start of this thesis, our working hypothesis was that HLA-DR, a human MHC II protein, could act as a receptor or co-receptor for human H2 IAVs. To test this, during my PhD I investigated further aspects of MHC II-mediated H2 entry. Specifically, we evidenced that HLA-DR could facilitate attachment of H2 A/Japan/57 independently of Sia and that, even in the presence of Sia, HLA-DR expression led to a modest increase in attachment of avian H2 A/Chicken/Jena/84. These data provided supporting evidence that HLA-DR could act as an authentic receptor for H2 IAVs. Moreover, we also investigated HLA-DR-mediated entry of avian H2 strains and noted that a subset of avian Eurasian H2N2 IAVs retained this trait, whereas North American strains exhibited a reduced HLA-DR-mediated entry competence. Since some recent H2 isolates can enter cells via HLA-DR, these data highlight the importance of continued surveillance of avian H2 IAVs and fully elucidating the significance of this entry pathway. Lastly, we assessed HLA-DR-dependent entry of avian H2 A/Chicken/Jena/84 of human M1-like macrophages, which can play a role in virus clearance, and noted that HLA-DR-dependent entry contributed to infection even in the presence of Sia. We previously hypothesized that further IAV receptors were to be discovered since pharmacological inhibition or reduction in expression of those proposed by others did not abolish virus infection (6). To identify them, in chapter three we performed TurboID-based proximity labelling focused on epsin 1, a host factor key for clathrin-mediated endocytosis (CME) of IAV (7), during virus uptake. Having initially corroborated expression and functionality of epsin 1-TurboID, we acquired the epsin 1 interactome in mock- and IAV-infected cells through liquid chromatography tandem mass spectrometry. Notably, potential interactors were enriched for gene ontology terms related to cellular components and biological processes associated with wildtype epsin 1 functionality and localization and also included 11 known epsin 1 interactors. As the transmembrane domain-containing Neogenin (Neo1) was enriched upon infection, we next assessed its potential as an IAV internalization receptor. In collaboration with the Alsteens lab from UCLouvain, we determined that human recombinant Neo1 interacts with H1 A/WSN/33 with a high-affinity in a Sia- and N-linked glycosylation-dependent manner via atomic force microscopy. Moreover, depletion of Neo1 expression through siRNA knockdown led to reduced replication of reporter H1 A/WSN/33 and A/Netherlands/602/09-Renilla viruses, as well as authentic IAV. Finally, we also observed through confocal microscopy that incoming IAV could co-localize with surface Neo1 and, in permeabilized samples, with Neo1 in conjunction with clathrin at early times post-infection. Due to the potential redundancy in receptor usage and endocytosis routes, in chapter four we aimed to create a tool to study IAV entry in a controlled manner. To do so, we generated an antibody-based receptor combining the cytoplasmic tail, transmembrane domain and stalk of the feline transferrin receptor type-1 and the single-chain variable fragment of 5J8, a neutralizing anti-H1 antibody (8), in collaboration with the Parrish lab from Cornell University. The antibody-based receptor was expressed in HEK293 and A549 Slc35a1 knockout cells, which do not carry sialylated glycans on proteins, to bias entry towards a single receptor species. We initially corroborated Sia depletion and expression of the antibody-based receptor through lectin staining and qPCR, respectively. Importantly, expression of the antibody-based receptor enabled recognition of an H1 A/California/09 HA-Fc probe and facilitated attachment of authentic H1 A/Netherlands/602/09. Moreover, it also rescued infection of A/California/07/09 and the reporter A/Netherlands/602/09-Renilla virus, but not A/PR8/34. Finally, treatment with pharmacological inhibitors revealed that viral uptake mediated by the antibody-based receptor relies on dynamin, CME and endosomal acidification in manner similar to Sia-dependent entry. Overall, in this thesis we explored multiple aspects of early IAV entry. We evidenced that some H2N2 IAVs possess dual receptor specificity and that MHC II entry can occur in primary infection sites. Furthermore, we identified Neo1 as an IAV receptor candidate and provide initial evidence for its role at early stages of virus infection. Finally, we developed and characterized an antibody-based receptor system that can rescue virus infection in the absence of Sia. This research contributes to our knowledge of IAV receptors, provides a list of factors potentially involved in IAV CME for future research and a platform to study virus entry.
Sempere Borau, Milagros