Viruses,
such as influenza A, typically bind to the plasma membrane
of their host by engaging multiple membrane receptors in parallel,
thereby forming so-called multivalent interactions that are created
by the collective action of multiple weak ligand–receptor bonds.
The overall interaction strength can be modulated by changing the
number of engaged receptors. This feature is used by viruses to achieve
a sufficiently firm attachment to the host’s plasma membrane
but also allows progeny viruses to leave the plasma membrane after
completing the virus replication cycle. Design of strategies to prevent
infection, for example, by disturbing these attachment and detachment
processes upon application of antivirals, requires quantification
of the underlying multivalent interaction in absence and presence
of antivirals. This is still an unresolved problem, as there is currently
no approach available that allows for determining the valency (i.e.,
of the number of receptors bound to a particular virus) on the level
of single viruses under equilibrium conditions. Herein, we track the
motion of single influenza A/X31 viruses (IAVs; interacting with the
ganglioside GD1a incorporated in a supported lipid bilayer) using
total internal reflection fluorescence microscopy and show that IAV
residence time distributions can be deconvoluted from valency effects
by taking the IAV mobility into account. The so-derived off-rate distributions,
expressed in dependence of an average, apparent valency, show the
expected decrease in off-rate with increasing valency but also show
an unexpected peak structure, which can be linked to a competition
in the opposing functionalities of the two influenza A virus spike
proteins, hemagglutinin (HA), and neuraminidase (NA). By application
of the antiviral zanamivir that inhibits the activity of NA, we provide
direct evidence, how the HA/NA balance modulates this virus-receptor
interaction, allowing us to assess the inhibition concentration of
zanamivir based on its effect on the multivalent interaction.
Interferon γ (IFNG) is a key host response regulator of intracellular pathogen replication, including that of Chlamydia spp The antichlamydial functions of IFNG manifest in a strictly host, cell-type and chlamydial strain dependent manner. It has been recently shown that the IFNG-inducible family of immunity-related GTPases (IRG) proteins plays a key role in the defense against nonhost adapted chlamydia strains in murine epithelial cells. In humans, IFN-inducible guanylate binding proteins (hGBPs) have been shown to potentiate the antichlamydial effect of IFNG; however, how hGBPs regulate this property of IFNG is unknown. In this study, we identified hGBP1/2 as important resistance factors against C. trachomatis infection in IFNG-stimulated human macrophages. Exogenous IFNG reduced chlamydial infectivity by 50 percent in wild-type cells, whereas shRNA hGBP1/2 knockdown macrophages fully supported chlamydial growth in the presence of exogenous IFNG. hGBP1/2 were recruited to bacterial inclusions in human macrophages upon stimulation with IFNG, which triggered rerouting of the typically nonfusogenic bacterial inclusions for lysosomal degradation. Inhibition of lysosomal activity and autophagy impaired the IFNG-mediated elimination of inclusions. Thus, hGBP1/2 are critical effectors of antichlamydial IFNG responses in human macrophages. Through their capacity to remodel classically nonfusogenic chlamydial inclusions and stimulate fusion with autophagosomes, hGBP1/2 disable a major chlamydial virulence mechanism and contribute to IFNG-mediated pathogen clearance.
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