Publikationsdatenbank

The role of a cholesterol consensus motif in HA and of an amphipathic helix in M2 for replication of influenza A virus (2019)

Art

Hochschulschrift

Autor

Hu, Bodan (WE 5)

Quelle

Berlin, 2019 — VI, 73 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/25504

Kontakt

Institut für Virologie

Robert-von-Ostertag-Str. 7-13
14163 Berlin
+49 30 838 51833
virologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

Influenza A virus are enveloped viruses with a spherical or filamentous morphology. In their membrane are two glycoproteins, the hemagglutinin and the neuraminidase as well as the unglycosylated proton channel M2. Virus entry into host cells depends on the fusion activity of HA and the proton conductance activity of M2, whereas virus assembly and budding is supposed to be initiated by accumulation of HA in cholesterol- and sphingolipid-enriched nanodomains at the plasma membrane. Furthermore, the amphipathic helix in the cytoplasmic tail of M2 is engaged in virus budding and membrane scission to release progeny virions from the host membrane. In this study the effect of mutations in a conserved cholesterol consensus motif (CCM) of HA on virus assembly and HA’s fusion activity was investigated. Phylogenetic group 2 HAs contain the conserved CCM motif Y-K-L-W in the transmembrane region. Studies previously done in our group reported that mutations in the CCM retarded intracellular transport of HA and reduced its association with cholesterol-rich nanodomains. Here I firstly analyzed whether cholesterol interacts with the CCM. The click-labeling with photocholesterol was significantly reduced when the whole CCM was substituted by alanine, both using immunoprecipitated HA and when HA was embedded in cellular membranes. In the content of virus replication, no virus was rescued if the whole motif is replaced (mutant YKLW4A); single (LA) or double (YK2A and LW2A) mutated virus exhibited reduced titers and a comparative fitness disadvantage. The apical transport of HA and apical budding of viruses in polarized cells were not disturbed by these mutations. However, reduced amounts of HA and cholesterol were incorporated into the viral membrane. Mutant viruses showed decreased fusion activity as demonstrated by hemolysis and fluorescence dequenching assays. Cell-cell fusion assay using dual-labelled erythrocytes and HA-expressing cells revealed that HA-YKLW4A can fuse with erythrocytes, but the number of events was reduced. Even after acidification unfused erythrocytes remained cell-bound, a phenomenon not observed in HA-WT expressing cells. Thus, cholesterol binding to HA has effects on membrane fusion, mainly on lipid mixing and possibly a preceding step. Being also crucial in virus assembly and budding, the amphipathic helix in the cytoplasmic tail of M2 was also investigated. Since a variety of amphipathic peptides mediate membrane deformation and induce their vesicularisation, I used reverse genetics to investigate whether they can substitute for M2’s helix. No virus could be generated if M2’s helix was deleted or exchanged to a peptide predicted not to form an amphipathic helix. In contrast, viruses could be rescued if the M2 helix was replaced by helices known to induce membrane curvature, but they showed only slightly decreased virus titers. Transmission EM of infected cells did not exhibit undetached mutant virions with a “bead-on-a-string” morphology, a hallmark of viruses with failed membrane scission. Nevertheless, individual mutant viruses exhibit other defects in M2, such as reduced surface expression and decreased incorporation into virions. The protein composition and specific infectivity was altered as well for most mutant virus particles. I conclude that the presence of an amphipathic helix in M2 is essential for virus replication, but it can be replaced by other curvature-inducing helices from cellular proteins.